Drug Resistant Neoplasms [1 ed.] 9781613244746, 9781607412557

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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS SERIES

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DRUG RESISTANT NEOPLASMS

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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS SERIES Cell Apoptosis and Cancer Albina W. Taylor (Editor) 2007. ISBN: 1-60021-506-8 Chronic Lymphocytic Leukemia Research Focus Chadi Nabhan (Editor) 2007. ISBN: 1-60021-526-2 Cervical Cancer Research Trends Eleanor P. Bankes (Editor) 2007. ISBN: 1-60021-648-x Lung Cancer in Women Varetta N. Torres (Editor) 2008. ISBN: 1-60021-659-5

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Cancer Research at the Leading Edge Ignatius K. Martakis (Editor) 2008. ISBN: 1-60021-728-1 Chronic Lymphocytic Leukemia: New Research Inès B. Moreau (Editor) 2008. ISBN: 978-1-60456-081-7 Cancer and Stem Cells Thomas Dittmar and Kurt S. Zander (Editors) 2008. ISBN: 978-1-60456-478-5 Cancer Prevention Research Trends Louis Braun and Maximilian Lange (Editors) 2008. ISBN: 978-1-60456-639-0

Clinical, Genetic and Molecular Precursor Features in Colorectal Neoplasia Kjetil Søreide and Håvard Søiland (Editors) 2008. ISBN: 978-1-60456-714-4 Human Polyomaviruses: Molecular Mechanisms for Transformation and their Association with Cancers Ugo Moens, Marijke Van Gheule and Mona Johannessen 2009. ISBN: 978-1-60692-812-7 Molecular Therapy of Breast Cancer: Classicism Meets Modernity Marc Lacroix 2009. ISBN: 978-1-60741-593-0 Aromatase Inhibitors: Types, Mode of Action and Indications Jean R. Lamonte (Editor) 2009. ISBN: 978-1-60741-711-8 Anticancer Drugs: Design, Delivery and Pharmacology Peter Spencer and Walter Holt (Editors) 2009. ISBN: 978-1-60741-004-1 Lung Cancer in Women Varetta N. Torres (Editor) 2008. ISBN: 978-1-60692-765-6 (Online book)

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Cancer Biology: An Updated Global Overview Tarek H. EL-Metwally 2009. ISBN: 978-1-60876-193-7

Drug Resisant Neoplasms Ethan G. Verrite (Editor) 2009. ISBN: 978-1-60741-255-7

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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS SERIES

DRUG RESISTANT NEOPLASMS

ETHAN G. VERRITE

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

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All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Drug resistant neoplasms / editor, Ethan G. Verrite. p. ; cm. ISBN 978-1-61324-474-6 (eBook) 1. Drug resistance in cancer cells. I. Verrite, Ethan G. [DNLM: 1. Drug Resistance, Neoplasm. QZ 267 D79395 2009] RC271.C5D778 2009 616.99'4061--dc22 2009026399

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Contents Preface Chapter I

Chapter II

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Chapter III

Chapter IV

Chapter V

Chapter VI

xiv Therapeutic Implications of the Intrinsic and Acquired Resistance of Cancer Stem/Progenitor Cells in Inefficacy of Current Cancer Treatments and Disease Relapse Murielle Mimeault and Surinder K. Batra Role of O6-Methyl Guanine-DNA Methyl Transferase and the Effect of O6-Benzylguanine in Cancer Chemotherapy Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa, Masao Yamada, Susumu Kokeguchi, Hitoshi Nagatsuka, Tatsuo Yamamoto and You-Jin Lee The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy Drug Resistance: The Potential for Its Modulation to Achieve Therapeutic Gain A. Weickhardt and M. Michael Inherent and Microenvironment-Mediated Mechanisms of Drug Resistance Malathy P. V. Shekhar Studies on the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitor Gefitinib in NSCLC Cell Lines: Evidence that Ligand-Induced Endocytosis of EGFR via the Early/Late Endocytic Pathway is Associated with Gefitinib Sensitivity of NSCLC Cell Line Yukio Nishimura Mechanisms of Resistance to EGF Receptor-Tyrosine Kinase Inhibitor in NSCLC Cell Lines: Gefitinib Sensitivity is Closely Correlated with Ligand-Induced Endocytosis of Phosphorylated EGF Receptor Yukio Nishimura, Kiyoko Yoshioka and Kazuyuki Itoh

1

33

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viii Chapter VII

Targeting Adverse Features of Hormone-Resistant Breast Cancer Stephen Hiscox, Liam Morgan, Nicola Jordan, Julia Gee and Robert I. Nicholson

Chapter VIII

Systematic Analysis of Patterns of Cross Resistance between Anticancer Agents Britta Stordal and Ross Davey

165

Molecular Structure and Energy: Clinical Importance in DrugResistant Neoplasms Viroj Wiwanitkit

175

Chapter IX

Chapter X

Treating Drug-Resistant Malignancy Viroj Wiwanitkit

Chapter XI

Overcoming Ovarian Cancer Drug Resistance with Phytochemicals and other Compounds Marion M. Chan and Dunne Fong

Chapter XII

Index

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Contents

New Research Communications on Cancer Drug Resistance, Assessment of Cancer Drug Resistance with Nuclear Medicine Images Seigo Kinuya

149

181

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Preface One of the main causes of failure in the treatment of cancer is the development of drug resistance by the cancer cells. The design of cancer chemotherapy has become increasingly sophisticated, yet there is no cancer treatment that is 100% effective against disseminated cancer. Resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors, even those from the same tissue of origin. Frequently, resistance is intrinsic to the cancer, but as therapy becomes more and more effective, acquired resistance has also become common. The most common reason for acquisition of resistance to a broad range of anticancer drugs is expression of one or more energy-dependent transporters that detect and eject anticancer drugs from cells. Studies on the mechanisms of cancer drug resistance have yielded important information about how to circumvent this resistance to improve cancer chemotherapy and its implications for pharmacokinetics of many commonly used drugs. This book presents new and important research in this field. Chapter I - Recent progress in cancer research has provided an accumulating body of experimental evidence that the malignant transformation of tissue-resident adult stem/progenitor cells into cancer stem/progenitor cells may be at the origin of the most human aggressive and recurrent cancers. Small subpopulations of highly tumorigenic cancer stem/progenitor cells have been identified and isolated from primary and secondary malignant neoplasms including brain tumors and the majority of epithelial cancers. These immature and multipotent cancer-initiating cells were able to give rise to the bulk mass of abnormally differentiated cancer cells resembling to the original patient’s tumors. Importantly, the unique intrinsic properties of cancer stem/progenitor cells, including their high levels of expression and/or activity of diverse anti-apoptotic factors, multidrug ATPbinding cassette (ABC) transporters and DNA repair and detoxifying enzymes may contribute to their resistance to current anti-cancer therapies and disease relapse. Moreover, the acquisition of a more malignant behavior by tumorigenic cancer stem/progenitor cells including a migratory phenotype during cancer progression may also promote the development of more invasive and metastatic cancer subtypes. Therefore, the molecular targeting of the oncogenic signaling elements that may contribute to the sustained growth, survival and treatment resistance of highly tumorigenic and migrating cancer stem/progenitor cells is of great therapeutic interest. These novel targeting approaches should lead to the

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elimination of the tumor- and metastasis-initiating cells and their further differentiated progenies, and thereby improve the efficacy of current therapeutic regimens against the aggressive, metastatic, recurrent and lethal cancers. Chapter II - Drug resistance is a major problem in the chemotherapy of human cancers. It is essential to identify the key targets of chemotherapy for cancer, and molecular-based studies need to be conducted to provide a better understanding of drug sensitivity. In this chapter, the authors focus on the new candidate molecular targets for drug sensitivity. Their studies indicate that the DNA repair enzyme O6-methyl guanine-DNA methyl transferase (MGMT) and its inhibitor O6-benzylguanine (O6-BG) are the preferential molecular targets for alkylating agents and other anticancer drugs. MGMT is a DNA repair enzyme that rapidly repairs adducts at the O6-position of guanine, and its expression is known to modulate the effectiveness to alkylating agents. Alkylating agents may generate DNA adducts and may produce suicide inactivation of MGMT. The addition of O6-BG to anticancer drugs (i.e. alkylating agents, cisplatin and 5-FU) invariably enhanced their sensitivities in comparison with the response to those drugs when they are used singly. Their results provide valuable information on the relationship between MGMT expression and drug sensitivity in cancer chemotherapy. These findings will allow clinicians to identify the patients most likely to benefit from chemotherapy and potentially spare many patients from unnecessary therapy. Chapter III - Cancer cells grow within a host microenvironment that supports and nourishes their growth, enabling them to invade locally and metastasize to distant sites [1]. These supports can be either cellular in nature, comprising surrounding fibroblasts, pericytes and smooth muscles, or physical in nature due to the content and structure of the surrounding extracellular matrix with its associated interstitial hypoxia as well as high tumour interstitial fluid pressures (TIFP)—a direct consequence of abnormal tumoural vasculature. These components of the tumoural microenvironment can all contribute to difficulties in targeting and therapeutically inhibiting the growth of such tumours, quite distinct to the well-defined tumoural intracellular mechanisms of drug resistance [2]. Using drugs that target the cancer cells directly alone fails to account for this supporting tumoural microenvironment and can lead to therapeutic failure. The “seed and soil” hypothesis first proposed by Paget in 1889 suggests that both seed (the cancer cell) and soil (the tumour microenvironment) need to be targeted to achieve therapeutic success. The tumour microenvironment is therefore fertile ground for drug discovery, with many different agents currently in pre-clinical, early phase 1 and 2 trials, and with some drugs already being used successfully in practice. This review will discuss the pathophysiology of the tumoural microenvironment and the current success and research that aims to take advantage of this for therapeutic gain. Chapter IV - Current treatments are successful at debulking disease; however, development of resistance to treatment made evident by the recurrence of a primary tumor or distant metastasis is, sadly, a frequent occurrence. An important question is whether drug resistant neoplasms represent expansion(s) of residual primary tumor subpopulations that fail to respond to treatment, or represent tumor subpopulations that are activated or rejuvenated by the treatment. Eradication of primary and metastatic disease requires intervention strategies that will target both the tumor cells as well as its tumor microenvironment. In this chapter, the authors will focus on the roles of mechanisms that are inherent to tumor cells and the tumor microenvironment as contributors to the evolution of drug-resistant neoplasms. The

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Preface

xi

rationale for targeting the DNA damage tolerance or postreplication repair pathway as a novel tool for overcoming drug resistance is discussed. The need for carrying out drug sensitivity evaluations in clinically relevant model systems that take into account the threedimensional organization and in vivo relationship of a tumor with its microenvironment is also addressed. It is anticipated that such integrative efforts will yield a more global understanding of the tumor- and microenvironment-derived mechanisms involved in drug resistance as well as provide novel intervention targets that will abrogate interactions between a tumor’s cells and its microenvironment. Chapter V - The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, the author examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, the author provides novel evidence that efficient endocytosis of EGF-EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because, even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, the author demonstrates that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. Chapter VI - Gefitinib is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that functions by competing with ATP for binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells. It has exhibited significant anti-tumor activity against a broad range of mouse tumor xenograft models in vivo and non-small cell lung cancer (NSCLC) cell lines in vitro. The authors recently demonstrated that gefitinib-sensitive NSCLC cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were

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transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. In contrast, gefitinib-resistant NSCLC cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. To further investigate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, the authors examined the endocytic trafficking of phosphorylated EGFR (pEGFR) using confocal immunofluorescence microscopy in the absence or presence of gefitinib. In the gefitinib-sensitive PC9 cells and the gefitinib-resistant QG56 cells without EGF stimulation, a large number of pEGFRpositive small vesicular structures were not colocalized with late endosomes/lysosomes, but spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Moreover, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, the authors confirmed their previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Taken together, their data provides novel evidence that extensive impairment in pEGFR endocytosis via the early/late endocytic pathway might confer gefitinib-resistance in QG56 cells. Chapter VII - Endocrine therapy is the treatment of choice in hormone receptor-positive breast cancer. However, the effectiveness of anti-estrogenic agents is limited by the development of drug resistance, ultimately leading to disease progression and patient mortality. Cell models of endocrine resistance have demonstrated a role for altered growth factor signalling in the development of an endocrine-insensitive phenotype. Significantly, recent studies have revealed that the acquisition of endocrine resistance in breast cancer is also accompanied by the development of an adverse cellular phenotype, with such resistant cells exhibiting altered adhesive interactions, enhanced migratory and invasive behaviour and the ability to promote angiogenic responses in vitro. Elucidation of the molecular mechanisms underlying these adverse tumour cell features and their subsequent targeting may provide a means of limiting tumour spread in vivo and may ultimately improve the outcome for breast cancer patients on endocrine therapy. Chapter VIII - The cross resistance relationship between two chemotherapy agents is often decided by testing a new chemotherapy agent in cells resistant to an older agent. This can be very useful in determining the role for the newer agent and indeed be part of the process to approve the newer agent for use in the clinic. However, if only a few cell models are analysed, this can lead to incorrect conclusions about the activity of a given agent. The

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Preface

xiii

most rigorous method to evaluate the pattern of cross resistance between two chemotherapy agents is to perform a systematic review of all drug-resistant cell models reporting toxicity data for the two agents in question. This systematic review process must be updated regularly and fulfill certain criteria to be a valid analysis of the cross-resistance relationship between two agents. The authors have performed systematic reviews examining the cross-resistance relationships between the platinums (cisplatin, carboplatin and oxaliplatin) and the taxanes (paclitaxel and docetaxel). In this process they have observed three broad patterns of crossresistance relationships: complete cross resistance, incomplete cross resistance and inverse resistance. These different patterns can give insight into mechanisms of resistance and suggest the sequence in which drugs should be administered in the clinical treatment of cancer. Chapter IX - Drug-resistant neoplasms are a significant problem in oncology related to the deterioration in physiological function as well as some specific pathological disorders at the molecular level. In this chapter, the author will briefly review and discuss this aspect. Analyses of the structural component will be demonstrated and presented. In addition, details on the clinical importance of those analyses will be reported. Chapter X - A malignant tumor is an undesirable disorder. Growth without control is the nature of cancer. Drug-resistant cancer can be due to several causes; one common etiology is a defect at the molecular genetic level. How to treat drug-resistant cancer is still a main problem for oncologists at present. The concept of cancer treatment is eliminating the root cause—the neoplastic cells. Due to advents in biotechnology, there are many new options for the treatment of drug-resistant malignancy, by tackling problematic cancer at the molecular genetic level or expanding to the expression level. Some facts on reported data for drugresistant cancer will be discussed. Chapter XI - Ovarian cancer is the most lethal of all gynecologic malignancies in the western world. Worldwide there will be almost 200,000 cases diagnosed per year and approximately 115,000 deaths. Sixty percent of patients are diagnosed with an already advanced disease. Drug resistance and relapse frequently occur within 2 years of initial treatment. Current treatment is surgery followed by chemotherapy, usually a regimen of platinum/taxane combination. Platinum analogs such as cisplatin act by forming intrastrand cross-links with DNA, whereas taxanes such as paclitaxel act by binding to the cytoskeletal tubulin proteins. The two drugs are administered intravenously, although the intraperitoneal route has recently been shown to improve patient survival, in spite of demonstrated drug toxicity. Hence, searching for less toxic chemotherapeutic agents and strategies of drug resistance reversal are urgently needed, as well as novel therapeutic approaches based on concepts to prevent or circumvent drug resistance. Resistance can involve decreased drug uptake, increased drug efflux, increased repair of DNA damaged by chemotherapy, or reduced ability to undergo apoptosis. Drugs for platinum/taxane-resistant ovarian cancer cells are currently undergoing various stages of development, from cell culture studies to clinical trials. For example, TLK-286 (Telcyta), a glutathione S-transferase pi-activated glutathione analog prodrug, shows 15% response in a phase II trial. Monoclonal antibodies (against targets such as vascular epithelial growth factor) and RNA interference techniques (small interfering RNA against ATP-binding cassette transporter) are being tested. Other approaches include inhibition of the transcription factor nuclear factor kappa B, with BAY 11-7085, to

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increase the efficacy of cisplatin, and the use of a nitro derivative of aspirin, NCX-4016, to reverse cisplatin resistance. Many dietary phytochemicals have bioactivity as cancer chemopreventive agents. We discovered that curcumin (in the spice turmeric), quercetin (in fruits and vegetables such as apples and onions), and epigallocatechin-3-gallate (EGCG in green tea) enhance cisplatin susceptibility of both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Another group has shown that resveratrol (in grapes) also improves the effectiveness of cisplatin. In our view, EGCG, the green tea compound is the most promising, because it acts on a variety of cellular pathways and targets. An epidemiological study has concluded that tea consumption leads to a reduced risk of ovarian cancer. These phytochemicals are potentially useful in combination therapy to combat drug resistance in ovarian cancer. Chapter XII - Nuclear medicine images are sensitive methods for imaging cellular and molecular processes. Diverse mechanisms are involved in resistance of tumors to chemotherapy: poor delivery of drugs, cellular metabolism that reduces drug viability, tumor environment such as blood flow, angiogenesis, tissue oxygenation, drug efflux due to cell membrane transporters such as P-gp and MRP, degree of tumor proliferation and induction likelihood of cell apoptosis. All of these factors have been targeted in developing in vivo diagnosis of tissue characteristics regarding cancer drug resistance. A variety of positron emission tomography (PET) tracers has been designed. PET affords quantitative analysis that may measure picomolar levels of ligands in vivo. Although single photon emission tomography (SPET) is less sensitive to detect signals and its spatial resolution is lower than PET, development of SPET tracers is also important because of its availability of facility. In this paper, recent knowledge concerning nuclear detection of cancer drug resistance of tumors is summarized.

In: Drug Resistant Neoplasms Editor: Ethan G. Verrite

ISBN 978-1-60741-255-7 © 2009 Nova Science Publishers, Inc.

Chapter I

Therapeutic Implications of the Intrinsic and Acquired Resistance of Cancer Stem/Progenitor Cells in Inefficacy of Current Cancer Treatments and Disease Relapse Murielle Mimeault∗ and Surinder K. Batra∗ Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA

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Abstract Recent progress in cancer research has provided an accumulating body of experimental evidence that the malignant transformation of tissue-resident adult stem/progenitor cells into cancer stem/progenitor cells may be at the origin of the most human aggressive and recurrent cancers. Small subpopulations of highly tumorigenic cancer stem/progenitor cells have been identified and isolated from primary and secondary malignant neoplasms including brain tumors and the majority of epithelial cancers. These immature and multipotent cancer-initiating cells were able to give rise to the bulk mass of abnormally differentiated cancer cells resembling to the original patient’s tumors. Importantly, the unique intrinsic properties of cancer stem/progenitor cells, including their high levels of expression and/or activity of diverse anti-apoptotic factors, multidrug ATP-binding cassette (ABC) transporters and DNA repair and detoxifying enzymes may contribute to their resistance to current anti-cancer therapies and disease relapse. Moreover, the acquisition of a more malignant behavior by tumorigenic cancer stem/progenitor cells including a migratory phenotype during cancer progression may also promote the development of more invasive and metastatic cancer ∗

Phone: (402) 559-5455; Fax: (402) 559-6650; E-mail: [email protected] and [email protected]

Murielle Mimeault and Surinder K. Batra

2

subtypes. Therefore, the molecular targeting of the oncogenic signaling elements that may contribute to the sustained growth, survival and treatment resistance of highly tumorigenic and migrating cancer stem/progenitor cells is of great therapeutic interest. These novel targeting approaches should lead to the elimination of the tumor- and metastasis-initiating cells and their further differentiated progenies, and thereby improve the efficacy of current therapeutic regimens against the aggressive, metastatic, recurrent and lethal cancers.

Keywords: cancer stem/progenitor cells, cancer progression, epithelial-mesenchymal transition, metastasis, drug resistance, cancer therapies, molecular targeting

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Abbreviations ABC ALDH BTSCs CDK COX-2 CXCR4 EGF EGFR Fzd KIT HA MAPKs MEK MDR MMPs MGMT NF-κB NSCs PI3K PTCH PTEN Rb RTK SDF-1 SHH SMO TERT TK TR uPA

ATP-binding cassette; aldehyde dehydrogenase; brain tumor stem cells; cyclin-dependent kinase; clyooxygenase 2; chemokine receptor 4; epidermal growth factor; epidermal growth factor receptor; Frizzeled receptor; stem cell factor receptor; hyaluronan; mitogen-activated protein kinase; extracellular signal-related kinase kinase; multidrug resistance; matrix metalloproteinases; O6-methylguanine DNA methyltransferase; nuclear factor-kappa B; neural stem cells; phosphoinositide 3'-kinase; hedgehog patched receptor; phosphatase and tensin homolog deleted on chromosome 10; retinoblastoma; receptor tyrosine kinase; stromal cell-derived factor-1; sonic hedgehog ligand; smoothened co-receptor; telomerase reverse transcriptase; tyrosine kinase; telomere RNA component; urokinase-type plasminogen activator;

Therapeutic Implications of Cancer Stem/Progenitor Cells VEGF VEGFR Wnt

3

vascular endothelial growth factor; vascular endothelial growth factor receptor Wingless ligand

Introduction

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Major advances in adult stem/progenitor cell biology have allowed researchers to identify certain specific physiological functions of these immature cells and their early progenies endowed with a self-renewal and multi-differentiation potential [1-8]. The tissueresident adult stem/progenitor cells with a long longevity generally provide critical roles in the replenishment of cells in homeostatic conditions and after intense injuries along the lifespan of individuals (Figure 1A) [1-8]. In counterpart, recent lines of experimental evidence have revealed that the accumulation of genetic and/or epigenetic alterations in adult stem/progenitor cells may result in their malignant transformation into cancer stem/progenitor cells also designated as cancer- or tumor-initiating cells (Figure 1B and 2) [4,5,9-15].

Figure 1. Hierarchical model of the clonal expansion and differentiation of adult stem/progenitor cells during epithelial tissue regeneration in physiological conditions and cancer initiation and progression through their malignant transformation. This scheme shows (A) the symmetric or asymmetric division of normal tissue-resident adult stem cells (SC) into transit-amplifying (TA)/ intermediate cells that in turn can regenerate the bulk mass of further differentiated cells in the tissue from the origin in homeostatic conditions or after tissue injury. Moreover, this scheme also shows (B) the malignant transformation of adult stem/progenitor cells into tumorigenic cancer stem/progenitor cells (CSCs), which may be induced through the genetic and/or epigenetic alterations in these immature cells and the changes in their local microenvironments including the activated stromal cells. The cancer stem/progenitor cells endowed with an aberrant differentiation potential can give rise to the malignant TA cells that in turn can generate the bulk mass of further differentiated cancer cells.

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Murielle Mimeault and Surinder K. Batra

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The highly tumorigenic and migrating cancer stem/progenitor cells can provide critical roles for primary tumor growth, metastases at distant tissues and organs, resistance to current conventional therapies and disease relapse [4,5,9-15]. In support with the major implication of cancer stem/progenitor cells in cancer initiation and progression, the small subpopulations of immature cancer stem/progenitor cells with stem cell-like properties, comprising about 0.1–3% of total tumor cell mass, have been identified in most human cancers [5,10,14,16-49]. The cancer stem/progenitor cells typically express several specific stem cell-like markers such as telomerase, aldehyde dehydrogenase (ADLH), CD133, CD44, ATP binding-cassette (ABC) multidrug transporters and/or CXC chemokine receptor-4 (CXCR4) but lack differentiation marker expression [5,10,14,16-50]. The highly tumorigenic cancer stem/progenitor cells were able to give rise in vitro and in vivo to further differentiated tumor cells expressing phenotypes of the patient’s original tumors. In addition, it has been shown that cancer development is generally associated with the occurrence of distinct molecular events in cancer stem/progenitor cells and their progenies that may contribute to their acquisition of a more malignant behavior [10,51-58]. The inactivation of diverse tumor suppressor gene products combined with a stimulation of diverse oncogenic signaling elements may lead to cancer initiation (Figure 2) [10,22,59-64].

Figure 2. Model of epithelial cancer initiation and progression mediated through tumorigenic and migrating cancer stem/progenitor cells. The scheme shows the cancer initiation through the accumulation of genetic abnormalities in tissue-resident adult stem cells. The asymmetric division of cancer stem cells localized in the basal compartment into transit-amplifying (TA) cancer progenitor cells can generate the bulk mass of differentiated cancer cells constituting the solid tumor. Furthermore, the transformation of tumorigenic stem/progenitor cells into migrating cancer progenitor cells, which may be induced by the sustained activation of distinct growth factor signaling during the epithelialmesenchymal transition (EMT) program, is also shown. The invasion of tumorigenic and migrating cancer stem/progenitor cells in the activated stroma which may lead to their dissemination through the peripheral circulation at distant sites and metastases is also illustrated. The new cancer therapies by molecular targeting of tumorigenic and migrating cancer stem/progenitor cells to counteract cancer progression and metastases at distant sites are also indicated.

Therapeutic Implications of Cancer Stem/Progenitor Cells

5

Additionally, the changes in the local microenvironment of cancer stem/progenitor cells including the release of soluble factors by host activated stromal cells during epithelialmesenchymal transition (EMT) process may also promote the development of the cancer (Figure 2) [10,55-58]. Importantly, the results from numerous recent studies have also revealed that the tumorigenic and migrating cancer stem/progenitor cells may be more resistant than their differentiated progenies to current clinical cancer therapies [7,10,28,29,32,35-44,48,57,6569]. Consequently, the persistence of cancer-initiating cells at the primary and secondary malignant neoplasms after treatment initiation may be responsible for the tumor re-growth and disease relapse. In this matter, we describe here the molecular oncogenic events that frequently occur during the early and late stages of cancer progression. The emphasis is on the deregulated gene products in tumorigenic and migrating cancer stem/progenitor cells that can contribute to the treatment resistance and cancer recurrence and new targeting therapies. The provided information on potential biomarkers and molecular therapeutic targets in tumor-initiating cells and their progenies should help to develop more effective diagnostic and prognostic tests and therapeutic treatments against the aggressive, recurrent and lethal cancers.

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Functions of Tumorigenic and Migrating Cancer Stem/Progenitor Cells in Cancer Initiation, Progression and Metastases Numerous factors may influence the risk of developing a cancer including inherited or somatic DNA mutations, intense oxidative stress, tobacco smoking, environmental carcinogens and chronic inflammatory and fibrotic atrophies and age of individuals [5,9,10,12-14,50,70-72]. Although the precise etiological causes responsible of cancer initiation remain not precisely established, the cancer development is usually associated with a cumulative genotoxic stress in cells which may cause chromosomal instability leading to the changes in the expression levels and/or activity of many deregulated gene products [9,10,50,72-86]. The most of human cancers appear to originate from sequential genetic abnormalities in tissue-resident adult stem/progenitor cells concomitant with the changes in their microenvironments that lead to their malignant transformation in cancer stem/progenitor cells (Figure 1) [14,55-57,87]. These molecular transforming events in adult stem/progenitor cells may promote their sustained proliferation and aberrant differentiation, and thereby disrupt the normal mechanisms of tissue regeneration. Numerous recent studies suggest that the cancer development may derive from the clonal expansion and aberrant differentiation from cancer stem cells (CSCs) and/or their early progenies endowed with a self-renewal capacity that trigger the tumor growth [14,55-57,87]. In analogy with the normal tissue regeneration process, CSCs can generate, through an asymmetric division, the daughter cells designated as transit amplifying (TA) progenitor cells with a malignant phenotype (Figs. 1 and 2) [14,55-57,87]. The malignant TA cells, in turn, can give rise to a heterogeneous population of poorly-, moderately- and terminally-differentiated cells with aberrant functions [14,55-57,87]. In support with the critical functions of tumorigenic cancer stem/progenitor

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cells in cancer development, recent investigations have led to identification and isolation of different cancer-initiating cells in the most of common human solid tumors and established cancer cell lines [14,55-57,87]. Among the malignant tissues harboring a very small subpopulation of cancer stem/progenitor cells, there are different brain tumor types and a variety of epithelial cancers including skin, head and neck, thyroid, lung, cervical, renal, hepatic, esophageal, gastrointestinal, colon, bladder, pancreatic, prostatic, mammary and ovarian cancers [5,10,14,16-49]. It has been shown that the small subpopulations of isolated cancer stem/progenitor cells with stem cell-like properties displayed a greater clonogenic potential in vitro and can generate tumors or metastasize with a higher incidence as compared to their differentiated progenies in animal model in vivo [5,10,14,16-49]. Moreover, the cancer progression is also associated with the acquisition of a more malignant behavior by tumorigenic stem/progenitor cells and their progenies.

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Molecular Transforming Events in Cancer Stem/Progenitor Cells and Their Progenies Associated with Tumor Development The transition of non-malignant hyperproliferative lesions to well established cancers and disease progression to locally invasive and metastatic states has been associated with the occurrence of some transforming events in cancer cells and their microenvironment [14,50,55-58,60,87]. The accumulation of genetic and/or epigenetic alterations in cancer stem/progenitor cells and their progenies may lead to an aberrant expression and/or activation of a complex network of cellular signaling elements. The transforming events include the activation of telomerase, inactivating mutations in numerous tumor suppressor gene products [p16INK4A, pRb, p53 and/or PTEN] or constitutive activation of diverse growth factors and oncogenic signaling products [Ras, Myc, NF-κB, PI3K/Akt/mTOR, Bcl-2, survivin and/or fusion proteins resulting from chromosomal rearrangements] (Figure 2) [9,10,61,63,73-75,7780,82-86,88]. Particularly, the telomere shortening during chronological aging may promote the chromosomal instability in tissue-resident adult stem cells, which in turn may trigger the early stage of carcinogenesis [74,75,89-93]. A subsequent up-regulation of telomerase activity and down-regulation of cell cycle checkpoint pathways may allow adult stem cells and their progenies to bypass the senescence process or programmed cell death by apoptosis, and thereby induce a level permissive for cancer initiation and progression. In support with this model of age-related carcinogenesis, it has been observed that the cancer cells in precancerous lesions, such as ductal carcinoma in situ for breast cancer and prostatic, cervical and pancreatic intraepithelial neoplasias (PINs), are characterized by shortened telomeres [92,94]. Moreover, it has been reported that a re-activation of telomerase activity may lead to an immortalization of human cells while the subsequent up-regulated expression and/or activity of diverse hormones, growth factors, cytokines and/or their cognate receptors may result in cancer development [74,75,92,95]. Particularly, the sustained activation of diverse developmental cascades including hedgehog, epidermal growth factor (EGF)-EGFR system, Wnt/β-catenin, Notch, stromal cell-derived factor-1 (SDF-1)-chemokine receptor 4 (CXCR4) and/or polycomb group (PcG) protein signaling pathways in tumorigenic cancer stem/progenitor cells may give to them a more malignant behavior [10,13,14,22,44,54-

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56,61,66,87,96-99]. The stimulation of these distinct tumorigenic signaling pathways may contribute to the sustained growth, survival and invasion of cancer cells during cancer progression [10,13,14,44,52,54-56,61,66,87,96-99]. In general, the cancer development is also accompanied by an enhanced glycolysis in cancer cells including cancer stem/progenitor cells [58,100-102]. This phenomenon known as Warburg effect may contribute to the resistance of cancer cells to oxidative stress as well as their survival in intratumoral hypoxic conditions [58,100-102]. Moreover, the epithelial-mesenchymal transition (EMT) phenomenon, which occurs during embryonic development and wound healing, is also reactivated during the progression from numerous aggressive cancers such as brain, skin, prostate, ovarian, mammary, hepatic, gastrointestinal, pancreatic and colorectal carcinomas [51-54,103-109].

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Molecular Events Associated With the EMT Process, Invasion and Metastasis of Cancer Stem/Progenitor Cells The acquisition of a more malignant behavior by cancer cells during EMT process is a determinant factor that may contribute to cancer progression to locally invasive forms [5154,103-109]. The frequent gene products altered in the cancer cells during EMT program include a decreased expression of E-cadherin concomitant with an up-regulation of different signaling elements such as N-cadherin, vimentin, snail, slug, twist, β-catenin, CXCR4 and anti-apoptotic factors that may contribute to their invasive phenotype and enhanced treatment resistance [51-54,103-109]. Particularly, the acquisition of a migratory phenotype by tumorigenic cancer stem/progenitor cells during EMT process, may result to the their migration to distant sites and progression from organ-confined cancers to metastatic disease states (Figure 2) [51-54,103-109]. In addition, the cancer progression is also accompanied by an extensive tumor stromal remodeling of the extracellular matrix (ECM) components and changes in the gene expression patterns in the tumor-associated activated stromal cells including myofibroblasts and/or stellate cells as well as infiltrating circulating endothelial progenitor cells (EPCs) and immune cells such as macrophages [6,7,10,14,55-57,103,110-118]. The tumor stromal cells can secrete a variety of soluble growth factors and cytokines, such as EGF, insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and TGF-β as well as matrix metalloproteinases (MMPs) and urokinase plasminogen (uPA) in reactive stroma. These soluble factors may promote, of a paracrine manner, the malignant transformation of cancer stem/progenitor cells and their progenies during the EMT process (Figure 2) [6,7,10,14,53,55-57,103,118-125]. Moreover, the secretion of diverse angiogenic factors by myofibroblasts may stimulate the tumor neovascularization process and invasion of tumorigenic and migrating cancer stem/progenitor cell into reactive stroma and metastasis at distant sites (Figure 2) [6,7,10,14,55-57,111,118,125]. Hence, the persistence of highly tumorigenic and migrating cancer stem/progenitor cells at the primary and secondary neoplasms after treatment initiation may provide critical roles for the re-growth of tumors, and disease relapse. We are reporting here the phenotypic and functional properties common

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to several cancer stem/progenitor cells that may contribute to their intrinsic and/or acquired resistance to current therapeutic treatments and disease recurrence.

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Intrinsic and Acquired Phenotypes of Cancer Stem/Progenitor Cells Associated with Their Resistance to Current Cancer Treatments Numerous lines of experimental evidence indicated that the tumorigenic cancer stem/progenitor cells may be intrinsically resistance to certain chemotherapeutic drugs at start of treatment and acquire an enhanced multidrug resistance (MDR) phenotype with cancer development [10,35,57,65,66,87,99,126-130]. Several intrinsic properties of cancer stem/progenitor cells common with their normal counterpart, adult stem/progenitor cells, may notably contribute to their resistance to current clinical therapeutic treatments and disease recurrence. These phenotypic features include their slow division, high expression levels of anti-apoptotic factors (Bcl-2, survivin and NF-kB), ATP-binding cassette (ABC) multidrug transporters and DNA repair and intracellular detoxifying enzymes (ALDH and O6methylguanine DNA methyltransferase “MGMT”) [10,35,57,64-66,72,87,99,126-138]. Particularly, the primitive cancer stem/progenitor cells like adult stem/progenitor cells may exist under a quiescent and less metabolically active state, and thereby they may be more resistance than their differentiated progenies to cytotoxic drugs targeting the proliferative cancer cells [10,56,57]. This quiescent state of tumor-initiating cells may explain, at least in part, the dormancy phenomenon associated with their persistence at the primary and metastatic neoplasms and disease recurrence following the re-activation of their proliferation. Furthermore, the large family of ABC multidrug transporters comprises diverse transmembrane proteins constituted of one or two transmembrane spanning domains (TMDs) involved in substrate binding and one or two cytoplasmic nucleotide (ATP)-binding domains (NBDs) [57,139-148]. Among the ABC multidrug transporters frequently expressed on primitive cancer stem/progenitor cells, there are multidrug resistance 1 (MDR1/ABCB1) encoding P-glycoprotein (P-gp), breast cancer resistant protein (BCRP-1/ABCB2) and multidrug resistance associated proteins (MRPs) (Figure 2) [57,144-146,149-151]. The ABC transporters localized at the plasmic membrane may protect these immature cancer cells from cytotoxic effects induced by diverse structurally and functionally non-related chemotherapeutic drugs and in this manner contribute to their MDR phenotype (Figure 2) [57,65,131,146,148,149]. In fact, the ABC efflux pumps can activately remove the intracellular cytotoxic agents outside from the cells at the expanse of the hydrolysis of ATP molecules. Certain ABC transporter types, including ABCA3 and ABCB2, are also localized in endolysosomal compartment in cancer cells including cancer stem/progenitor cells (Figure 2) [142,152,153]. These intracellular ABC transporters found in lysosomal membrane can mediate a sequestration of intracellular toxic compounds, including the chemotherapeutic drugs, and thereby induce a decrease of the intracellular drug concentration that promotes the MDR phenomenon [142,152,153]. In the same pathway, the high expression levels of DNAmismatch repair and detoxifying enzymes in cancer stem/progenitor cells may be responsible in part for their resistance to certain chemotherapeutic drugs or ionizing radiation. For

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instance, a high expression level of MGMT in cancer stem/progenitor cells may protect them against the cytotoxic effects induced by methylating and chloroethylating agents [carmustine, 1,3-bis (2-chloroethyl)-1-nitrosourea or BCNU, methyl methanesulfonate (MMS) and temozolomide (TMZ)] by repairing alkyl adducts at the O6-position of guanine in DNA [65,66,132,154-156]. Furthermore, the cancer-initiating cells found in retinoblastoma, brain, breast, pancreatic and colorectal cancers also express a high level of ALDH activity which may contribute to the detoxification of a variety of compounds such as the active metabolite of an alkylating agent, cyclophosphamide and treatment resistance [28,134,157-160]. In addition, the acquisition of a more malignant phenotype by cancer stem/progenitor cells during cancer progression has also been associated with the occurrence of more aggressive cancers displaying a high rate of growth, metastasis and therapy resistance. In respect with this, we discuss accumulating lines of evidence suggesting that the acquisition of distinct phenotypes by cancer-initiating cells during cancer progression may lead to the formation of different cancer subtypes that differently respond to current cancer treatments.

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Heterogeneity of Cancers Derived from Distinct Tumorigenic and Migrating Cancer Stem/Progenitor Cells and Their Differential Response to Current Clinical Treatments Numerous investigations revealed that the occurrence of different malignant transforming events in adult stem/progenitor cells during cancer initiation as well as an accumulation of distinct genetic and/or epigenetic alterations in cancer stem/progenitor cells along cancer progression may lead to the development of different cancer subtypes [6,7,10,14,20,25,26,5356,58,59,83,117,118,161-187]. This heterogeneity of cancers has important repercussion since these distinct cancer subtypes may differentially respond to current cancer therapies. For instance, the expression of mesenchymal genes and the acquisition of a migratory phenotype by poorly- or moderately-differentiated tumorigenic cancer stem/progenitor cells during the EMT program may result in the formation of highly invasive cancer subtypes characterized by a poorly- to moderately-differentiated state [55,185-187]. In contrast, the tumorigenic cancer stem/progenitor cells that do not undertake the EMT transition could rather give rise to weakly invasive cancer subtypes [55]. In support with this model of heterogeneity of cancers, at least five subtypes of breast cancer have been identified based on the gene expression signatures of cancer cells and their invasive phenotype. The classification of breast cancer includes basal-like (CK5/6+, estrogen receptor “ERα-”, progesterone receptor “PR-”, erbB2-/low, EGFR+, vimentin+ and KIT+); erbB2/HER2+ overexpressing (ERα- and PR-); luminal A (ERα+ and/or PR+ and erbB2-); luminal B (ERα+ and/or PR+ and erbB2+) and normal breast cancer subtype (high expression of normal epithelium genes and low expression of luminal epithelial gene products) [161,185-194]. The ERα- basal-like and erbB2-overexpressing subtypes, which are among the most aggressive and recurrent breast cancers, are generally associated with a poor prognosis and patient survival relative to differentiated ERα+ breast cancer subtypes [188,190-192,195-198]. Collectively, these observations suggest that the malignant transformation of poorly differentiated ERα- progenitor cells in basal compartment of breast epithelium may result in

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the most aggressive breast cancer subtypes that are less responsive to current clinical chemotherapies. In support with this, it has been reported that the targeted expression of stabilized β-catenin in the basal myoepithelial cells of mouse mammary epithelium resulted in an enhanced proliferation of basal-type cell-like progenitors possessing an abnormal differentiation potential, whose oncogenic event led to the development of invasive basaltype carcinomas [165]. The ERα- breast cancer cells, which did not express the metastasisassociated gene 3 (MTA3) that inhibits snail transcriptional activity, also possessed a lower level of E-cadherin and displayed a higher migratory capacity than the ERα+ breast cancer cells [199]. Moreover, an increased expression of NF-kB in ERα- breast cancer cells may lead to an induction of EMT program throughout the stimulation of transcription factor RelB and an enhanced expression of the anti-apoptotic protein Bcl-2 that may contribute to treatment resistance [200]. In this matter, a differently expressed gene pattern, designated as invasiveness 186-gene signature (IGS), has also been detected in CD44+CD24−/low tumorigenic breast cancer stem/progenitor cells relative to that of normal breast epithelial cells and associated with a poor overall survival of patients with breast cancer [201]. Among the genes expressed in this very little population of CD44+CD24−/low tumor-initiating cells, there are the gene products associated with the NF-κB and MAPK pathways, and epigenetic control of gene expression [201]. The results from another study have also revealed that the TGF-β pathway may be specifically activated in CD44+ breast cancer cells and its inhibition induced a more epithelial phenotype, suggesting that the targeting of TGF-β signaling could represent a potent therapeutic strategy to prevent the EMT process [202]. In this same pathway, the glioblastoma multiformes (GBMs), which represent a heterogeneous population of cancer cells, may also arise from the malignant transformation of neural stem cells (NSCs) that acquire the mesenchymal properties like mesenchymal stem cells and give rise to further differentiated progenies [19,20,54]. Primary GBMs, which are aggressive brain cancers that are frequently accompanied by the overexpression of EGFR, typically progress rapidly without evidence of a transitory step of lower-grade tumor. In this regard, it has been reported that CD133+ cells found in three primary cell lines established from glioblastoma patients, expressed high mRNA levels of BCRP1/ABCG2 multidrug efflux pump and anti-apoptotic products [66]. The isolated CD133+ glioblastoma cells were also more resistant to chemotherapeutic agents, such as temozolomide, carboplatin, etoposide and paclitaxel, as compared to the CD133- cell fraction [66]. Importantly, the higher levels of CD133 stem cell-like surface marker were also expressed in recurrent GBM tissues obtained from five patients relative to their respective newly diagnosed tumors [66]. On the opposite end, the secondary or progressive GBMs, which are often characterized by mutations in the p53 suppressor gene, appear to derive from low-grade tumors that did not show the changes in the gene expression pattern that are usually associated with the EMT program [54]. Hence, the clinical management of these two different brain cancer subtypes with different aggressivity and phenotypic markers generally require distinct types of therapeutic treatments. In addition, the changes in the local microenvironment of tumorigenic cancer stem/progenitor cells and their progenies including their localization within hypoxic zones of solid tumors may result in the expression of a different subset of oncogenic gene products during cancer development and be responsible, at least in part, for the intratumoral

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heterogeneity [55,58]. This line of thought is well-supported by the observations that have indicated that certain invasive cancer types, such as mammary, ovarian, prostatic, pancreatic, gastric, colorectal and squamous cell carcinomas, may harbor an intratumoral heterogeneity [14,20,32,53-57,87,181]. These invasive cancer subtypes typically display distinct proliferating and differentiating regions, including a preferential localization of migrating cancer stem/progenitor cells at the intratumoral hypoxic zones and invasive front (Figure 2) [14,20,32,53-58,87,181]. For instance, the data from immunohistochemical analyses of pancreatic adenocarcinoma specimens from patients have revealed the presence of two different subpopulations of pancreatic cancer stem/progenitor cells, including tumorigenic CD133+ cells and migrating CD133+/CXCR4+ localized in the bulk mass and invasive front of pancreatic tumor, respectively [32]. The migrating CD133+/CXCR4+ cells may then correspond to a more malignant cell subpopulation than tumorigenic CD133+ cells, which may have acquired a migratory phenotype during the EMT program, and thereby they may be involved in invasion and metastases to distant sites. Consistently, it has been observed that the depletion of CD133+/CXCR4+ migrating cancer stem/progenitor cells effectively abrogated the metastatic capacity of pancreatic tumors without altering their tumorigenic potential [32]. In light of these observations, it appears that different cancer subtypes may originate from distinct tumorigenic cancer stem/progenitor cells that acquire a specific oncogenic gene profile during cancer development. Therefore, the management of these cancer subtypes may require different therapeutic strategies. In respect with this, we describe the potential implications of cancer stem/progenitor cells to inefficacy of current cancer therapies and disease relapse as well as new targeting approaches that have been developed to eradicate the total cancer cell mass including tumor-and metastasis-initiating cells and their differentiated progenies.

Current Cancer Therapies Major progress in developing of earlier diagnostic tests for the cancer patients in the few last years has led to a significant improvement of curative rate in the treatment of localized cancers by surgical resection, anti-hormonal therapy, radiation and/or chemotherapy [7,11,13,14,57,87,203]. Unfortunately, the rapid progression of organ-confined cancers to locally invasive or metastatic disease stages may however results to the disease relapse and death of patients [7,11,13,14,57,87,203]. In fact, although the current therapies may be effective to eradicate the bulk mass of cancer cells, the resistance of cancer stem/progenitor cells to current therapeutic treatments may lead to their persistence at the primary and secondary neoplasms and disease recurrence (Figure 2) [7,10,11,13,14,28,29,32,3544,48,57,65-69,87,203]. This inefficacy of current treatments against aggressive and metastatic cancers underlines the importance to target the tumor- and metastasis-initiating cells to counteract the cancer progression and disease relapse.

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Novel Cancer Therapies by Targeting Tumor- and Metastasis-Initiating Cells and Their Microenvironment The development of locally advanced, invasive and metastatic cancers that are resistant to current therapies represents one of the major causes of disease recurrence and cancerrelated deaths [7,11,13,14,57,87,203]. Therefore, the molecular targeting of oncogenic products in cancer cells involved cancer progression into locally invasive and metastatic disease stages offers great promise for developing new therapeutic options against aggressive and recurrent cancers. Since recent studies revealed that only a small subpopulation of tumorigenic and/or migrating cancer stem/progenitor cells could possess a high potential to drive tumor growth and metastases at distant sites, the molecular targeting of deregulated signaling elements in these tumor-initiating cells must then be considered (Figure 2) [7,11,13,14,57,87,203]. Of particular interest, we review the novel targeting strategies design to eradicate the total cancer cell mass consisting of tumorigenic and migrating cancer stem/progenitor cells and their progenies, and which could be used for improving the current treatments against the locally invasive, metastatic and recurrent cancers.

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Molecular Targeting of Tumorigenic and Migrating Cancer Stem/Progenitor Cells The molecular targeting of the deregulated signaling elements that mediate the transforming events occurring in tumorigenic and migrating cancer stem/progenitor cells during cancer progression, and more particularly during the EMT process, represents new promising therapeutic strategies to improve the current cancer therapies [10,13,14,5557,87,203]. Among the potential molecular targets often altered in tumor-initiating cells, there are diverse developmental signaling pathways. These deregulated pathways include hedgehog, EGFR, Wnt/β-catenin, Notch, hyaluronan (HA)/CD44, interleukin (IL-4)/IL-4Rα, BMI-1, stem cell factor (SCF)/KIT, extracellular matrix (ECM)/integrin and/or SDF1/CXCR4 signaling elements (Figure 3) [10,13,14,55,56,68,87,98,158,203,203-211]. It has been reported that the blockade of these tumorigenic pathways by using specific inhibitor or antagonists, monoclonal antibody (mAb) or antisense oligonucleotides (As) led to a growth inhibition, apoptotic cell death and/or a reduction of invasiveness or metastatic spread of tumor-initiating cells and their progenies in vitro or in animal models in vivo (Table 1; Figure 3) [10,13,14,55,56,,68,87,98,158,203,203-211]. For instance, the inhibition of smoothened (SMO) hedgehog signaling element by cyclopamine has been observed to eliminate the total cancer cell mass including tumor-initiating cells and improve the cytotoxic and antimetastatic effects induced by the current chemotherapeutic drugs on gliomasphere cells as well as prostatic and pancreatic cancer cell lines in vitro and/or in vivo [158,208,212].

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Table 1. Potential cancer therapeutic targets involved in sustained growth, survival, invasion and drug resistance of cancer stem/progenitor cells

ALDH, aldehyde dehydrogenase; As, antisense; BCRP, brain cancer resistance protein; COX-2, cyclooxygense; CXCR4, CXC chemokine receptor 4; DAPT, N-(N-3,5-difluorophenacetyl)-Lalanyl]-S-phenylglycine t-butyl ester; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FTI, farnesyl transferase; IkBα, inhibitor of nuclear factor-kBα; mAb, monoclonal antibody; MDR-1, multidrug resistance 1; MGMT, O6methylguanine DNA methyltransferase; MRP-1, multidrug resistance protein 1; NF-κB, nuclear factor-kappaB; P-gp, P-glycoprotein; PI3K, phosphatidylinositol-3’ kinase; RT, reverse transcriptase; SDF-1, stromal cell-derived factor-1; SMO, smoothened; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; WIF-1, Wingless inhibitory factor-1 and Wnt, Wingless ligand.

The molecular targeting of IL-4, whose cytokine may protect the tumorigenic CD133+ cancer stem/progenitor cells from human colon carcinoma of apoptotic death, by using IL4Rα antagonist or anti-IL-4 neutralizing antibody, also sensitized these tumor-initiating cells to the anti-tumor effects induced by standard chemotherapeutic drugs [68]. Furthermore, the blockade of the SDF-1/CXCR4 axis by using a monoclonal antibody directed against SDF-1

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or CXCR4 or selective CXCR4 antagonists, such as 14-mer peptide (TN14003) and AMD 3100, has also given promising results to counteract the migration of cancer stem/progenitor cells to distant sites and metastases (Figure 3) [6,7,10,14,55,56,118,203,213-222].

Figure 3. New cancer therapies by molecular targeting distinct signaling pathways involved in aggressive behavior and multidrug resistance phenotype of cancer stem/progenitor cells. The inhibitory effects induced by diverse pharmacological agents such as the selective inhibitors of receptor tyrosine kinase (RTK) activity, smoothened (SMO) hedgehog signaling element (cyclopamine) and telomerase on cancer stem/progenitor cells are indicated. Moreover, the anti-carcinogenic effects induced by a monoclonal antibody (mAb) directed against IL-4 ligand, CD44 receptor, Wnt ligand, SDF-1 or CXCR4 antagonist are shown. Particularly, the anti-proliferative, anti-invasive and apoptotic effects induced by these pharmacological agents in cancer stem/progenitor cells through the down-regulation of the expression levels of numerous gene products are indicated. In addition, the potent inhibitory effect mediated by the specific inhibitors of the ABC multidrug transporters on drug efflux concomitant with intracellular drug accumulation is also illustrated.

Other potential molecular targets also comprise the gene products that are frequently involved in sustained growth, enhanced survival and invasion during the EMT process and/or drug resistance of cancer stem/progenitor cells and their progenies (Table 1) [6,7,13,14,55,57,65,98,118,158,200,223-225]. These cellular signaling effectors include telomerase, Cripto-1, tenacin C, NF-kB, PI3K/Akt/mTOR, Bcl-2, survivin, snail, slug, twist and/or ABC multidrug efflux pumps as well as deregulated apoptotic signaling elements such as ceramide and caspases. In addition, the induction of the differentiation of cancer stem/progenitor cells by using agents such as retinoic acid and its synthetic analogues, interferons (IFNs) or histone deacetylase inhibitor, also may represent a promising adjuvant therapeutic strategy [226-230]. For instance, it has been reported that the IFN-α treatment

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caused a dramatic reduction in verapamil-sensitive SP cell fraction from diverse ovarian cancer cell lines [230].

Molecular Targeting of the Local Microenvironment of Tumorigenic and Migrating Cancer Stem/Progenitor Cells Since the local microenvironment of cancer stem/progenitor cells also plays an active role in cancer progression, well efforts are being made to counteract cancer progression by targeting the host stromal cells. In particular, the targeting of the myofibroblasts and immune cells that support the malignant transformation of cancer stem/progenitor cells as well as the use of anti-angiogenic agents may constitute an adjuvant treatment for counteracting cancer progression to metastatic and lethal disease states [6,7,10,13,55-57,112,114,118,222,231238]. More specifically, the combined use of the cytotoxic agents targeting cancer stem/progenitor cells plus a selective inhibitor of the angiogenic process, such as cyclooxygenase-1 or 2 (COX-1 or -2), NF-kB and/or VEGF-VEFGR, may represent the effective strategies to prevent disease relapse (Table 1) [239-251]. As a matter of fact, it has been observed that the treatment of the mice-bearing orthotopic U87 glioma cell xenografts with an anti-VEGF monoclonal antibody called bevacizumab noticeably reduced the microvasculature density and tumor growth. This anti-carcinogenic effect was also accompanied by a decrease of the number of vessel-associated self-renewing CD133+/nestin+ tumor-initiating cells [236].

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Conclusions Recent advances in basic and clinical oncology have revealed that tumorigenic and migrating cancer stem/progenitor cells provide critical functions in tumor formation, metastases treatment resistance and disease relapse. Consequently, the molecular targeting of tumor- and metastasis-initiating cells and their microenvironment may represent a potential strategy for improving the efficacy of current cancer treatments. Future investigations are still essential to more precisely determine the specific biomarkers and altered gene products occurring in tumor- and metastasis-initiating cells and the changes in their niches during cancer development. These studies should lead to identification of new biomarkers and molecular therapeutic targets that could be exploited to develop new diagnostic and prognostic methods and preventive and therapeutic approaches for treating and even curing patients diagnosed with locally advanced, invasive, metastatic and recurrent cancers.

Acknowledgments The authors of this work are supported by grants from the National Institutes of Health (CA78590, CA111294, CA133774 and CA131944). We thank Ms. Kristi L. Berger for editing the manuscript.

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References [1] [2] [3] [4]

[5]

[6] [7]

[8] [9]

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[10]

[11] [12]

[13]

[14]

[15]

[16]

Bryder D., Rossi D.J. and Weissman I.L. (2006). Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am. J. Pathol. 169: 338-346. Wilson A. and Trumpp A. (2006). Bone-marrow haematopoietic-stem-cell niches. Nat. Rev. Immunol. 6: 93-106. Arai F. and Suda T. (2007). Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche. Ann. N. Y. Acad. Sci. 1106: 41-53. Rizo A., Vellenga E., de Haan G., and Schuringa J.J. (2006). Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum. Mol. Genet. 15 Spec No 2: R210-R219. Kim C.F., Jackson E.L., Woolfenden A.E., Lawrence S., Babar I., Vogel S., Crowley D., Bronson R.T., and Jacks T. (2005). Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121: 823-835. Mimeault M. and Batra S.K. (2006). Recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 24: 2319-2345. Mimeault M., Hauke R., and Batra S.K. (2007). Stem cells -- A revolution in therapeutics--Recent advances on the stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin. Pharmacol. Ther. 82: 252-264. Zhao C., Deng W., and Gage F.H. (2008). Mechanisms and functional implications of adult neurogenesis. Cell 132: 645-660. Vaish M. (2007). Mismatch repair deficiencies transforming stem cells into cancer stem cells and therapeutic implications. Mol. Cancer 6: 26. Mimeault M., Hauke R., Mehta P.P., and Batra S.K. (2007). Recent advances on cancer stem/progenitor cell research: therapeutic implications for overcoming resistance to the most aggressive cancers. J. Mol. Cell. Med. 11: 981-1011. Mimeault M. and Batra S.K. (2008). Recent progress on tissue-resident adult stem cell biology and their therapeutic implications. Stem Cell Rev. 4: 27-49. Nijhof J.G., van Pelt C., Mulder A.A., Mitchell D.L., Mullenders L.H., and de Gruijl F.R. (2007). Epidermal stem and progenitor cells in murine epidermis accumulate UV damage despite NER proficiency. Carcinogenesis 28: 792-800. Mimeault M. and Batra S.K. (2008). Recent progress on normal and malignant pancreatic stem/progenitor cell research: therapeutic implications for the treatment of type 1 or 2 diabetes mellitus and aggressive pancreatic cancer. Gut 57: 1456-1468. Mimeault M., Mehta P.P., Hauke R., and Batra S.K. (2008). Functions of normal and malignant prostatic stem/progenitor cells in tissue regeneration and cancer progression and novel targeting therapies. Endocr. Rev. 29: 234-252. Sengupta A., Banerjee D., Chandra S., Banerji S.K., Ghosh R., Roy R., and Banerjee S. (2007). Deregulation and cross talk among Sonic hedgehog, Wnt, Hox and Notch signaling in chronic myeloid leukemia progression. Leukemia 21: 949-955. Singh S.K., Clarke I.D., Terasaki M., Bonn V.E., Hawkins C., Squire J., and Dirks P.B. (2003). Identification of a cancer stem cell in human brain tumors. Cancer Res. 63: 5821-5828.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

17

[17] Singh S.K., Hawkins C., Clarke I.D., Squire J.A., Bayani J., Hide T., Henkelman R.M., Cusimano M.D., and Dirks P.B. (2004). Identification of human brain tumour initiating cells. Nature 432: 396-401. [18] Hemmati H.D., Nakano I., Lazareff J.A., Masterman-Smith M., Geschwind D.H., Bronner-Fraser M., and Kornblum H.I. (2003). Cancerous stem cells can arise from pediatric brain tumors. Proc. Natl. Acad. Sci. U. S. A 100: 15178-15183. [19] Yuan X., Curtin J., Xiong Y., Liu G., Waschsmann-Hogiu S., Farkas D.L., Black K.L., and Yu J.S. (2004). Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23: 9392-9400. [20] Galli R., Binda E., Orfanelli U., Cipelletti B., Gritti A., De V.S., Fiocco R., Foroni C., Dimeco F., and Vescovi A. (2004). Isolation and characterization of tumorigenic, stemlike neural precursors from human glioblastoma. Cancer Res. 64: 7011-7021. [21] Baehring J.M. (2005). An update on oligodendroglial neoplasms. Curr. Opin. Neurol. 18: 639-644. [22] Nicolis S.K. (2007). Cancer stem cells and "stemness" genes in neuro-oncology. Neurobiol. Dis. 25: 217-229. [23] Yu S.C., Ping Y.F., Yi L., Zhou Z.H., Chen J.H., Yao X.H., Gao L., Wang J.M., and Bian X.W. (2008). Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87. Cancer Lett. 265: 124-134. [24] Salmaggi A., Boiardi A., Gelati M., Russo A., Calatozzolo C., Ciusani E., Sciacca F.L., Ottolina A., Parati E.A., La P.C. et al. (2006). Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 54: 850-860. [25] Al-Hajj M. and Clarke M.F. (2004). Self-renewal and solid tumor stem cells. Oncogene 23: 7274-7282. [26] Ponti D., Costa A., Zaffaroni N., Pratesi G., Petrangolini G., Coradini D., Pilotti S., Pierotti M.A., and Daidone M.G. (2005). Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 65: 5506-5511. [27] Ginestier C., Hur M.H., Charafe-Jauffret E., Monville F., Dutcher J., Brown M., Jacquemier J., Viens P., Kleer C.G., Liu S. et al. (2007). ALDH1 Is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1: 555-567. [28] Wright M.H., Calcagno A.M., Salcido C.D., Carlson M.D., Ambudkar S.V., and Varticovski L. (2008). Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res. 10: R10. [29] Fillmore C.M. and Kuperwasser C. (2008). Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 10: R25. [30] Eramo A., Lotti F., Sette G., Pilozzi E., Biffoni M., Di Virgilio A., Conticello C., Ruco L., Peschle C., and De Maria R. (2008). Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death. Differ. 15: 504-514.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

18

Murielle Mimeault and Surinder K. Batra

[31] Yang Z.F., Ho D.W., Ng M.N., Lau C.K., Yu W.C., Ngai P., Chu P.W., Lam C.T., Poon R.T., and Fan S.T. (2008). Significance of CD90(+) cancer stem cells in human liver cancer. Cancer Cell 13: 153-166. [32] Hermann P.C., Huber S.L., Herrler T., Aicher A., Ellwart J.W., Guba M., Bruns C.J., and Heeschen C. (2007). Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1: 313-323. [33] Zhang S., Balch C., Chan M.W., Lai H.C., Matei D., Schilder J.M., Yan P.S., Huang T.H., and Nephew K.P. (2008). Identification and characterization of ovarian cancerinitiating cells from primary human tumors. Cancer Res. 68: 4311-4320. [34] Mimeault M., Mehta P.P., Hauke R., Moore E., Henichart J.P., Depreux P., and Batra S.K. (2007). Improvement of cytotoxic effects of mitoxantrone on hormone-refractory metastatic prostate cancer cells by co-targeting epidermal growth factor receptor and hedgehog signaling cascades. Growth Factors 25: 400-416. [35] Hirschmann-Jax C., Foster A.E., Wulf G.G., Nuchtern J.G., Jax T.W., Gobel U., Goodell M.A., and Brenner M.K. (2004). A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc. Natl. Acad. Sci. U. S. A. 101: 1422814233. [36] She J.J., Zhang P.G., Wang Z.M., Gan W.M., and Che X.M. (2008). Identification of side population cells from bladder cancer cells by dyecycle violet staining. Cancer Biol. Ther 7. [37] Huang D., Gao Q., Guo L., Zhang C., Wei J., Li H., Jing W.J., Han X., Shi Y., and Shih H.L. (2009). Isolation and identification of cancer stem-like cells in esophageal carcinoma cell lines. Stem Cells Dev. 18: 465-473. [38] Chiba T., Miyagi S., Saraya A., Aoki R., Seki A., Morita Y., Yonemitsu Y., Yokosuka O., Taniguchi H., Nakauchi H. et al. (2008). The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma. Cancer Res. 68: 7742-7749. [39] Shi G.M., Xu Y., Fan J., Zhou J., Yang X.R., Qiu S.J., Liao Y., Wu W.Z., Ji Y., Ke A.W. et al. (2008). Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials. J. Cancer Res. Clin. Oncol. 134: 1155-1163. [40] Sung J.M., Cho H.J., Yi H., Lee C.H., Kim H.S., Kim D.K., bd El-Aty A.M., Kim J.S., Landowski C.P., Hediger M.A. et al. (2008). Characterization of a stem cell population in lung cancer A549 cells. Biochem. Biophys. Res. Commun. 371: 163-167. [41] Friel A.M., Sergent P.A., Patnaude C., Szotek P.P., Oliva E., Scadden D.T., Seiden M.V., Foster R., and Rueda B.R. (2008). Functional analyses of the cancer stem celllike properties of human endometrial tumor initiating cells. Cell Cycle 7: 242-249. [42] Loebinger M.R., Giangreco A., Groot K.R., Prichard L., Allen K., Simpson C., Bazley L., Navani N., Tibrewal S., Davies D. et al. (2008). Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade. Br. J. Cancer 98: 380-387. [43] Zhou J., Wang C.Y., Liu T., Wu B., Zhou F., Xiong J.X., Wu H.S., Tao J., Zhao G., Yang M. et al. (2008). Persistence of side population cells with high drug efflux capacity in pancreatic cancer. World J. Gastroenterol. 14: 925-930.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

19

[44] Haraguchi N., Utsunomiya T., Inoue H., Tanaka F., Mimori K., Barnard G.F., and Mori M. (2006). Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24: 506-513. [45] Ricci-Vitiani L., Lombardi D.G., Pilozzi E., Biffoni M., Todaro M., Peschle C., and De Maria R. (2007). Identification and expansion of human colon-cancer-initiating cells. Nature 445: 111-115. [46] Prince M.E., Sivanandan R., Kaczorowski A., Wolf G.T., Kaplan M.J., Dalerba P., Weissman I.L., Clarke M.F., and Ailles L.E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl. Acad. Sci. U. S. A. 104: 973-978. [47] Mitsutake N., Iwao A., Nagai K., Namba H., Ohtsuru A., Saenko V., and Yamashita S. (2007). Characterization of side population in thyroid cancer cell lines: cancer stemlike cells are enriched partly but not exclusively. Endocrinology 148: 1797-1803. [48] Maitland N.J., Bryce S.D., Stower M.J., and Collins A.T. (2006). Prostate cancer stem cells: a target for new therapies. Ernst. Schering. Found. Symp. Proc. 5: 155-179. [49] Bapat S.A., Mali A.M., Koppikar C.B., and Kurrey N.K. (2005). Stem and progenitorlike cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res. 65: 3025-3029. [50] Gumucio D.L., Fagoonee S., Qiao X.T., Liebert M., Merchant J.L., Altruda F., Rizzetto M., and Pellicano R. (2008). Tissue stem cells and cancer stem cells: potential implications for gastric cancer. Panminerva Med. 50: 65-71. [51] Larue L. and Bellacosa A. (2005). Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways. Oncogene 24: 74437454. [52] Thiery J.P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2: 442-454. [53] Brabletz T., Jung A., Spaderna S., Hlubek F., and Kirchner T. (2005). Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression. Nat. Rev. Cancer 5: 744-749. [54] Tso C.L., Shintaku P., Chen J., Liu Q., Liu J., Chen Z., Yoshimoto K., Mischel P.S., Cloughesy T.F., Liau L.M. et al. (2006). Primary glioblastomas express mesenchymal stem-like properties. Mol. Cancer Res. 4: 607-619. [55] Mimeault M. and Batra S.K. (2007). Interplay of distinct growth factors during epithelial-mesenchymal transition of cancer progenitor cells and molecular targeting as novel cancer therapies. Ann. Oncol. 18: 1605-1619. [56] Mimeault M. and Batra S.K. (2007). Functions of tumorigenic and migrating cancer progenitor cells in cancer progression and metastasis and their therapeutic implications. Cancer Metastasis Rev. 26: 203-214. [57] Mimeault M., Hauke R., and Batra S.K. (2008). Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies. Clin. Pharmacol. Ther 83: 673-691. [58] Das B., Tsuchida R., Malkin D., Koren G., Baruchel S., and Yeger H. (2008). Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction. Stem Cells 26: 1818-1830.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

20

Murielle Mimeault and Surinder K. Batra

[59] Liu S., Dontu G., Mantle I.D., Patel S., Ahn N.S., Jackson K.W., Suri P., and Wicha M.S. (2006). Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 66: 6063-6071. [60] Li L. and Neaves W.B. (2006). Normal stem cells and cancer stem cells: the niche matters. Cancer Res. 66: 4553-4557. [61] Sato M., Shames D.S., Gazdar A.F., and Minna J.D. (2007). A translational view of the molecular pathogenesis of lung cancer. J. Thorac. Oncol. 2: 327-343. [62] Gray-Schopfer V., Wellbrock C., and Marais R. (2007). Melanoma biology and new targeted therapy. Nature 445: 851-857. [63] Ma S., Lee T.K., Zheng B.J., Chan K.W., and Guan X.Y. (2008). CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27: 1749-1758. [64] Rich J.N. (2007). Cancer stem cells in radiation resistance. Cancer Res. 67: 8980-8984. [65] Dean M., Fojo T., and Bates S. (2005). Tumour stem cells and drug resistance. Nat. Rev. Cancer 5: 275-284. [66] Liu G., Yuan X., Zeng Z., Tunici P., Ng H., Abdulkadir I.R., Lu L., Irvin D., Black K.L., and Yu J.S. (2006). Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol. Cancer 5: 67. [67] Zhang X., Komaki R., Wang L., Fang B., and Chang J.Y. (2008). Treatment of radioresistant stem-like esophageal cancer cells by an apoptotic gene-armed, telomerase-specific oncolytic adenovirus. Clin. Cancer Res. 14: 2813-2823. [68] Todaro M., Mileidys Perez Alea M.P., Di Stefano A.B., Cammareri P., Vermeulen L., Iovino F., Tripodo C., Russo A., Gulotta G., Medema J.-P. et al. (2007). Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1: 389-402. [69] Wang J., Guo L.P., Chen L.Z., Zeng Y.X., and Lu S.H. (2007). Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res. 67: 3716-3724. [70] Catassi A., Servent D., Paleari L., Cesario A., and Russo P. (2008). Multiple roles of nicotine on cell proliferation and inhibition of apoptosis: implications on lung carcinogenesis. Mutat. Res. 659: 221-231. [71] Aubert G. and Lansdorp P.M. (2008). Telomeres and aging. Physiol Rev 88: 557-579. [72] Widera D., Kaus A., Kaltschmidt C., and Kaltschmidt B. (2008). Neural stem cells, inflammation and NF-kappaB: basic principle of maintenance and repair or origin of brain tumours? J. Cell. Mol. Med. 12: 459-470. [73] Schwarze S.R., DePrimo S.E., Grabert L.M., Fu V.X., Brooks J.D., and Jarrard D.F. (2002). Novel pathways associated with bypassing cellular senescence in human prostate epithelial cells. J. Biol. Chem. 277: 14877-14883. [74] Deng Y. and Chang S. (2007). Role of telomeres and telomerase in genomic instability, senescence and cancer. Lab. Invest. 87: 1071-1076. [75] Shin J.S., Hong A., Solomon M.J., and Lee C.S. (2006). The role of telomeres and telomerase in the pathology of human cancer and aging. Pathology 38: 103-113. [76] Tchernev G. and Orfanos C.E. (2007). Downregulation of cell cycle modulators p21, p27, p53, Rb and proapoptotic Bcl-2-related proteins Bax and Bak in cutaneous

Therapeutic Implications of Cancer Stem/Progenitor Cells

[77]

[78]

[79]

[80]

[81] [82] [83]

[84]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[85] [86] [87]

[88]

[89]

[90] [91] [92] [93]

21

melanoma is associated with worse patient prognosis: preliminary findings. J. Cutan. Pathol. 34: 247-256. Pacifico A., Goldberg L.H., Peris K., Chimenti S., Leone G., and Ananthaswamy H.N. (2008). Loss of CDKN2A and p14ARF expression occurs frequently in human nonmelanoma skin cancers. Br. J. Dermatol. 158: 291-297. Maurelli R., Zambruno G., Guerra L., Abbruzzese C., Dimri G., Gellini M., Bondanza S., and Dellambra E. (2006). Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment. FASEB J. 20: 1516-1518. Jarrard D.F., Modder J., Fadden P., Fu V., Sebree L., Heisey D., Schwarze S.R., and Friedl A. (2002). Alterations in the p16/pRb cell cycle checkpoint occur commonly in primary and metastatic human prostate cancer. Cancer Lett. 185: 191-199. Shiras A., Chettiar S.T., Shepal V., Rajendran G., Prasad G.R., and Shastry P. (2007). Spontaneous transformation of human adult nontumorigenic stem cells to cancer stem cells is driven by genomic instability in a human model of glioblastoma. Stem Cells 25: 1478-1489. Serakinci N., Graakjaer J., and Kolvraa S. (2008). Telomere stability and telomerase in mesenchymal stem cells. Biochimie 90: 33-40. Shay J.W. and Wright W.E. (2005). Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 26: 867-874. Zhou Z., Flesken-Nikitin A., and Nikitin A.Y. (2007). Prostate cancer associated with p53 and Rb deficiency arises from the stem/progenitor cell-enriched proximal region of prostatic ducts. Cancer Res. 67: 5683-5690. Marusyk A. and DeGregori J. (2007). Replicational stress selects for p53 mutation. Cell Cycle 6: 2148-2151. Bell D.R. and Van Zant G. (2004). Stem cells, aging, and cancer: inevitabilities and outcomes. Oncogene 23: 7290-7296. DePinho R.A. (2000). The age of cancer. Nature 408: 248-254. Mimeault M. and Batra S.K. (2008). Targeting of cancer stem/progenitor cells plus stem cell-based therapies: the ultimate hope for treating and curing aggressive and recurrent cancers. Panminerva Med. 50: 3-18. Marusyk A. and DeGregori J. (2008). Declining cellular fitness with age promotes cancer initiation by selecting for adaptive oncogenic mutations. Biochim. Biophys. Acta 1785: 1-11. Rajaraman S., Choi J., Cheung P., Beaudry V., Moore H., and Artandi S.E. (2007). Telomere uncapping in progenitor cells with critical telomere shortening is coupled to S-phase progression in vivo. Proc. Natl. Acad. Sci. U. S. A. 104: 17747-17752. Shay J.W. and Wright W.E. (2002). Telomerase: a target for cancer therapeutics. Cancer Cell 2: 257-265. Lansdorp P.M. (2008). Telomeres, stem cells, and hematology. Blood 111: 1759-1766. Shay J.W. and Wright W.E. (2007). Hallmarks of telomeres in ageing research. J. Pathol. 211: 114-123. Campisi J. (2005). Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 120: 513-522.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

22

Murielle Mimeault and Surinder K. Batra

[94] Meeker A.K., Hicks J.L., Iacobuzio-Donahue C.A., Montgomery E.A., Westra W.H., Chan T.Y., Ronnett B.M., and De Marzo A.M. (2004). Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin. Cancer Res. 10: 33173326. [95] Rudolph K.L., Millard M., Bosenberg M.W., and DePinho R.A. (2001). Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Nat Genet. 28: 155-159. [96] Katoh M. (2007). Networking of WNT, FGF, Notch, BMP, and Hedgehog signaling pathways during carcinogenesis. Stem Cell Rev. 3: 30-38. [97] Glinsky G.V. (2008). "Stemness" genomics law governs clinical behavior of human cancer: implications for decision making in disease management. J. Clin. Oncol. 26: 2846-2853. [98] Beachy P.A., Karhadkar S.S., and Berman D.M. (2004). Tissue repair and stem cell renewal in carcinogenesis. Nature 432: 324-331. [99] Bao S., Wu Q., McLendon R.E., Hao Y., Shi Q., Hjelmeland A.B., Dewhirst M.W., Bigner D.D., and Rich J.N. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444: 756-760. [100] Olivotto M. and Dello S.P. (2008). Environmental restrictions within tumor ecosystems select for a convergent, hypoxia-resistant phenotype of cancer stem cells. Cell Cycle 7: 176-187. [101] Gerlee P. and Anderson A.R. (2008). A hybrid cellular automaton model of clonal evolution in cancer: the emergence of the glycolytic phenotype. J. Theor. Biol. 250: 705-722. [102] Kondoh H. (2008). Cellular life span and the Warburg effect. Exp. Cell Res. 314: 19231928. [103] Gotzmann J., Mikula M., Eger A., Schulte-Hermann R., Foisner R., Beug H., and Mikulits W. (2004). Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis. Mutat. Res. 566: 9-20. [104] Eastham A.M., Spencer H., Soncin F., Ritson S., Merry C.L., Stern P.L., and Ward C.M. (2007). Epithelial-mesenchymal transition events during human embryonic stem cell differentiation. Cancer Res. 67: 11254-11262. [105] Behr R., Heneweer C., Viebahn C., Denker H.W., and Thie M. (2005). Epithelialmesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation. Stem Cells 23: 805-816. [106] Barrow K.M., Ward C.M., Rutter J., Ali S., and Stern P.L. (2005). Embryonic expression of murine 5T4 oncofoetal antigen is associated with morphogenetic events at implantation and in developing epithelia. Dev. Dyn. 233: 1535-1545. [107] Ullmann U., In't V.P., Gilles C., Sermon K., De R.M., van d., V, Van S.A., and Liebaers I. (2007). Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Mol Hum. Reprod. 13: 21-32. [108] Derycke L.D. and Bracke M.E. (2004). N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. Int. J. Dev. Biol. 48: 463-476.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

23

[109] Shih I. and Kurman R.J. (2004). Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am. J. Pathol. 164: 1511-1518. [110] Kopfstein L. and Christofori G. (2006). Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment. Cell Mol. Life Sci. 63: 449-468. [111] Kleeff J., Beckhove P., Esposito I., Herzig S., Huber P.E., Lohr J.M., and Friess H. (2007). Pancreatic cancer microenvironment. Int. J. Cancer 121: 699-705. [112] Rafii S., Lyden D., Benezra R., Hattori K., and Heissig B. (2002). Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat. Rev. Cancer 2: 826-835. [113] Orimo A. and Weinberg R.A. (2006). Stromal fibroblasts in cancer: A novel tumorpromoting cell type. Cell Cycle 5: 1597-1601. [114] Ganss R. (2006). Tumor stroma fosters neovascularization by recruitment of progenitor cells into the tumor bed. J. Cell. Mol. Med. 10: 857-865. [115] Kaplan R.N., Riba R.D., Zacharoulis S., Bramley A.H., Vincent L., Costa C., MacDonald D.D., Jin D.K., Shido K., Kerns S.A. et al. (2005). VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438: 820-827. [116] Bruno S., Bussolati B., Grange C., Collino F., Graziano M.E., Ferrando U., and Camussi G. (2006). CD133+ renal progenitor cells contribute to tumor angiogenesis. Am. J. Pathol. 169: 2223-2235. [117] Mimeault M. and Batra S.K. (2006). Recent advances on multiple tumorigenic cascades involved in prostatic cancer progression and targeting therapies. Carcinogenesis 27: 122. [118] Mimeault M. and Batra S.K. (2008) Stem cell applications in disease research: Recent advances on stem cell and cancer stem cell biology and their therapeutic implications. In Progress in Stem Cell Applications, eds. Allen V. Faraday and Jonathon T. Dyer, Nova Publishers, Inc. 57-98. [119] Savagner P. (2001). Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays 23: 912-923. [120] Bissell M.J. and Radisky D. (2001). Putting tumours in context. Nat. Rev. Cancer 1: 46-54. [121] Yang J., Mani S.A., and Weinberg R.A. (2006). Exploring a new twist on tumor metastasis. Cancer Res. 66: 4549-4552. [122] Bergers G. and Coussens L.M. (2000). Extrinsic regulators of epithelial tumor progression: metalloproteinases. Curr. Opin. Genet. Dev. 10: 120-127. [123] Radisky D.C. and Bissell M.J. (2006). Matrix metalloproteinase-induced genomic instability. Curr. Opin. Genet. Dev. 16: 45-50. [124] Reiss K., Ludwig A., and Saftig P. (2006). Breaking up the tie: Disintegrin-like metalloproteinases as regulators of cell migration in inflammation and invasion. Pharmacol. Ther. 111: 985-1006. [125] Radisky E.S. and Radisky D.C. (2007). Stromal induction of breast cancer: inflammation and invasion. Rev. Endocr. Metab. Disord. 8: 279-287.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

24

Murielle Mimeault and Surinder K. Batra

[126] Ho M.M., Ng A.V., Lam S., and Hung J.Y. (2007). Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 67: 4827-4833. [127] de Jonge-Peeters S.D., Kuipers F., de Vries E.G., and Vellenga E. (2007). ABC transporter expression in hematopoietic stem cells and the role in AML drug resistance. Crit. Rev. Oncol. Hematol. 62: 214-226. [128] Salmaggi A., Boiardi A., Gelati M., Russo A., Calatozzolo C., Ciusani E., Sciacca F.L., Ottolina A., Parati E.A., La P.C. et al. (2006). Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 54: 850-860. [129] Frank N.Y., Margaryan A., Huang Y., Schatton T., Waaga-Gasser A.M., Gasser M., Sayegh M.H., Sadee W., and Frank M.H. (2005). ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res. 65: 43204333. [130] Kang M.K. and Kang S.K. (2007). Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma. Stem Cells Dev. 16: 837-847. [131] Gottesman M.M., Fojo T., and Bates S.E. (2002). Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2: 48-58. [132] Johannessen T.C., Bjerkvig R., and Tysnes B.B. (2008). DNA repair and cancer stemlike cells - Potential partners in glioma drug resistance? Cancer Treat. Rev. 34: 558567. [133] Matsui W., Wang Q., Barber J.P., Brennan S., Smith B.D., Borrello I., McNiece I., Lin L., Ambinder R.F., Peacock C. et al. (2008). Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res. 68: 190-197. [134] Dylla S.J., Beviglia L., Park I.K., Chartier C., Raval J., Ngan L., Pickell K., Aguilar J., Lazetic S., Smith-Berdan S. et al. (2008). Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. PLoS. ONE. 3: e2428. [135] Shervington A. and Lu C. (2008). Expression of multidrug resistance genes in normal and cancer stem cells. Cancer Invest. 26: 535-542. [136] Xu J.X., Morii E., Liu Y., Nakamichi N., Ikeda J., Kimura H., and Aozasa K. (2007). High tolerance to apoptotic stimuli induced by serum depletion and ceramide in sidepopulation cells: high expression of CD55 as a novel character for side-population. Exp. Cell Res. 313: 1877-1885. [137] Chen Y.C., Hsu H.S., Chen Y.W., Tsai T.H., How C.K., Wang C.Y., Hung S.C., Chang Y.L., Tsai M.L., Lee Y.Y. et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS. ONE. 3: e2637. [138] Birnie R., Bryce S.D., Roome C., Dussupt V., Droop A., Lang S.H., Berry P.A., Hyde C.F., Lewis J.L., Stower M.J. et al. (2008). Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions. Genome Biol. 9: R83. [139] Borst P. and Elferink R.O. (2002). Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 71: 537-592.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

25

[140] Choudhuri S. and Klaassen C.D. (2006). Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters. Int. J. Toxicol. 25: 231-259. [141] Ho R.H. and Kim R.B. (2005). Transporters and drug therapy: implications for drug disposition and disease. Clin. Pharmacol. Ther. 78: 260-277. [142] Kaminski W.E., Piehler A., and Wenzel J.J. (2006). ABC A-subfamily transporters: structure, function and disease. Biochim. Biophys. Acta 1762: 510-524. [143] Teodori E., Dei S., Martelli C., Scapecchi S., and Gualtieri F. (2006). The functions and structure of ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR). Curr. Drug Targets. 7: 893-909. [144] Krishnamurthy P. and Schuetz J.D. (2006). Role of ABCG2/BCRP in biology and medicine. Annu. Rev. Pharmacol. Toxicol. 46: 381-410. [145] Mao Q. and Unadkat J.D. (2005). Role of the breast cancer resistance protein (ABCG2) in drug transport. AAPS. J. 7: E118-E133. [146] Deeley R.G., Westlake C., and Cole S.P. (2006). Transmembrane transport of endoand xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol. Rev. 86: 849-899. [147] Szakacs G., Paterson J.K., Ludwig J.A., Booth-Genthe C., and Gottesman M.M. (2006). Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5: 219-234. [148] Higgins C.F. (2007). Multiple molecular mechanisms for multidrug resistance transporters. Nature 446: 749-757. [149] Ozben T. (2006). Mechanisms and strategies to overcome multiple drug resistance in cancer. FEBS Lett. 580: 2903-2909. [150] Gerk P.M. and Vore M. (2002). Regulation of expression of the multidrug resistanceassociated protein 2 (MRP2) and its role in drug disposition. J. Pharmacol. Exp. Ther. 302: 407-415. [151] Han B. and Zhang J.T. (2004). Multidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the half ATP-binding cassette transporter ABCG2. Curr. Med. Chem. Anticancer Agents 4: 31-42. [152] Boonstra R., Timmer-Bosscha H., van Echten-Arends J., van der Kolk D.M., van den B.A., de J.B., Tew K.D., Poppema S., and de Vries E.G. (2004). Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation. Br. J. Cancer 90: 2411-2417. [153] Mack J.T., Beljanski V., Tew K.D., and Townsend D.M. (2006). The ATP-binding cassette transporter ABCA2 as a mediator of intracellular trafficking. Biomed. Pharmacother. 60: 587-592. [154] Rabik C.A., Njoku M.C., and Dolan M.E. (2006). Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy. Cancer Treat. Rev. 32: 261-276. [155] Cai S., Xu Y., Cooper R.J., Ferkowicz M.J., Hartwell J.R., Pollok K.E., and Kelley M.R. (2005). Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents. Cancer Res. 65: 3319-3327.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

26

Murielle Mimeault and Surinder K. Batra

[156] Juillerat A. and Juillerat-Jeanneret L. (2007). S-alkylthiolation of O6-methylguanineDNA-methyltransferase (MGMT) to sensitize cancer cells to anticancer therapy. Expert. Opin. Ther. Targets. 11: 349-361. [157] Seigel G.M., Campbell L.M., Narayan M., and Gonzalez-Fernandez F. (2005). Cancer stem cell characteristics in retinoblastoma. Mol. Vis. 11: 729-737. [158] Feldmann G., Dhara S., Fendrich V., Bedja D., Beaty R., Mullendore M., Karikari C., Alvarez H., Iacobuzio-Donahue C., Jimeno A. et al. (2007). Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers. Cancer Res. 67: 2187-2196. [159] Bar E.E., Chaudhry A., Lin A., Fan X., Schreck K., Matsui W., Piccirillo S., Vescovi A.L., Dimeco F., Olivi A. et al. (2007). Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 25: 2524-2533. [160] Douville J., Beaulieu R., and Balicki D. (2008). ALDH1 as a functional marker of cancer stem and progenitor cells. Stem Cells Dev. [161] Sorlie T., Wang Y., Xiao C., Johnsen H., Naume B., Samaha R.R., and Borresen-Dale A.L. (2006). Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms. BMC. Genomics 7: 127. [162] Al-Hajj M., Wicha M.S., ito-Hernandez A., Morrison S.J., and Clarke M.F. (2003). Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 100: 3983-3988. [163] Dontu G., El-Ashry D., and Wicha M.S. (2004). Breast cancer, stem/progenitor cells and the estrogen receptor. Trends Endocrinol. Metab. 15: 193-197. [164] Liu S., Dontu G., and Wicha M.S. (2005). Mammary stem cells, self-renewal pathways, and carcinogenesis. Breast Cancer Res. 7: 86-95. [165] Teuliere J., Faraldo M.M., Deugnier M.A., Shtutman M., Ben-Ze'ev A., Thiery J.P., and Glukhova M.A. (2005). Targeted activation of beta-catenin signaling in basal mammary epithelial cells affects mammary development and leads to hyperplasia. Development 132: 267-277. [166] Clarke R.B., Spence K., Anderson E., Howell A., Okano H., and Potten C.S. (2005). A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev. Biol. 277: 443-456. [167] Du Z., Podsypanina K., Huang S., McGrath A., Toneff M.J., Bogoslovskaia E., Zhang X., Moraes R.C., Fluck M., Allred D.C. et al. (2006). Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models. Proc. Natl. Acad. Sci. U. S. A. 103: 17396-17401. [168] Woodward W.A., Chen M.S., Behbod F., and Rosen J.M. (2005). On mammary stem cells. J. Cell Sci. 118: 3585-3594. [169] Tang P., Wang X., Schiffhauer L., Wang J., Bourne P., Yang Q., Quinn A., and Hajdu S. (2006). Expression patterns of ER-alpha, PR, HER-2/neu, and EGFR in different cell origin subtypes of high grade and non-high grade ductal carcinoma in situ. Ann. Clin. Lab. Sci. 36: 137-143. [170] Fodde R. and Brabletz T. (2007). Wnt/beta-catenin signaling in cancer stemness and malignant behavior. Curr. Opin. Cell Biol. 19: 150-158.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

27

[171] Moraes R.C., Zhang X., Harrington N., Fung J.Y., Wu M.F., Hilsenbeck S.G., Allred D.C., and Lewis M.T. (2007). Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia. Development 134: 1231-1242. [172] Chung L.W., Baseman A., Assikis V., and Zhau H.E. (2005). Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J. Urol. 173: 10-20. [173] Langley R.R. and Fidler I.J. (2007). Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis. Endocr. Rev 28: 297-321. [174] Carver B.S. and Pandolfi P.P. (2006). Mouse modeling in oncologic preclinical and translational research. Clin Cancer Res. 12: 5305-5311. [175] Chen Z., Trotman L.C., Shaffer D., Lin H.K., Dotan Z.A., Niki M., Koutcher J.A., Scher H.I., Ludwig T., Gerald W. et al. (2005). Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436: 725-730. [176] Wang S., Gao J., Lei Q., Rozengurt N., Pritchard C., Jiao J., Thomas G.V., Li G., RoyBurman P., Nelson P.S. et al. (2003). Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4: 209-221. [177] Wang S., Garcia A.J., Wu M., Lawson D.A., Witte O.N., and Wu H. (2006). Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc. Natl. Acad. Sci. U. S. A. 103: 1480-1485. [178] Bruxvoort K.J., Charbonneau H.M., Giambernardi T.A., Goolsby J.C., Qian C.N., Zylstra C.R., Robinson D.R., Roy-Burman P., Shaw A.K., Buckner-Berghuis B.D. et al. (2007). Inactivation of Apc in the mouse prostate causes prostate carcinoma. Cancer Res. 67: 2490-2496. [179] Nikitin A.Y., Matoso A., and Roy-Burman P. (2007). Prostate stem cells and cancer. Histol. Histopathol. 22: 1043-1049. [180] Reiner T., de Las P.A., Parrondo R., and Perez-Stable C. (2007). Progression of prostate cancer from a subset of p63-positive basal epithelial cells in FG/Tag transgenic mice. Mol. Cancer Res. 5: 1171-1179. [181] Kalluri R. and Zeisberg M. (2006). Fibroblasts in cancer. Nat. Rev. Cancer 6: 392-401. [182] Laakso M., Tanner M., Nilsson J., Wiklund T., Erikstein B., Kellokumpu-Lehtinen P., Malmstrom P., Wilking N., Bergh J., and Isola J. (2006). Basoluminal carcinoma: a new biologically and prognostically distinct entity between basal and luminal breast cancer. Clin. Cancer Res. 12: 4185-4191. [183] Simon R. (2006). Development and evaluation of therapeutically relevant predictive classifiers using gene expression profiling. J. Natl. Cancer Inst. 98: 1169-1171. [184] Leonard G.D. and Swain S.M. (2004). Ductal carcinoma in situ, complexities and challenges. J. Natl. Cancer Inst. 96: 906-920. [185] Buyse M., Loi S., van't V.L., Viale G., Delorenzi M., Glas A.M., d'Assignies M.S., Bergh J., Lidereau R., Ellis P. et al. (2006). Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J. Natl. Cancer Inst. 98: 1183-1192. [186] Anderson W.F. and Matsuno R. (2006). Breast cancer heterogeneity: a mixture of at least two main types? J. Natl. Cancer Inst. 98: 948-951.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

28

Murielle Mimeault and Surinder K. Batra

[187] Asselin-Labat M.L., Shackleton M., Stingl J., Vaillant F., Forrest N.C., Eaves C.J., Visvader J.E., and Lindeman G.J. (2006). Steroid hormone receptor status of mouse mammary stem cells. J. Natl. Cancer Inst. 98: 1011-1014. [188] Nielsen T.O., Hsu F.D., Jensen K., Cheang M., Karaca G., Hu Z., Hernandez-Boussard T., Livasy C., Cowan D., Dressler L. et al. (2004). Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 10: 5367-5374. [189] Birnbaum D., Bertucci F., Ginestier C., Tagett R., Jacquemier J., and Charafe-Jauffret E. (2004). Basal and luminal breast cancers: basic or luminous? (review). Int. J. Oncol. 25: 249-258. [190] Livasy C.A., Karaca G., Nanda R., Tretiakova M.S., Olopade O.I., Moore D.T., and Perou C.M. (2006). Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod. Pathol. 19: 264-271. [191] Carey L.A., Perou C.M., Livasy C.A., Dressler L.G., Cowan D., Conway K., Karaca G., Troester M.A., Tse C.K., Edmiston S. et al. (2006). Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295: 2492-2502. [192] Jumppanen M., Gruvberger-Saal S., Kauraniemi P., Tanner M., Bendahl P.O., Lundin M., Krogh M., Kataja P., Borg A., Ferno M. et al. (2007). Basal-like phenotype is not associated with patient survival in estrogen receptor negative breast cancers. Breast Cancer Res. 9: R16. [193] Lacroix M. (2006). Significance, detection and markers of disseminated breast cancer cells. Endocr. Relat. Cancer 13: 1033-1067. [194] Calza S., Hall P., Auer G., Bjohle J., Klaar S., Kronenwett U., Liu E.T., Miller L., Ploner A., Smeds J. et al. (2006). Intrinsic molecular signature of breast cancer in a population-based cohort of 412 patients. Breast Cancer Res. 8: R34. [195] van de Rijn M., Perou C.M., Tibshirani R., Haas P., Kallioniemi O., Kononen J., Torhorst J., Sauter G., Zuber M., Kochli O.R. et al. (2002). Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am. J. Pathol. 161: 1991-1996. [196] Sorlie T., Tibshirani R., Parker J., Hastie T., Marron J.S., Nobel A., Deng S., Johnsen H., Pesich R., Geisler S. et al. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc. Natl. Acad. Sci. U. S. A. 100: 8418-8423. [197] Potemski P., Kusinska R., Watala C., Pluciennik E., Bednarek A.K., and Kordek R. (2005). Prognostic relevance of basal cytokeratin expression in operable breast cancer. Oncology 69: 478-485. [198] Rakha E.A., El-Sayed M.E., Green A.R., Lee A.H., Robertson J.F., and Ellis I.O. (2007). Prognostic markers in triple-negative breast cancer. Cancer 109: 25-32. [199] Fujita N., Jaye D.L., Kajita M., Geigerman C., Moreno C.S., and Wade P.A. (2003). MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113: 207-219. [200] Wang X., Belguise K., Kersual N., Kirsch K.H., Mineva N.D., Galtier F., Chalbos D., and Sonenshein G.E. (2007). Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2. Nat Cell Biol. 9: 470-478.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

29

[201] Liu R., Wang X., Chen G.Y., Dalerba P., Gurney A., Hoey T., Sherlock G., Lewicki J., Shedden K., and Clarke M.F. (2007). The prognostic role of a gene signature from tumorigenic breast-cancer cells. N. Engl. J. Med. 356: 217-226. [202] Shipitsin M., Campbell L.L., Argani P., Weremowicz S., Bloushtain-Qimron N., Yao J., Nikolskaya T., Serebryiskaya T., Beroukhim R., Hu M. et al. (2007). Molecular definition of breast tumor heterogeneity. Cancer Cell 11: 259-273. [203] Mimeault M. and Batra S.K. (2008). Recent advances on the development of novel anti-cancer drugs targeting cancer stem/progenitor cells. Drug Develop. Res.69: 415430. [204] Misra S., Toole B.P., and Ghatak S. (2006). Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells. J. Biol. Chem. 281: 34936-34941. [205] Ghatak S., Misra S., and Toole B.P. (2002). Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3kinase/Akt cell survival pathway. J. Biol. Chem. 277: 38013-38020. [206] Bourguignon L.Y., Peyrollier K., Xia W., and Gilad E. (2008). Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J. Biol. Chem. 283: 17635-17651. [207] Rubin L.L. and de Sauvage F.J. (2006). Targeting the Hedgehog pathway in cancer. Nat. Rev. Drug Discov. 5: 1026-1033. [208] Karhadkar S.S., Bova G.S., Abdallah N., Dhara S., Gardner D., Maitra A., Isaacs J.T., Berman D.M., and Beachy P.A. (2004). Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431: 707-712. [209] Chen J.S., Pardo F.S., Wang-Rodriguez J., Chu T.S., Lopez J.P., Aguilera J., Altuna X., Weisman R.A., and Ongkeko W.M. (2006). EGFR regulates the side population in head and neck squamous cell carcinoma. Laryngoscope 116: 401-406. [210] Sims-Mourtada J., Izzo J.G., Ajani J., and Chao K.S. (2007). Sonic Hedgehog promotes multiple drug resistance by regulation of drug transport. Oncogene 26: 5674-5679. [211] Villanueva A., Newell P., Chiang D.Y., Friedman S.L., and Llovet J.M. (2007). Genomics and signaling pathways in hepatocellular carcinoma. Semin. Liver Dis. 27: 55-76. [212] Clement V., Sanchez P., de Tribolet N., Radovanovic I., and Altaba A. (2007). HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell selfrenewal, and tumorigenicity. Curr. Biol. 17: 165-172. [213] Kang H., Watkins G., Douglas-Jones A., Mansel R.E., and Jiang W.G. (2005). The elevated level of CXCR4 is correlated with nodal metastasis of human breast cancer. Breast 14: 360-367. [214] Rubin J.B., Kung A.L., Klein R.S., Chan J.A., Sun Y., Schmidt K., Kieran M.W., Luster A.D., and Segal R.A. (2003). A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc. Natl. Acad. Sci. U. S. A. 100: 1351313518.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

30

Murielle Mimeault and Surinder K. Batra

[215] Liang Z., Wu T., Lou H., Yu X., Taichman R.S., Lau S.K., Nie S., Umbreit J., and Shim H. (2004). Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4. Cancer Res. 64: 4302-4308. [216] Kulbe H., Levinson N.R., Balkwill F., and Wilson J.L. (2004). The chemokine network in cancer--much more than directing cell movement. Int. J. Dev. Biol. 48: 489-496. [217] Balkwill F. (2004). Cancer and the chemokine network. Nat. Rev. Cancer 4: 540-550. [218] Kucia M., Reca R., Miekus K., Wanzeck J., Wojakowski W., Janowska-Wieczorek A., Ratajczak J., and Ratajczak M.Z. (2005). Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1CXCR4 axis. Stem Cells 23: 879-894. [219] Hart C.A., Brown M., Bagley S., Sharrard M., and Clarke N.W. (2005). Invasive characteristics of human prostatic epithelial cells: understanding the metastatic process. Br. J. Cancer 92: 503-512. [220] Neiva K., Sun Y.X., and Taichman R.S. (2005). The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis. Braz. J. Med. Biol. Res. 38: 1449-1454. [221] Engl T., Relja B., Marian D., Blumenberg C., Muller I., Beecken W.D., Jones J., Ringel E.M., Bereiter-Hahn J., Jonas D. et al. (2006). CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins. Neoplasia 8: 290-301. [222] Onoue T., Uchida D., Begum N.M., Tomizuka Y., Yoshida H., and Sato M. (2006). Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells. Int. J. Oncol. 29: 1133-1138. [223] Chen B.Y., Liu J.Y., Chang H.H., Chang C.P., Lo W.Y., Kuo W.H., Yang C.R., and Lin D.P. (2007). Hedgehog is involved in prostate basal cell hyperplasia formation and its progressing towards tumorigenesis. Biochem. Biophys. Res. Commun. 357: 10841089. [224] Strizzi L., Bianco C., Normanno N., and Salomon D. (2005). Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis. Oncogene 24: 57315741. [225] Hu X.F. and Xing P.X. (2005). Cripto as a target for cancer immunotherapy. Expert Opin. Ther. Targets. 9: 383-394. [226] Sell S. (2004). Stem cell origin of cancer and differentiation therapy. Crit. Rev. Oncol. Hematol. 51: 1-28. [227] Sano T., Kagawa M., Okuno M., Ishibashi N., Hashimoto M., Yamamoto M., Suzuki R., Kohno H., Matsushima-Nishiwaki R., Takano Y. et al. (2005). Prevention of rat hepatocarcinogenesis by acyclic retinoid is accompanied by reduction in emergence of both TGF-alpha-expressing oval-like cells and activated hepatic stellate cells. Nutr. Cancer 51: 197-206. [228] Massard C., Deutsch E., and Soria J.C. (2006). Tumour stem cell-targeted treatment: elimination or differentiation. Ann. Oncol. 17: 1620-1624. [229] Perabo F.G. and Muller S.C. (2006). New agents for treatment of advanced transitional cell carcinoma. Ann. Oncol. 18: 835-843. [230] Moserle L., Indraccolo S., Ghisi M., Frasson C., Fortunato E., Canevari S., Miotti S., Tosello V., Zamarchi R., Corradin A. et al. (2008). The side population of ovarian

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Therapeutic Implications of Cancer Stem/Progenitor Cells

31

cancer cells is a primary target of IFN-alpha antitumor effects. Cancer Res. 68: 56585668. [231] Davidoff A.M., Ng C.Y., Brown P., Leary M.A., Spurbeck W.W., Zhou J., Horwitz E., Vanin E.F., and Nienhuis A.W. (2001). Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express an angiogenesis inhibitor, can restrict tumor growth in mice. Clin. Cancer Res. 7: 2870-2879. [232] Rafii S., Lyden D., Benezra, R., Hattori K. and Heissig B. (2002) Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat. Rev. Cancer 2: 826-835. [233] Byrne A.M., Bouchier-Hayes D.J., and Harmey J.H. (2005). Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J. Cell Mol. Med. 9: 777-794. [234] Moreira I.S., Fernandes P.A., and Ramos M.J. (2007). Vascular endothelial growth factor (VEGF) inhibition—a critical review. Anticancer Agents Med. Chem. 7: 223245. [235] Rosell R., Cecere F., Cognetti F., Cuello M., Sanchez J.M., Taron M., Reguart N., and Jablons D. (2006). Future directions in the second-line treatment of non-small cell lung cancer. Semin. Oncol. 33: S45-S51. [236] Calabrese C., Poppleton H., Kocak M., Hogg T.L., Fuller C., Hamner B., Oh E.Y., Gaber M.W., Finklestein D., Allen M. et al. (2007). A perivascular niche for brain tumor stem cells. Cancer Cell 11: 69-82. [237] Folkins C., Man S., Xu P., Shaked Y., Hicklin D.J., and Kerbel R.S. (2007). Anticancer therapies combining antiangiogenic and tumor cell cytotoxic effects reduce the tumor stem-like cell fraction in glioma xenograft tumors. Cancer Res. 67: 3560-3564. [238] Yang Z.J. and Wechsler-Reya R.J. (2007). Hit 'em where they live: targeting the cancer stem cell niche. Cancer Cell 11: 3-5. [239] Sun S.Y., Hail N., Jr., and Lotan R. (2004). Apoptosis as a novel target for cancer chemoprevention. J. Natl. Cancer Inst. 96: 662-672. [240] Miller J.C. and Sorensen A.G. (2005). Imaging biomarkers predictive of disease/therapy outcome: ischemic stroke and drug development. Prog. Drug Res. 62: 319-356. [241] Arora A. and Scholar E.M. (2005). Role of tyrosine kinase inhibitors in cancer therapy. J. Pharmacol. Exp. Ther. 315: 971-979. [242] Zhong H. and Bowen J.P. (2006). Antiangiogenesis drug design: multiple pathways targeting tumor vasculature. Curr. Med. Chem. 13: 849-862. [243] Meric J.B., Rottey S., Olaussen K., Soria J.C., Khayat D., Rixe O., and Spano J.P. (2006). Cyclooxygenase-2 as a target for anticancer drug development. Crit. Rev. Oncol. Hematol. 59: 51-64. [244] Aggarwal B.B., Shishodia S., Sandur S.K., Pandey M.K., and Sethi G. (2006). Inflammation and cancer: How hot is the link? Biochem. Pharmacol. 72: 1605-1621. [245] Kobayashi H. and Lin P.C. (2006). Antiangiogenic and radiotherapy for cancer treatment. Histol. Histopathol. 21: 1125-1134.

32

Murielle Mimeault and Surinder K. Batra

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[246] Grosch S., Maier T.J., Schiffmann S., and Geisslinger G. (2006). Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J. Natl. Cancer Inst. 98: 736-747. [247] Cairns R., Papandreou I., and Denko N. (2006). Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment. Mol. Cancer Res. 4: 61-70. [248] Lu H., Ouyang W., and Huang C. (2006). Inflammation, a key event in cancer development. Mol. Cancer Res. 4: 221-233. [249] Lyden D., Hattori K., Dias S., Costa C., Blaikie P., Butros L., Chadburn A., Heissig B., Marks W., Witte L. et al. (2001). Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat. Med. 7: 1194-1201. [250] Vosseler S., Mirancea N., Bohlen P., Mueller M.M., and Fusenig N.E. (2005). Angiogenesis inhibition by vascular endothelial growth factor receptor-2 blockade reduces stromal matrix metalloproteinase expression, normalizes stromal tissue, and reverts epithelial tumor phenotype in surface heterotransplants. Cancer Res. 65: 12941305. [251] Van Beijnum J., Dings R.P., van der L.E., Zwaans B.M., Ramaekers F.C., Mayo K.H., and Griffioen A.W. (2006). Gene expression of tumor angiogenesis dissected; specific targeting of colon cancer angiogenic vasculature. Blood 108: 2339-2348.

In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter II

Role of O6-Methyl Guanine-DNA Methyl Transferase and the Effect of O6Benzylguanine in Cancer Chemotherapy Jun Murakami*1, Jun-ichi Asaumi1, Hidetsugu Tsujigiwa2, Masao Yamada2, Susumu Kokeguchi3, Hitoshi Nagatsuka4, Tatsuo Yamamoto5, and You-Jin Lee6 1

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4

Department of Oral and Maxillofacial Radiology, 2Virology, 3Oral Microbiology, Oral Pathology, 5Preventive Dentistry, Graduate Schools of Medicine and Dentistry, Okayama University, Okayama, Japan 6 Jeonnam Biotechnology Research Center, Korea

Abstract Drug resistance is a major problem in the chemotherapy of human cancers. It is essential to identify the key targets of chemotherapy for cancer, and molecular-based studies need to be conducted to provide a better understanding of drug sensitivity. In this chapter, we focus on the new candidate molecular targets for drug sensitivity. Our studies indicate that the DNA repair enzyme O6-methyl guanine-DNA methyl transferase (MGMT) and its inhibitor O6-benzylguanine (O6-BG) are the preferential molecular targets for alkylating agents and other anticancer drugs. MGMT is a DNA repair enzyme that rapidly repairs adducts at the O6-position of guanine, and its expression is known to modulate the effectiveness to alkylating agents. Alkylating agents may generate DNA adducts and may produce suicide inactivation of MGMT. The addition of O6-BG to anticancer drugs (i.e. alkylating agents, cisplatin and 5-FU) invariably enhanced their sensitivities in comparison with the response to those drugs when they are used singly. * Address correspondence to: Jun-ichi Asaumi and Jun Murakami, Department of Oral and Maxillofacial Radiology, Field of Tumor Biology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1, Shikata-cho, Okayama 700-8525, Japan. E-mail: [email protected] and [email protected] Telephone number: +81-86-235-6621 Fax number: +81-86-235-6709

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Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al. Our results provide valuable information on the relationship between MGMT expression and drug sensitivity in cancer chemotherapy. These findings will allow clinicians to identify the patients most likely to benefit from chemotherapy and potentially spare many patients from unnecessary therapy.

Alkylating Agents in Cancer Chemotherapy

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Among alkylating agents, methylating and chloroethylating agents are used in the therapy of various kinds of cancer, including brain cancer, melanoma, lymphoma, and gastrointestinal cancers (1-4). Methylating agents (e.g., procarbazine, dacarbazine, streptozotocin and temozolomide) and chloroethylating agents (e.g., carmustine, lomustine, semustine and fotemustine) alkylate DNA at various sites, among them the O6-position of guanine in DNA. For example, Figure 1 shows the methyl adduct at the O6-position of guanine generated by a methylating agent. The resulting DNA lesions (i.e., O6-methylguanine and O6-chloroethylguanine) are believed to be important for the mutagenic and carcinogenic actions of such compounds (5), thus acting as a trigger of cytotoxicity and apoptosis (6-9). Cytotoxicity and apoptosis caused by O6-methylguanine cause cytotoxicity and apoptosis via mismatch repair (MMR) (10, 11). Another DNA adduct, O6-chloroethylguanine, is capable of rearranging through an intermediate to produce a DNA-interstrand crosslink between N1 of guanine and N3 of cytosine (12-14). The formation of this DNA-interstrand cross link is supposed to block DNA replication and trigger the cytotoxic response.

Figure 1. O6-methylguanine. Methylating agents (e.g., procarbazine, dacarbazine, streptozotocin and temozolomide) and chloroethylating agents (e.g., carmustine, lomustine, semustine and fotemustine) alkylate DNA at various sites, among them the O6 position of guanine. DNA adducts induced at the O6position of guanine by alkylating agents are believed to be important for the mutagenic, carcinogenic and cytotoxic actions of such compounds. Methylating agents are used to treat a variety of human tumors.

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To protect cells from DNA damage induced by alkylating agents, several DNA repairing systems are involved (15), such as direct reversal of DNA repair enzyme MGMT, nucleotide excision repair, MMR, and base excision repair (BER). Among those repair mechanisms, MGMT plays a role as the primary defense against the lethal effects of O6-alkylguanines in DNA. In this chapter, we focus on MGMT and its inhibitor O6-benzylguanine (O6-BG) as molecular targets for alkylating agents and other anticancer agents.

O6-Methyl Guanine-DNA Methyl Transferase (MGMT)

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MGMT is a highly important DNA repair protein involved in the repair of O6alkylguanine adducts (16). The human MGMT protein consists of 208 amino acids and has a molecular weight of 22 kDa (17). MGMT was found in both the nucleus and the cytoplasm in cells (18-20) (Figures 2 and 3). MGMT plays a role as the primary defense against the lethal effects of O6-alkylguanines in DNA. Among O6-alkylguanines, O6-methylguanine and O6chloroethylguanine are subject to repair by MGMT. Interestingly, the DNA-interstrand cross links generated by O6-chloroethylguanine are not subject to repair by MGMT; however, repair of the initial O6-chloroethylguanine adduct by MGMT is of particular significance in preventing cross-link formation (21, 22).

Figure 2. Pathological features. Tissue samples embedded in paraffin wax block were obtained from a patient with oral SCC who underwent curative surgery at the Okayama University Hospital, Okayama, Japan. (a) Haematoxylin and eosin (H&E) staining. Serial sections were cut from each paraffin wax block, and H&E staining was carried out for histopathological diagnosis. Original magnification, x4. (b) Immunohistochemistry of MGMT in oral SCC patient. The sample was probed with monoclonal antibody MT 3.1 specific for human MGMT (NeoMarkers, Fremont CA, USA). MGMT was stained in the cancer cells and corresponding normal epithelium. Original magnification, x4. (c) Immunohistochemistry of MGMT in oral SCC patient. Original magnification, x10. (d) Immunohistochemistry of MGMT in oral SCC patient. Original magnification, x20. >From [Murakami J et al. (2008) NOVASCIENCE], with permission.

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Figure 3. Immunocytochemistry of MGMT in HSC3 and HSC4 cells with rich expression of MGMT. The protein expressions of MGMT were monitored with mouse anti-MGMT monoclonal antibody. The cells were fixed with 10% formaldehyde and processed with the standard avidin-biotin peroxidase complex. DAB was used as chromogen. (a) HSC3 (b) HSC4. Original magnification, x40. Both cell lines expressed MGMT ubiquitously. MGMT protein was localized in the nucleus and cytoplasm. >From [Murakami J et al. (2008) NOVASCIENCE], with permission.

In the repair of alkylguanine adducts, MGMT transfers the alkyl adduct from the O6position of guanine onto an internal cysteine residue in MGMT (Figure 4). This methylation of the MGMT cysteine residue prevents any further repair by the transferase and likely induces a conformational change leading to rapid ubiquitin-mediated degradation (23). Thus, guanine in the DNA is restored and the MGMT is inactivated in “suicidal” way. Due to the suicide mechanism of action, a cell’s ability to repair O6-alkylguanines with MGMT depends on the initial number of MGMT molecules per cell and the rate of resynthesis of MGMT (5, 24).

Figure 4. MGMT removes alkyl adducts from the O6-position of guanine in DNA. MGMT removes alkyl adducts from the O6-position of guanine and transfers the alkyl adducts from DNA to a reactive cysteine residue within MGMT (a suicidal repair protein). MGMT protects cells from the cytotoxic and genotoxic effects of O6-alkylguanine-generating agents.

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In the clinical setting, MGMT is an important protein that contributes to the resistance of human cancers to clinically relevant alkylating agents (25-31). In determining responses to chemotherapy with alkylating agents, the level of MGMT has been clearly demonstrated to be a prognostic marker (32–34). The level of MGMT has been determined in a broad range of different human cancers (Figure 5 and Table 1) and compared with the corresponding normal tissues (35). Interestingly, MGMT levels vary according to the tissue or cancer cell type, and are often higher in cancer cells than in the surrounding tissue (36-38).

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Figure 5. Western blot analysis for MGMT protein. MGMT activity is closely correlated with the spontaneous MGMT protein expression detected by the immuno-reactive method, including Western blotting analysis or immunohistochemical study (92, 191). We studied the MGMT protein expression status of two colorectal cancer and four oral cancer cell lines by Western blotting. Whole cell lysates from the indicated cell lines were loaded onto a 10% SDS-PAGE gel and electrophoresed. Proteins were electroblotted onto a PVDF membrane which was probed with monoclonal antibody specific for human MGMT. Each of the blots shown was demonstrated to have equal protein loading by reprobing with the monoclonal antibody for beta-actin (beta actin AC-15-ab6276, Abcam Limited, Cambridge, UK). A high level of MGMT expression was observed in 2 of the CRC cell lines (LoVo and RPMI4788) and 2 of the oral cancer cell lines (HSC4 and HSC3). On the other hand, low expression levels were observed in 2 of the oral cancer cell lines (SAS and Hep2). >From [Murakami J et al. (2007) Oncol Rep. 17(6):1461-1467.], with permission.

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Figure 6. Sensitivity to anticancer agents in oral cancer cell lines. To confirm the contribution of MGMT expression status to the cellular sensitivity to alkylating agents, we treated each of the oral cancer cell lines with varying concentrations of the alkylating agents N-methyl-N’-nitro-N nitrosoguanidine (MNNG) (Nacalai Tosque, Inc. Kyoto, Japan) and N-methyl-N-nitrosourea (MNU) (Nacalai Tosque, Inc. Kyoto, Japan) and the non-alkylating chemotherapeutic agent bleomycin (BLM) (Nippon Kayaku Co., Ltd., Tokyo). MNNG, BLM and MNU, diluted in water, were added to MDEM to the final concentration indicated in each treatment. First, 5 x 105 cells were seeded in 5 ml MDEM in a flask (Nalge Nunc International, Roslilde, Denmark). Then, 24 h after seeding, the medium was exchanged for one containing an appropriate agent, and the flask was immersed in a 37°C water bath (Taitec, Co., Ltd., Saitama, Japan). Following drug treatment for 1 h, the cells were rinsed three times with drug-free medium and their survival rates were determined. Cell survivals were assayed by measuring the colony-forming ability of the cells in triplicate. Only colonies containing more than 50 cells were counted. After treatment with drugs, the cells were dispersed with trypsin, seeded at adequate concentrations, and incubated at 37°C in a CO2 incubator. Surviving cells were fixed in 10% formaldehyde and stained with 10% Giemsa stain solution. Cell-survival rates were corrected for the seeding efficacy of untreated controls. (a): Cellular sensitivities against alkylating agent MNNG. HSC4 and HSC3 cells with high MGMT expression were resistant to the lethal effects of MNNG, while SAS and Hep2 cells with low MGMT expression were sensitive. (b): Cellular sensitivities against BLM. HSC3 cells with high MGMT expression were the most sensitive to the lethal effects of BLM, whereas the 3 other lines proved to be much less sensitive. We could find no evidence of a relationship between the MGMT expression status of the 4 cell lines and their sensitivity to BLM. (c): Cellular sensitivities against alkylating agent MNU. Two of the four cell lines expressing relatively large amounts of MGMT protein (HSC4 and HSC3) appeared to be resistant to the lethal effects of MNU, depending on the concentrations used. In contrast, growth of the cell lines expressing low levels of MGMT protein (SAS and Hep2) was inhibited in a dose-dependent manner by MNU (p < 0.05). Each point is the average of three independent experiments, each performed in triplicate; bars, SD. The significance of the differences was tested by the Student’s t-test. Symbols: HSC4, ◆; HSC3, ▲; SAS, □; Hep2, △. >From [Maki Y et al. (2005) Oral Oncol. 41(10):984-93. ] and [Murakami J et al. (2007) Oncol Rep. 17(6):1461-1467.], with permission.

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MGMT is expressed in all normal cell lines and tissues with a few possible exceptions (39). Whereas all normal human tissues and cultured cancer cell lines that constitutively express MGMT are referred to as the Mer methyl repair+ (Mer+) phenotype, a subset of cancer cell lines that appear to be totally MGMT-deficient is referred to as the Mer− phenotype (16, 32, 40-42). Cells expressing MGMT are more resistant to the cytotoxic activities of alkylating agent-based chemotherapy compared with cells deficient in MGMT. In Figure 6, to confirm the contribution of MGMT expression status to cellular sensitivity to the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N’-nitro-Nnitrosoguanidine (MNNG), we treated each of the oral cancer cell lines considered here with varying concentrations of MNNG and MNU and with a non-alkylating chemotherapeutic agent, bleomycin (BLM). The cell lines with high MGMT expression (HSC4 and HSC3) were resistant to the effects of MNNG and MNU, whereas the cell lines with low MGMT expression (SAS and Hep2) exhibited higher sensitivity. In contrast, our group also found no evidence of a relationship between MGMT expression status and BLM sensitivity in the 4 cell lines studied here (Figure 6).

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Loss of MGMT: Epigenetics In Mer+ cells, MGMT protein levels generally correlate well with mRNA levels (43-45). Interestingly, in Mer- cells, the MGMT gene is present with no gross rearrangements or deletions; however, both the protein and mRNA are essentially undetectable. This lack of MGMT expression is presumed to be associated with a defect in transcription (43, 46, 47), most likely through an epigenetic mechanism rather than a mutation. Among several epigenetic mechanisms in mammalian cells, methylation of cytosine in CpG dinucleotides is the main epigenetic modification of DNA (48, 49). CpG methylation is clearly involved in gene activation/inactivation (50-59), genomic imprinting and differentiation (48, 49, 58). Approximately 60% of all human genes contain GC-rich regions of DNA known as CpG islands at their 5’ ends “promoter” region (59). CpG islands are characterized by an abundance of binding sites for ubiquitous transcription factors (such as Sp1), an open chromatin structure, and a lack of cytosine methylation (60). Hypermethylation in CpG islands is intimately linked with a tightly packaged chromatin conformation (50, 61). Given that DNA methylation is a common mechanism of inactivation of tumor suppressor genes in cancers, the alterations in DNA methylation patterns may contribute to carcinogenesis. Several studies suggest that the expression of MGMT is highly influenced by the methylation status of the corresponding gene (62, 63). The MGMT gene is located on chromosome 10 (64), and the 5′ flanking region of the gene includes the promoter. The MGMT promoter contains a typical CpG island which is subject to methylation and which is responsible for silencing the promoter and down-regulation of MGMT activity (62, 63). The aberrant inactivation of the MGMT gene caused by increased methylation of the CpG island is associated with the loss of open chromatin structure (65). To date, the difference between Mer+ and Mer− cells relates to DNA methylation of MGMT gene (66). Specifically, the CpG island at the 5′ flanking region of the MGMT gene is

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highly methylated in Mer− cells, whereas it is totally methylation free in Mer+ cells (67). In Figure 7, we examined the CpG island methylation status of the MGMT promoter region plus neighboring sequences using a bisulfite sequencing technique in seven oral cancer cell lines (Figure 7). We were able to classify the cell lines into two distinct groups because of their somewhat different methylation patterns. The first group consisted of the SAS, Hep2, and HO-1-u-1 cell lines, all of which were hypermethylated in the MGMT promoter region, and a second group consisted of the HSC3, HSC4, HSC2, and KB cell lines, for which there was little or no evidence of hypermethylation. Three oral cancer cell lines with fully methylated sequences of the MGMT gene (SAS, Hep2 and HO-1-u-1) produced significantly reduced levels of MGMT, whereas copious amounts of MGMT were produced by the four cell lines with poorly methylated sequences (HSC4, HSC3, HSC2 and KB) (Figure 5 and Table 1).

Figure 7. Methylation map of the CpG island in the 5’ region of the MGMT gene (-284 to +203) CpGs. | bars represent CpG dinucleotides in the MGMT promoter. Closed circles on the bars represent methylated cytosine. Bars with open circles show no methylation of the CpG cytosine. From [Murakami J et al. (2004) Oncol Rep. 12(2):339-345.], with permission.

Loss of MGMT and/or DNA Mismatch Repair in Alkylation Tolerance The understanding of the DNA repair mechanisms has grown tremendously in recent years. Mammalian DNA MMR is implicated in the repair of DNA base mismatches and the induction of apoptosis (68-71). MMR is one likely factor limiting the clinical efficacy of alkylating agents. Resistance to alkylating agents is a consequence of an abrogated long patch MMR via tolerance of DNA O6-alkylguanine. O6-methylguanine-thymine generated by alkylating agents, as well as various other lesions such as 1,2-intrastrand d(GpG) cross-links generated by cisplatin, are subject of repair by MMR (72, 73) (Figure 8). The repair of O6methylguanine-thymine mismatches is initiated by the binding of a protein complex

Role of O6-Methyl Guanine-DNA Methyl Transferase …

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(designated MutSα) to the mismatch (74). Thus, the MutSα complex (hMSH2 and hMSH6) is able to bind to base-base mismatches and to insertion/deletion mismatches (75-77) in contrast to the MutSβ complex (hMSH2 and hMSH3), which is capable of binding to insertion/deletion mismatches. Since DNA adduct at the O6-position of guanine (i.e., O6methylguanine and O6-chloroethylguanine) is a very efficient trigger of apoptotic signaling (9, 11), the treatment with an alkylating agent can lead to cell death in MGMT-deficient cancer cells (likely via apoptosis) if the cells have intact MMR. However, if MMR fails to recognize or repair base mismatches, the cancer cell develops mutations and resistance to alkylating agents Furthermore, the absence of MMR activity results in resistance to a number of anticancer agents, including cisplatin and doxorubicin (78-80). Previously Lage et al. revealed that the acquired resistance of a melanoma cell line (MeWo) to cisplatin, etoposide and vindesine, but not to fotemustine, was accompanied by down-regulation of the MMR proteins MSH2 and MSH6 (81).

Figure 8. MGMT is required to prevent O6-methylguanine-induced mutations. DNA adducts induced at the O6-position of guanine by alkylating agents are highly pro-mutagenic and pro-carcinogenic and act as a trigger of cytotoxicity and apoptosis. MGMT is able to remove methyl and chloroethyl groups from the O6-position of guanine in a one-step reaction. If not repaired, O6-methylguanine can mispair with thymine during DNA replication. O6-methylguanine mispaired with thymine is subject to repair by the mismatch repair (MMR) system. If the cell has an intact MMR system, MMR can lead to cell death (likely via apoptosis). If the cell fails to recognize or repair base mismatches, O6-methylguanine mispaired with thymine causes GC to AT transition mutations during DNA replication.

Loss of MGMT in G to a Transition Mutation Mer- cancers are unable to repair O6-methylguanine in the treatment with a methylating agent (24, 82). If those adducts are not repaired in the absence of MGMT and MMR activity, O6-methylguanine mispairs with thymine instead of cytosine and, during DNA replication, causes GC to AT transition mutations (83-86). Jackson et al. reported that low MGMT activity was associated with increased mutations in K-ras involving G:C to A:T transitions, but not G to C/T transversions in human colonic mucosa from individuals with colon cancer (87). Interestingly, Esteller et al. reported that MGMT promoter hypermethylation is

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associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis (88).

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O6-Benzylguanine, a Nearly Non-Toxic MGMT Inhibitor MGMT is the major mechanism of resistance of cancer cells to O6-alkylating agents, and its depletion in cancers has become a therapeutic target for sensitizing cells to O6-alkylating agents (89). To deplete MGMT in cancers, attempts have been made to inactivate it by pretreatment with a methylating agent to induce O6-methylguanine (90) or by using specific MGMT inhibitors (25). Inactivation of the MGMT protein reduces its efficiency in repairing O6-alkylguanine adducts, and drug resistance due to MGMT expression can be overcome. Recently, MGMT inhibitors have been developed (91). Given that a variety of O6alkylguanines were substrates for the MGMT protein, the inhibitors of MGMT, such as O6benzyl-2'-deoxyguanosine (dBG) and O6-benzylguanine (O6-BG), have been designed to produce O6-substituted guanine adducts (91). O6-BG is one such specific, rationally designed MGMT inhibitor that produces suicidal inactivation of MGMT with a restoration of sensitivity to chloroethylators or methylators. O6-BG is highly effective at depleting MGMT in the organism upon systemic administration. An advantage of O6-BG over other modulators is that it has been used clinically and does not exhibit significant cytotoxicity as a single agent (92-96). Fishel et al. reported that O6-BG was nearly non-toxic, even at doses as high as 100 M, in the cell lines (97). In this review, we also confirmed that the cytocidal effect on cancer cells induced by O6-BG pre-treatment alone was minimal, as reported previously (Figure 9a), given that the cell survival in the O6-BG-treated groups was always higher than 80% of the survival in the untreated groups the untreated groups. Furthermore, O6-BG is capable of rapidly inactivating high levels of MGMT for prolonged time periods at relatively low concentrations (98). O6-BG inhibited MGMT activity for several hours in animals bearing a MGMT-positive, BCNU-resistant human cancer, and during that time, the

a 120 100 80 60 40 20 0

NT O6- NTO6BG BG HSC4 HSC3

Figure 9. (Continued)

NT O6- NTO6- NT O6- NTO6BG BG BG BG LoVo RPMI4788 SAS Hep2

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Figure 9. Effects of O6-benzylguanine (O6-BG) in cancer cell lines. (a): The cytotoxicity for O6-BG in cancer cell lines. We evaluated the effect of an MGMT inhibitor, O6-BG, on cell growth. O6-BG (Sigma Chemical Co., St. Louis, MO, USA), diluted in water, was added to MDEM to the final concentration indicated in each treatment. For the O6-BG-treated groups, 1×105 cells incubated in medium containing 75 μM of O6-BG for 4 days were rinsed three times with fresh medium, and then the cells were seeded into 96-well plates. Eight replicate wells per assay condition were seeded at a density of 1.5×104 cells in 0.1 ml of medium containing the appropriate amount of O6-BG (37.5 or 75μM). To serve as O6-BG-untreated control groups, cells were also seeded into 96-well plates at the same density in medium lacking O6-BG. The cells were then incubated for 24 h at 37°C. The cells were incubated for an additional 38 h. The alteration of O6-BG sensitivity for each condition was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay. The MTT assay was carried out using the MTT Cell Growth Kit (Chemicon International, Inc. Temecula, CA) according to the manufacturer’s instructions. At the end of the exposure to O6-BG, 10 μl of MTT (5 mg/ml) were added to each well for 4 h at 37℃ to allow MTT to form formazan crystals by reaction with metabolically active cells. Next, 100 μl of color development solution (isopropanol with 0.04 N HCl) were added to each well. Within one hour, the absorbance of each well was measured in a microplate reader (Corona microplate reader MTP-120, Corona Electric Co., Ltd, Japan) with a test wavelength of 570 nm. The percentage of cell growth inhibition was calculated by comparing the absorbance readings from treated versus untreated control cells under each experimental condition. Given that the cell survival in the O6-BG-treated groups was always higher than 80% compared to the untreated groups, the cytotoxicity of O6-BG was confirmed as minimum. Each column is the average of more than five wells; bars, SD. Closed columns represent cells with no O6-BG treatment. Open columns represent cells treated with O6-BG. (b): Western blot analysis of MGMT protein before and after O6BG treatment. The levels of MGMT protein were then examined. Depletions of MGMT protein were observed in all cell lines treated with O6-BG. (c): The restoration of MNNG sensitivity by O6-BG in HSC4 cells. The alteration of MNNG sensitivity for each condition was evaluated using MTT assay. The HSC4 cell line showing resistance to the alkylating agent was treated with our O6-BG combined regimen with the alkylating agent MNNG. Significant enhancement of cellular sensitivity to the MNNG was observed. Closed symbols represent cells with no O6-BG treatment. Open symbols represent cells treated with O6-BG. The significance of the differences was tested by the Student’s t-test: p < 0.05. Each point is the average of more than five wells; bars, SD. >From [Murakami J et al. (2007) Oncol Rep. 17(6):1461-1467.], with permission.

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cancer became highly sensitive to BCNU (99,100). O6-BG has an ED50 of From [Maki Y et al. (2005) Oral Oncol. 41(10):984-93. ], with permission.

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10 to 100 μM. The combined treatment with O6-BG and CDDP produced supra-additive effects compared to the result obtained with CDDP alone. Interestingly, 2 cell lines with low MGMT expression levels (SAS and Hep2) also showed restored sensitivities to the cytocidal effects of CDDP by pre-treatment with O6-BG. Fishel et al. (97) also previously reported that O6-BG enhanced the cytotoxicity of platinum agents against head and neck cancer cell lines. In addition to CDDP, O6-BG-enhanced cytotoxicity was also observed with carboplatin, indicating that O6-BG may be an effective adjuvant for chemotherapy with platinum agents or cross-linking agents in general. From their results (97), pretreatment of a series of head and neck cancer cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon cancer cells with O6-BG (100 μM for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED50 of CDDP and a concomitant increase in the percentage of cells undergoing apoptosis. In future clinical settings, the combination of O6-BG and CDDP may result in increased response rates to CDDP in patients with CDDP-resistant as well as CDDP-sensitive cancers.

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6). How does CDDP Attenuate MGMT Expression? The mechanism of this MGMT attenuation was unclear. D’Atri et al. (153) reported that this was not due to the direct inactivation of MGMT by CDDP or the indirect inactivation via the formation of platinum adducts in DNA because neither platinum nor platinated DNA was able to inactivate purified rhMGMT in vitro. In contrast, Wang and Setlow (152) suggested that the MGMT protein can be inactivated by reaction with platinum adducts in DNA. Interestingly, Kokkinakis et al. (155) observed in brain cancer and nonsmall cell lung cancer cells that when these cancer cells were deprived of methionine (Met) in vitro, their MGMT was markedly down-regulated. Recently, CDDP was shown to affect Met metabolism in cancer cells. Scanlon et al. (156, 157) and Mineura et al. (158) demonstrated that CDDP interfered with Met transport and acted as an inhibitor of amino acid entry. Scanlon et al. (156, 159) proposed that CDDP inhibited the transport of natural amino acids, including Met, into cultured L-1210 cells. Shirasaka et al. (160) also confirmed CDDP inhibition of -Met incorporation into ascitic Yoshida sarcoma cells. Furthermore, CDDP induced the intracellular reduced folate levels (i.e., CH2FH4 and FH4) and Met synthase activity via inhibition of cellular uptake of Met (156, 159-161). This occurs because CDDP inhibits the transport of -Met and indirectly causes the elevation of intracellular CH2FH4 and FH4 levels in cancer cells in vitro and in vivo. Considering their results, CDDP might indirectly attenuate MGMT expressions via its inhibitory effect on Met transport.

7). Which Pathways are Involved in O6-Benzylguanine Enhanced Cytotoxicity of CDDP? The mechanism by which O6-BG enhances CDDP cytotoxicity is not fully understood. Fishel et al. proposed 2 possibilities for this O6-BG-enhanced cytotoxicity of platinum agents. One explanation is that O6-BG is converted to a benzylated nucleotide and misincorporated

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into DNA while undergoing NER or disrupts the pool of unaltered nucleosides required for repair. Another explanation is that O6-BG inhibits the removal of interstrand and/or intrastrand cross-links formed in DNA by the NER pathway. However, O6-BG enhanced the cytotoxicity of CDDP even in a series of cell lines deficient in NER (162), suggesting that O6-BG-mediated enhancement of platinum agents-induced cytotoxicity is independent of NER capacity. Interestingly, given that O6-BG dramatically enhanced the CDDP cytotoxicity not only in cell lines with high MGMT activity (HT29 cells) but also without MGMT activity (CHO cells), they considered that its enhancement was also independent of MGMT status, and that O6-BG was working through a mechanism independent of MGMT. As mentioned above, the metabolism of amino acids (i.e., Met, glutathione) plays a key role in CDDP sensitivity in cancer chemotherapy. We hypothesized that O6-BG might induce glutathione (GSH) and/or Met depletion, causing an increase in CDDP cytotoxicity. Scanlon et al. (156, 157) also reported that the efficacy of CDDP was enhanced by Met depletion (156-158). Hoshiya et al. have shown previously that a Met-depleted diet enhanced the efficacy of CDDP against the human MX-1 breast cancer in nude mice (163). Poirson-Bichat et al. reported that Met depletion was induced by a Met-deprived diet and ethionine to decrease the cellular ATP and GSH content and to enhance the anticancer effects of chemotherapeutic agents on drug-resistant cancers (164). Another amino acid, GSH, as well as Met metabolism, is correlated with cellular sensitivity to anticancer agents. The GSH content is decreased by Met depletion, irreversibly blocks cells in the S phase and G2 of the cell cycle, and induces apoptosis (165). In particular, GSH has been suggested to be of great importance in the metabolism of CDDP, causing alterations in the rate of drug uptake and elimination. In CDDP-resistant human ovarian cancer lines, GSH levels were increased 13- to 50-fold (166). Because GSH plays a protective role against drug cytotoxicity (167-170), the decrease of GSH levels via treatment with the -glutamylcysteine synthetase inhibitor, -Buthionine-(S,R)-sulfoximine (BSO), could enhance the activity of melphalan, CDDP, and other anticancer agents in vitro as well as in vivo (169, 171, 172-174).

O6-Benzylguanine/ MGMT in Relation to 5-Fluorouracil (5-FU) Treatment 1). The Widely used Anticancer Drug 5-FU In this section, we focused on the relationship between MGMT expression and responsiveness to 5-Fluorouracil (5-FU). 5-FU is among the most commonly used antineoplastic agents applied to a variety of malignancies, including colon and oral cancers. As shown in Figure 13, 5-FU is a fluoropyrimidine whose metabolites can cause both RNA- and DNA-directed cytotoxicities (175). Two main modes of action have been proposed for 5-FU through its active metabolites, FdUMP and 5-fluoro UTP (176). 5-fluoro-UTP is incorporated into cellular RNA, resulting in RNA dysfunction. FdUMP is thought to form a ternary complex with 5, 10-methylentetrahydrofolate and thymidylate synthetase (TS), which inhibits DNA synthesis (177). FdUMP inhibits the function of TS which catalyzes the conversion of

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deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), and is incorporated into DNA (178, 179). The catabolic enzymes of 5-FU in cancer tissue, including TS and dihydropyrimidine dehydrogenase (DPD), have been assessed for their effectiveness in clinical practice; however, this effectiveness remains controversial (180, 181). The mismatch repair (MMR) system is also related to the effectiveness of 5-FU. It has been previously shown that cells deficient in MMR are resistant to some cytotoxic agents that act through causing DNA damage. This phenomenon has been proven in response to a variety of chemotherapeutic agents for a number of cell lines defective in MMR (182-185). Carethers et al. have demonstrated that colon cancer cell lines deficient in MMR activity (loss of hMLH1) are significantly more tolerant of the 5-FU effect than the MMR-proficient HCT116+chr3 cells established by chromosomal transfer (186). Accordingly, 5-FU responsiveness might be more complex than has been thought. \ We have recently shown that colorectal cancer (CRC) patients who receive 5-FU as adjuvant chemotherapy have a better prognosis if the tumor reveals hypermethylation in its MGMT promoter (187). These results indicate that CRC cells with low MGMT expression could be sensitive to the 5-FU treatment. MGMT is one of the DNA repair enzymes that act with the MMR system. Although its expression is known to modulate the effectiveness of the alkylating agents or CDDP (40, 41, 42, 150, 188), almost no study has focused on the relationship between MGMT activity and the 5-FU effect on cells.

Figure 13. Metabolism of 5-FU. 5-FU is a fluoropyrimidine whose metabolites can cause both RNAand DNA-directed cytotoxicities. Two main modes of action have been proposed for 5-FU, through its active metabolites, FdUMP and 5-fluoro UTP. 5-fluoro-UTP is incorporated into cellular RNA, resulting in RNA dysfunction. FdUMP is thought to form a ternary complex with 5, 10methylentetrahydrofolate and thymidylate synthetase (TS), which inhibits DNA synthesis. FdUMP inhibits the function of TS, which catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), and is incorporated into DNA.

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2). MGMT and O6-Benzylguanine in the Cytotoxicity of 5-FU

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We, therefore, sought to investigate whether a cell expressing low levels of MGMT is more sensitive to 5-FU in vitro by using human colon adenocarcinoma cell lines, which commonly express high levels of MGMT, and human oral cancer cell lines, which show a variety of MGMT expression (189). To observe the cellular response to 5-FU, four oral cancer cells with a variety of MGMT expression were treated with 5-FU. Interestingly, MGMT low-expressing cell lines (Hep2 and SAS) were sensitive to 5-FU, just as to MNU, in a dose-dependent manner (Figure 14). In contrast, the cell lines expressing a large amount of MGMT (HSC4 and HSC3) were not inhibited in their proliferation by the treatment with 5FU (p < 0.05, Figure 14). To examine the alteration of MGMT expression by the 5-FU treatment, we assessed the protein and mRNA expression levels by Western blotting and RTPCR after treating the cell lines with 5-FU in vitro (Figure 15). Interestingly, the 5-FU treatment uniformly reduced MGMT protein content in all the cell lines examined. The RTPCR results, however, revealed that the mRNA content was slightly degraded by the 5-FU treatment in all cell lines. These results suggested that the MGMT depletion was primarily a posttranslational consequence of 5-FU treatment. As shown in Figure 15, treatment with 5FU in combination with O6-BG revealed enhanced anticancr effects compared with the results obtained with 5-FU treatment alone (Figure 16). The survival rates of the cells treated with O6-BG and 5-FU significantly decreased compared with those treated with 5-FU alone, except for the points indicated by asterisks (p < 0.05). Among the four MGMT-expressed cell lines (HSC4, HSC3, LoVo, and RPMI4788), O6-BG especially enhanced the cytotoxicities of HSC3 and RPMI4788 to 5-FU. Interestingly, two 5-FU-sensitive cell lines with low expression of MGMT (SAS and Hep2) also increased their sensitivities to the cytocidal effects of 5-FU under the O6-BG/5-FU combined regimen.

3). Which Pathways are Involved in O6-Benzylguanine-Enhanced Cytotoxicity of 5-FU? The mechanism involved in the depletion of MGMT protein by 5-FU is not known. Since metabolites of 5-FU cause both RNA- and DNA-directed cytotoxicities (175), 5-FU may also generate DNA adducts (such as O6-alkylating DNA adducts generated by alkylating agents), and MGMT is consumed in the process. It remains to be determined in future attempts whether or not 5-FU generates such adducts. Bibby et al. have reported that molecular combination of 5-FU and alkylating agent 2chloroethyl-1-nitrosourea (CNU) results in a significant growth retardation of BCNUresistant murine colon cancer and human breast tumor xenografts in mice in vivo (89). We also took into account in this study that the up-regulation of the CNU effect by 5-FU might have been achieved by the depletion of MGMT caused by 5-FU. Yoshioka et al. (190) reported that Met depletion by methioninase (rMETase) potentiates the anticancer efficacy of 5-FU in the treatment of the Lewis lung carcinoma. rMETase is synergistic in combination with 5-FU, and has negligible toxicity. Considering their results,

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O6-BG might have caused Met depletion and enhanced the anticancer effect of 5-FU in our study. In summary, MGMT depletion uniformly occurs in response to 5-FU in colon and oral cancer cell lines, irrespective of the original expression levels of MGMT. The levels of MGMT expression were related to the sensitivity to 5-FU, and an enhancement of the anticancer effect of 5-FU was observed in response to MGMT depletion. Our result demonstrates that MGMT activity is one of the causes of 5-FU resistance, thus providing the first evidence for an essential involvement of MGMT in 5-FU-induced cellular toxicity. As such, the depletion of MGMT in cancer cells could become an important therapeutic target for sensitizing cells to 5-FU treatment. Our current in vitro findings, in addition to our previous clinical studies, suggest important clinical applications related to enhancing the 5FU anticancer effect in various types of cancers.

Figure 14. Sensitivities to 5-FU in oral cancer cell lines. 5-fluorouracil (5-FU) (Wako Pure Chemical Industries, Ltd. Osaka, Japan) diluted in water was added to the MDEM to the final concentration indicated in each treatment. First, 5 x 105 cells were seeded in 5 ml MDEM in a flask (Nalge Nunc International, Roslilde, Denmark). Then, 24 h after seeding, the medium was exchanged for one containing 5-FU, and the flask was immersed in a 37°C water bath. Following drug treatment for 1 h, the cells were rinsed three times with drug-free medium and their survival rates were determined by measuring the colony-forming ability of the cells in triplicate. MGMT low-expressing cell lines (Hep2 and SAS) were sensitive to 5-FU, just as to alkylating agents (shown in Figure 6), in a dose-dependent manner. In contrast, the cell lines expressing a large amount of MGMT (HSC4 and HSC3) were not inhibited in their proliferation by the treatment with 5-FU (p < 0.05). The significance of the differences was tested by Student’s t-test. Each point is the average of three independent experiments, each performed in triplicate; bars, SD. Symbols: HSC4, ◆; HSC3, ▲; SAS, □; Hep2, △. >From [Murakami J et al. (2007) Oncol Rep. 17(6):1461-1467.], with permission.

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Figure 15. Effects of 5-FU on MGMT expressions in colon and oral cell lines. In order to test whether or not MGMT expression was altered by 5-FU, we examined the levels of MGMT expression after treating the cell lines with 5-FU. For the 5-FU-treated groups, 1 x 105 cells were incubated in the medium containing 200 μM of 5-FU for 38 h. The protein and mRNA expressions of MGMT were measured by Western blotting or RT-PCR, and then the results were compared to those obtained with non-treated cells. (a): 5-FU treatment attenuated MGMT protein in all 6 cancer cell lines. A high level of MGMT expression was observed in 2 of the CRC cell lines (LoVo and RPMI4788) and 2 of the oral cancer cell lines (HSC4 and HSC3). On the other hand, low expression levels were observed in 2 of the oral cancer cell lines (SAS and Hep2). Interestingly, 5-FU treatment uniformly reduced MGMT protein content in all the cell lines examined. (b): 5-FU treatment attenuated MGMT mRNA in all 6 cancer cell lines. The RT-PCR results revealed that the mRNA content was slightly degraded by the 5-FU treatment in all cell lines. These results suggested that the MGMT depletion was primarily a posttranslational consequence of 5-FU treatment. (c) Intensities of the bands were quantified by the proportion of MGMT versus beta actin or GAPDH with Image J 1.33u as described above. The relative band intensity represents the intensity of 5-FU treated sample / 5-FU un-treated control sample. Closed columns represent cells with no 5-FU treatment. Open columns represent cells treated with 5-FU. The significance of the differences was tested by Student’s t-test: p < 0.05. Each column is the average of three measurements; bars, SD. The band intensities in groups treated with 5-FU were significantly different from those without 5-FU. >From [Murakami J et al. (2007) Oncol Rep. 17(6):1461-1467.], with permission.

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Figure 16. Effects of O6-BG on cellular sensitivity to 5-FU. To determine whether MGMT depletion may enhance the anti-tumor effect of 5-FU, we evaluated the combined effects of 5-FU and MGMT inhibitor O6-BG on cellular sensitivity in 4 oral and 2 colon cancer cell lines. For the O6-BG-treated groups, cells were exposed to 75 μM O6-BG for 4 days. Then, the cells were washed and seeded into 96-well plates with medium containing the appropriate concentrations of O6-BG (37.5 or 75 μM). After 24-h incubation, the cells were then exposed to 5-FU at various concentrations ranging from 100-800 μM for an additional 38 h. Alteration of the chemosensitivity for each condition was evaluated using an MTT assay. Closed symbols represent the survivals of 5-FU single-treated groups, while open symbols represent the survivals of O6-BG /5-FU combination-treated groups. Each point is the average of more than five wells; bars, SD. Treatment with 5-FU in combination with O6-BG revealed enhanced antitumor effects compared with the results obtained with 5-FU treatment alone. Interestingly, two 5-FUsensitive cell lines with low expression of MGMT (SAS and Hep2) also increased their sensitivities to the cytocidal effects of 5-FU under the O6-BG/5-FU combined regimen. The cell-survival rates in groups treated with 5-FU together with O6-BG were found to be significantly different (p < 0.05) from those without O6-BG by Student’s t-test, except for the asterisked groups.

References [1] [2]

[3]

Colvin M. The alkylating agents. In: Chabner B. (ed), Pharma-cological Principles of Cancer Treatment. (1985) Philadelphia: WB Saunders Co, pp.276-308. Weinkam R. J Shiba DA, Chabner BA. Nonclassical alkylating agents. In: Chabner B. (ed), Pharmacological Principles of Cancer Treatment. (1985) Philadelphia: WB Saunders Co, pp.340-362. Newlands ES, Blackledge GRP, Slack JA, Rustin GJS, Smith, DB, Stuart NSA, Quarterman CP, Hoffman R, Stevens MFG, Brampton MH, Gibson AC. Phase I trial of

58

[4]

[5]

[6]

[7]

[8]

[9] [10] [11]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[12]

[13]

[14] [15] [16] [17]

[18]

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al. temozolomide (CCRG81045: M&B39831: NSC362856). (1992) Br J Cancer. 65: 287291. Tew K, Colvin M, Chabner BA. Alkylating agents. In: Chabner BA, Longo DL. (ed), Cancer chemotherapy: principles and practice, 2nd edition. (1995) Philadelphia: JB Lippincott. Pegg AE. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. (1990) Cancer Res. 50: 6119-6129. Day III RS, Babich MA, Yarosh DB, Scudiero DA. The role of O6-methylguanine in human cell killing, sister chromatid exchange induction and mutagenesis: a review. (1987) J Cell Sci. 6: 333-353. Kaina B, Fritz G, Mitra S, Coquerelle T. Transfection and expression of human O6methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents. (1991) Carcinogenesis. 12: 1857-1867. Kaina B, Ziouta A, Ochs K, Coquerelle T. Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex-, Mex+ and methylationtolerant mismatch repair compromised cells: facts and models. (1997) Mutat Res. 381: 227-241. Meikrantz W, Bergom MA, Memisoglu A, Samson L. O6-Alkylguanine DNA lesions trigger apoptosis. (1998) Carcinogenesis. 19: 369-372. Hickman MJ, Samson LD. Role of mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. (1999) Proc Natl Acad Sci USA. 96: 10764-10769. Ochs K, Kaina B. Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/-3 regulated and Fas/caspase-8 independent. (2000) Cancer Res. 60: 58155824. Tong WP, Kirk MC, Ludlum DB. Formation of the cross-link 1-[N3-deoxycytidyl),2[N1-deoxyguanosinyl]ethane in DNA treated with N,N'-bis(2-chloroethyl)-Nnitrosourea. (1982) Cancer Res. 42: 3102-3105. Tong WP, Kirk, MC, Ludlum DB. Mechanism of action of the nitrosoureas--V. Formation of O6-(2-fluoroethyl)guanine and its probable role in the crosslinking of deoxyribonucleic acid. (1983) Biochem. Pharmacol. 13: 2011-2015. Ludlum DB. DNA alkylation by the haloethylnitrosoureas: nature of modifications produced and their enzymatic repair or removal. (1990) Mutat Res. 233: 117-126. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature (Lond.), 396: 643–649, 1998. Pegg AE. Byers TL. Repair of DNA containing O6-alkylguanine. (1992) FASEB J. 6: 2302-2310. Tano K, Shiota S, Collier J, Foote RS, Mitra S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. (1990) Proc nat Acad Sci (Wash). 87: 686-690. Ayi TC, Loh KC, Au RB, Li BF. Intracellular localization of human DNA repair enzyme methylguanine-DNA methyltransferase by antibodies and its importance. (1992) Cancer Res. 52: 6423-6430.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

59

[19] Brent TP, von Wronski MA. Edwards CC, Bromley M, Margison GP, Raffarty JA, Pegram CN, Bigner DD. Identification of nitrosourearesistant human rhabdomyosarcomas by in situ immunostaining of O6-methylguanine-DNA methyl transferase. (1993) Oncol Res. 5: 83-86. [20] Lee SM, Rafferty JA, Elder RH, Fan CY, Bromley M, Harris M, Thatcher N, Potter PM, Altermatt HJ, Perinat-Frey T, Cerny T, O'Connor PJ, Margison GP. Immunohistological examination of the inter- and intracellular distribution of O6alkylguanine DNA-alkyltransferase in human liver and melanoma. (1992) Br J Cancer. 66: 355-360. [21] Robins P, Harris AL, Goldsmith I, Lindahl T. Cross linking of DNA induced by chloroethylnitrosourea is prevented by O6-methylguanine-DNA methyltransferase. (1983) Nucleic Acid Res. 11: 7743-7758. [22] Brent TP. Suppression of cross-link formation in chloroethylnitrosourea treated DNA by an activity in extracts of human leukemic lymphoblasts. (1984) Cancer Res. 44: 1887-1892. [23] Srivenugopal KS, Yuan XH, Friedman HS. Ali-Osman F. Ubiquitination-dependent proteolysis of O6-methylguanine-DNA methyltransferase in human and murine tumor cells following inactivation with O6-benzylguanine or 1,3-bis(2-chloroethyl)-1nitrosourea. (1996) Biochemistry. 35:1328-1334. [24] Mitra S, Kaina B. Regulation of repair of alkylation damage in mammalian genomes. (1993) Prog Nucleic Acid Res Mol Biol. 44: 109-142. [25] Dolan ME, Pegg AE. O6-Benzylguanine and its role in chemotherapy. (1997) Clin Cancer Res. 3: 837-847. [26] Bobola MS, Blank A, Berger MS, Silber JR. Contribution of O6-methylguanine-DNA methyltransferase to monofunctional alkylating agent resistance in human brain tumorderived cell lines. (1995) Mol Carcinog. 13: 70-80. [27] Bobola MS, Berger MS, Silber JR. Contribution of O6-methylguanine-DNA methyltransferase to resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human brain tumor-derived cell lines. (1995) Mol Carcinog. 13: 81-88. [28] Bobola MS, Tseng SH, Blank A, Berger MS, Silber JR. Role of O6-methyl-guanineDNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin. (1996) Clin Cancer Res. 2: 735-741. [29] Kreklau EL, Kurpad C, Williams DA, Erickson LC. Prolonged inhibition of O6methylguanine DNA methyltransferase in human tumor cells by O6-benzylguanine in vitro and in vivo. (1999) J Pharmacol Exp. Ther. 291: 1269-1275. [30] Keir ST, Dolan ME, Pegg AE, Lawless A, Moschel RC, Bigner DD, Friedman HS. O6benzylguanine-mediated enhancement of nitrosourea activity in Mer- central nervous system tumor xenografts: implications for clinical trials. (2000) Cancer Chemother Pharmacol. 45: 437-440. [31] Srivenugopal KS, Shou J, Mullapudi SR, Lang FF Jr, Rao JS, Ali-Osman F. Enforced expression of wild-type p53 curtails the transcription of the O6-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents. (2001) Clin Cancer Res. 7: 1398-1409.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

60

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

[32] D’Incalci M, Citti L, Taverna P, Catapano CV. Importance of the DNA repair enzyme O6-alkyl guanine alkyltransferase (AT) in cancer chemotherapy. (1988) Cancer Treat Rev. 15: 279-292. [33] Brent TP, Houghton PJ, Houghton JA. O6-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea. (1985) Proc Natl Acad Sci USA. 82: 2985-2989. [34] Dumenco LL, Allay E, Norton K, Gerson SL. The prevention of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA alkyltransferase. (1993) Science. 259: 219-222. [35] Preuss I, Eberhagen I, Haas S, Eibl RH, Kaufmann M, von Minckwitz G, Kaina B. O6methylguanine-DNA methyltransferase activity in breast and brain tumors. (1995) Int J Cancer. 61: 321-326. [36] Gerson SL, Trey JE, Miller K, Berger NA. Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues. (1986) Carcinogenesis. 7: 745-749. [37] Preuss I, Haas S, Eichhorn U, Eberhagen I, Kaufmann M, Beck T, Eibl RH, Dall P, Bauknecht T, Hengstler J, Wittig BM, Dippold W, Kaina B. Activity of the DNA repair protein O6-methylguanine-DNA methyltransferase in human tumor and corresponding normal tissue. (1996) Cancer Detect Prev. 20: 130-136. [38] Silber JR, Mueller BA, Ewers TG, Berger MS. Comparison of O6-methylguanine-DNA methyltransferase activity in brain tumors and adjacent normal brain. (1993) Cancer Res. 53: 3416-3420. [39] Citron M, Decker R, Chen S, Schneider S, Graver M, Kleynerman L, Kahn LB, White A, Schoenhaus M, Yarosh D. O6-Methyiguarrine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary. (1991) Cancer Res. 51: 41314134. [40] Day RSIII, Ziolkowski CH, Scudiero DA, Meyer SA, Lubmiecki AS, Girardi AJ, Galloway SM, Bynum GD. Defective repair of alkylated DNA by human tumor and SV40-transformed human cell strains. (1980) Nature. 288: 724-727. [41] Tsujimura T, Zhang YP, Fujio C, Chang HR, Watatani M, Ishizaki K, Kitamura H, Ikenaga M. O6-methylguanine methyltransferase activity and sensitivity of Japanese tumor cell strains to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)- 3nitrosourea hydrochloride. (1987) Jpn J Cancer Res. 78: 1207-1215. [42] Sklar R, Strauss B. Removal of O6-methylguanine from DNA of normal and xeroderma pigmentosum-derived lymphoblastoki cell lines. (1981) Nature. 289: 417-420. [43] Ostrowski LE, von Wronski MA, Bigner SH, Rasheed A, Schold SC Jr, Brent TP, Mitra S, Bigner DD. Expression of O6-methylguanine-DNA methyltransferase in malignant human glioma cell lines. (1991) Carcinogenesis. 12(9): 1739-1744. [44] He X, Ostrowski LE, von Wronski MA, Friedman HS, Wikstrand CJ, Bigner SH, Rasheed A, Batra SK, Mitra S, Brent TP, Bigner DD. Expression of O6methylguanine-DNA methyltransferase in six human medulloblastoma cell lines. (1992) Cancer Res. 52: 1144-1148.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

61

[45] von Wronski MA, Harris LC, Tano K, Mitra S, Bigner DD, Brent TP. Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts. (1992) Oncol Res. 4: 167-174. [46] Yarosh DB, Foote RS, Mitra S, Day RS 3rd. Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains. (1983) Carcinogenesis. 4(2): 199-205. [47] Pieper RO, Futscher BW, Dong Q, Ellis TM, Erickson LC. Comparison of O-6methylguanine DNA methyltransferase (MGMT) mRNA levels in Mer+ and Merhuman tumor cell lines containing the MGMT gene by the polymerase chain reaction technique. (1990) Cancer Commun. 2(1): 13-20. [48] Bird AP. CpG-nch islands and the function of DNA methylation. (1986) Nature. 321: 209-213. [49] Jones PA. DNA methylation and cancer. (1986) CancerRes. 46: 461-466. [50] Lewis J, Bird A. DNA methylation and chromatin structure. (1991) FEBS Lett. 285(2): 155-159. [51] Pfeifer GP, Tanguay RL, Steigerwald SD, Riggs AD. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. (1990) Genes Dev. 4(8): 1277-2187. [52] Levine A, Cantoni BL, Razin A. Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism. (1992) Proc Natl Acad Sci U S A. 89: 10119-10123. [53] Antequera F, Macleod D, Bird AP. Specific protection of methylated CpGs in mammalian nuclei. (1989) Cell. 58(3): 509-517. [54] Wolf SF, Jolly DJ, Lunnen KD, Friedmann T, Migeon BR. Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation. (1984) Proc Natl Acad Sci U S A. 81(9): 2806-2810. [55] Stein R, Sciaky-Gallili N, Razin A, Cedar H. Pattern of methylation of two genes coding for housekeeping functions. (1983) Proc Natl Acad Sci U S A. 80(9): 24222426. [56] Razin A, Riggs AD. DNA methylation and gene function. (1980) Science. 210(4470): 604-610. [57] Stöger R, Kubicka P, Liu CG, Kafri T, Razin A, Cedar H, Barlow DP. Maternalspecific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. (1993) Cell. 73(1): 61-71. [58] Leonhardt H, Bestor TH. Structure, function and regulation of mammalian DNA methyltransferase. In: Jost JP, Saluz HP. (ed), DNA Methylation: Molecular Biology and Biological Significance. pp.109-119. Basel: Birkhäuser Verlag, 1993. [59] Larsen F, Gundersen G, Lopez R, Prydz H. CpG islands as gene markers in the human genome. (1992) Genomics. 13(4): 1095-1107. [60] Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. (1987) J Mol Biol. 196(2): 261-282.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

62

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

[61] Antequera F, Boyes J, Bird A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. (1990) Cell. 62: 503-514. [62] Harris LC, Potter PM, Tano K, Shiota S, Mitra S, Brent TP. Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. (1991) Nucleic Acids Res. 19: 6163-6167. [63] Qian XC, Brent TP. Methylation hot spots in the 5 flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene. (1997) Cancer Res. 57: 36723677. [64] Nakatsu Y, Hattori K, Hayakawa H, Shimizu K, Sekiguchi M. Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase. (1993) Mutat Res. 293(2): 119-132. [65] Costello JF, Futscher BW, Tano K, Graunke DM, Pieper RO. Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells. (1994) J Biol Chem. 269: 17228-17237. [66] Qian X, von Wronski MA, Brent TP. Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression. (1995) Carcinogenesis. 16: 1385-1390. [67] Graessmann M, Graessmann A. In: Jost JP, Saluz HP. (ed), DNA Methylation: Molecular Biology and Biological Significance. pp.404-424. Basel: Birkhäuser, 1993. [68] Modrich P. Strand-specific Mismatch Repair in Mammalian Cells. (1997) J Biol Chem. 272: 24727-24730. [69] Gong J, Constanzo A, Yang H-Q, Melino G, Kaelin WG, Levrero M, Wang JYJ. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. (1999) Nature (Lond.). 399: 806-809. [70] Anthoney DA, McIlwrath AJ, Gallagher WM, Edlin ARM, Brown R. Microsatellite instability, apoptosis, and loss of p53 function in drug-resistant tumor cells. (1996) Cancer Res. 56: 1374-1381. [71] Duckett DR, Bronstein M, Taya Y, Modrich P. hMutS alpha- and hMutL alphadependent phosphorylation of p53 in response to DNA methylator damage. (1999) Proc Natl Acad Sci USA. 96: 12384-12388. [72] Duckett DR, Drummond JT, Murchie AI, Reardon JT, Sancar A, Lilley DM, Modrich P. Human MutS alpha recognizes damaged DNA base pairs containing O6methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct. (1996) Proc Natl Acad Sci USA. 93: 6443-6447. [73] Yamada M, O'Regan E, Brown R, Karran P. Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins. (1997) Nucleic Acids Res. 25: 491-496. [74] Palombo F, Gallinari P, Iaccarino I, Lettieri T, Hughes M, D'Arrigo A, Truong O, Husuan JJ, Jiricny J. GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. (1995) Science. 268: 1912-1914. [75] Umar A, Boyer JC, Kunkel TA. DNA loop repair by human cell extracts. (1994) Science. 266: 814-816.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

63

[76] Littmann SJ, Fang W, Modrich P. Repair of Large Insertion/Deletion Heterologies in Human Nuclear Extracts Is Directed by a 5' Single-strand Break and Is Independent of the Mismatch Repair System. (1999) J Biol Chem. 274: 7474-7481. [77] Li G-M, Modrich P. Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. (1995) Proc Natl Acad Sci U S A. 92: 1950-1954. [78] Aebi S, Fink D, Gordon R, Kim HK, Zheng H, Fink JL, Howell SB. Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells. (1997) Clin Cancer Res. 3: 1763-1767. [79] Brown R, Hirst GL, Gallagher WM, McIlwrath AJ, Margison GP, van der Zee AGJ, Anthoney DA. hMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents. (1997) Oncogene. 15: 45-52. [80] Toft NJ, Winton DJ, Kelly J, Howard LA, Dekker M, Riele HT, Arends MJ, Wyllie AH, Margison GP, Clarke AR. Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine. (1999) Proc Natl Acad Sci USA. 96: 39113914. [81] Lage H, Christmann M, Kern MA, Dietel M, Pick M, Kaina B, Schadendorf D. Expression of DNA repair proteins hMSH2, hMSH6, hMLH1, O6-methylguanineDNA methyltransferase and N-methylpurine-DNA glycosylase in melanoma cells with acquired drug resistance. (1999) Int J Cancer. 80: 744-750. [82] Tsuzuki T, Sakumi K, Shiraishi A, Kawate H, Igarashi H, Iwakuma T, Tominaga Y, Zhang S, Shimizu S, Ishikawa T, Nakamura K, Nakao K, Katsuki M, Sekiguchi M. Targeted disruption of the DNA repair methyltransferase gene renders mice hypersensitive to alkylating agent. (1996) Carcinogenesis. 17: 1215-1220. [83] Yarosh DB. The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. (1985) Mutation Res. 145: 1-16. [84] Karran P, Bignami M. Self-destruction and tolerance in resistance of mammalian cells to alkylation damage. (1992) Nucleic Acids Res. 20: 2933-2940. [85] Hampson R, Humbert P, Macpherson P, Aquilina G, Karran P. Mismatch Repair Defects and O6-Methylguanine-DNA Methyltransferase Expression in Acquired Resistance to Methylating Agents in Human Cells. (1997) J Biol Chem. 272: 2859628606. [86] Loveless A. Possible Relevance of O-6 Alkylation of Deoxyguanosine to the Mutagenicity and Carcinogenicity of Nitrosamines and Nitrosamides. (1969) Nature. 223: 206-207. [87] Jackson PE, Hall CN, O'Connor PJ, Cooper DP, Margison GP, Povey AC. Low O6alkylguanine DNA-alkyltransferase activity in normal colorectal tissue is associated with colorectal tumours containing a GC->AT transition in the K-ras oncogene. (1997) Carcinogenesis. 18: 1299-1302. [88] Esteller M, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, Watkins N, Issa JP, Sidransky D, Baylin SB, Herman JG.. Inactivation of the DNA repair gene O6methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis. (2000) Cancer Res. 60: 2368-2371.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

64

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

[89] Bibby MC, Thompson MJ, Rafferty JA, Margison GP, McElhinney RS. Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2chloroethyl-1-nitrosourea in mice. (1999) Br J Cancer. 79(9-10): 1332-1339. [90] Gerard B, Aamdal S, Lee SM, Leyvraz S, Lucas C, D'Incalci M, Bizzari JP. Activity and unexpected lung toxicity of the sequential l administration of two alkylating agents - dacarbazine and fotemustine - in patients with melanoma. (1999) Eur J Cancer. 29A(5): 711-719. [91] Dolan ME, Moschel RC, Pegg AE. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-nenzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agenls. (1990) Proc Nati Acad Sci USA. 87: 5368-5372. [92] Dolan ME, Roy SK, Fasanmade AA, Paras PR, Schilsky RL, Ratain MJ. O6Benzylguanine in humans: metabolic, pharmacokinetic, and pharmacodynamic findings. (1998) J Clin Oncol. 16: 1803-1810. [93] Friedman HS, Kokkinakis DM, Pluda J, Friedman AH, Cokgor I, Haglund MM, Ashley DM, Rich J, Dolan ME, Pegg AE, Moschel RC, McLendon RE, Kerby T, Herndon JE, Bigner DD, Schold SC Jr. Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma. (1998) J Clin Oncol. 16: 3570-3575. [94] Spiro TP, Gerson SL, Liu L, Majka S, Haaga J, Hoppel CL, Ingalls ST, Pluda JM, Willson JK. O6-Benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair. (1999) Cancer Res. 59: 2402-2410. [95] Schilsky RL, Dolan ME, Bertucci D, Ewesuedo RB, Vogelzang NJ, Mani S, Wilson LR, Ratain MJ. Phase I clinical and pharmacological study of O6-benzylguanine followed by carmustine in patients with advanced cancer. (2000) Clin. Cancer Res. 6: 3025-3031. [96] Friedman HS, Pluda J, Quinn JA, Ewesuedo RB, Long L, Friedman A H, Cokgor I, Colvin OM, Haglund MM, Ashley DM, Rich JN, Sampson J, Pegg AE, Moschel RC, McLendon RE, Provenzale JM, Stewart ES, Tourt-Uhlig S, Garcia-Turner AM, Herndon JE, II Bigner DD, Dolan ME. Phase I trial of carmustine plus O6benzylguanine for patients with recurrent or progressive malignant glioma. (2000) J Clin Oncol. 18: 3522-3528. [97] Fishel ML, Delaney SM, Durtan LJ, Hansen RJ, Zuhowski EG, Moschel RC, Egorin MJ, Dolan ME. Enhancement of platinum-induced cytotoxicity by O6-benzylguanine. (2003) Mol Cancer Ther. 2: 633-640. [98] Kokkinakis DM, Ahmed M, Delgado R, Fruitwala M, Mohiuddin M, Albores-Saavedra J. Role of O6-methylguanine-DNA methyltransferase in the resistance of pancreatic tumors to DNA alkylating agents. (1997) Cancer Res. 57: 5360-5368. [99] Schold SC, Kokkinakis DM, Rudy J, Moschel RC, Pegg AE. Treatment of human brain xenografts with O6-benzyl-2'-deoxyguanosine and BCNU. (1996) Cancer Res. 56: 2076-2081.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

65

[100] Friedman H, Dolan EM, Moschel RC, Pegg AE, Felker GM, Rich J, Bigner DD, Schold SC. Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme. (1992) J Natl Cancer Inst. 84: 1926-1931. [101] Moschel RC, McDougall MG, Dolan ME, Stine L, Pegg AE. Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanineDNA alkyltransferase. (1995) J Med Chem. 35: 4486-4491. [102] Chen JM, Zhang YP, Moschel RC, Ikenaga M. Depletion of O6-methylguanine-DNA methyltransferase and potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea antitumor activity by O6-benzylguanine in vitro. (1993) Carcinogenesis (Lond.). 14: 1057-1060. [103] Dolan ME, Mitchell RB. Mummen C, Moschel RC, Pegg AE. Effect of O6benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents. (1991) CancerRes. 5: 3367-3372. [104] Felker GM, Friedman HS, Dolan ME, Moschel RC, Schold SC. Treatment of subcutaneous and intracranial brain tumor xenografts with O6-benzyl-guanine and l.3bis(2-chloroethyl)-l-nitrosourea. (1993) Cancer Chemother Pharmacol. 32: 471-476. [105] Gerson SL, Zborowska E, Norton K, Gordon NH, Wilson JK. Synergistic efficacy of (O6-benzylguanineand1.3-bis(2-chloroethyh-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone. (1993) Biochem Pharmacol. 45: 483-491. [106] Chen J, Zhang Y, Sui J, Chen Y. O6-Methylguanine-DNA methyltransferase activity and sensitivity of human tumor cell lines to bis-chloroethyl nitrosourea. (1992) Chin Med Sci J. 8: 187-190. [107] Pegg AE, Swenn K, Chae MY, Dolan ME, Moschel RC. Increased killing of prostate, breast, colon, and lung tumor cells by the combination of inactivators of O6alkylguanine-DNA alkyltransferase and N,N'-bis(2-chloroethyl)-N-nitrosourea. (1995) Biochem Pharmacol. 50: 1141-1148. [108] Magull SA, Zeller WJ. Sensitization of human colon tumor cell lines to carmustine by depletion of O6-alkylguanine-DNA alkyltransferase. (1995) J Cancer Res Clin Oncol. 121: 225-229. [109] Kurpad SN, Dolan ME, McLendon RE, Archer GE, Moschel RC, Pegg AE, Bigner DD, Friedman HS. Intra-arterial O6-benzylguanine enables the specific therapy of nitrosourea resistant human glioma xenografts in athymic rats with N,N'-bis(2chloroethyl)-1-nitrosourea. (1997) Cancer Chemother Pharmacol. 39: 307-316. [110] Gross DS, Garrard TW. Nuclease hypersensitive sites in chromatin. (1988) Annu Rev Biochem. 57: 159-197. [111] Gibson AE, Arris CE, Bentley J, Boyle FT, Curtin NJ, Davies TG, Endicott JA, Golding BT, Grant S, Griffin RJ, Jewsbury P, Johnson LN, Mesguiche V, Newell DR, Noble ME, Tucker JA, Whitfield HJ. Probing the ATP ribose-binding domain of cyclin-dependent kinases 1 and 2 with O(6)-substituted guanine derivatives. (2002) J Med Chem. 45(16): 3381-3393. [112] Toogood PL. Cyclin-dependent kinase inhibitors for treating cancer. (2001) Med Res Rev. 21(6): 487-498. [113] Middleton MR, Kelly J, Thatcher N, Donnelly DJ, McElhinney RS, McMurry TB, McCormick JE, Margison GP. O(6)-(4-Bromothenyl)guanine improves the therapeutic

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

66

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

index of temozolomide against A375M melanoma xenografts. (2000) Int J Cancer. 85: 248-252. [114] Mitchell RB, Dolan ME. Effect of temozolomide and dacarbazine on O6-alkylguanineDNA transferase activity and sensitivity of human tumor cells and xenografts to 1,3bis(2-chloroethyl)-1-nitrosourea. (1993) Cancer Chemother Pharmacol. 32: 59-63. [115] Roy SK, Gupta E, Dolan ME. Pharmacokinetics of O6-benzylguanine in rats and its metabolism by rat. (1995) Drug Metab Dispos. 23: 1394-1399. [116] Hassan AB, Cook PR. Visualisation of replication sites in unfixed cells. (1993) J Cell Sci. 105: 541-550. [117] Hozak P, Jackson DA, Cook PR. Replication factories and nuclear bodies: the ultrastructural characterisation of replication sites during the cell cycle. (1994) J Cell Sci. 107: 2191-2202. [118] Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ Jr. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. (1992) Cell 71: 587-597. [119] Lawley PD. In: Glover PL. (ed.), Chemical carcinogens and DNA. (1979) Monograph 182. Boca Raton, Fla.: CRC Press, Inc, pp.325-484. [120] Kleibl K, Margison GP. Increasing DNA repair capacity in bone marrow by gene transfer as a prospective tool in cancer therapy. (1998) Neoplasma. 45: 181-186. [121] Loktionova NA, Xu-Welliver M, Crone TM, Kanugula S, Pegg AE. Protection of CHO cells by mutant forms of O6-alkylguanine-DNA alkyltransferase from killing by 1,3bis-(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine or O6-benzyl-8oxoguanine. (1999) Biochem Pharmacol. 58: 237-244. [122] Marathi UK, Harris LC, Venable CC, Brent TP. Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity. (1997) Clin Cancer Res. 3: 301-307. [123] Reese JS, Davis BM, Liu L, Gerson SL. Simultaneous protection of G156A methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and sensitization of tumor cells using O6-benzylguanine and temozolomide. (1999) Clin Cancer Res. 5: 163-169. [124] Chinnasamy N, Rafferty JA, Hickson I, Lashford LS, Longhurst SJ, Thatcher N, Margison GP, Dexter TM, Fairbairn LJ. Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase. (1998) Gene Ther. 5: 842-847. [125] Kokkinakis DM, Moschel RC, Pegg AE, Schold SC. Eradication of human medulloblastoma tumor xenografts with a combination of O6-benzyl-2'deoxyguanosine and 1,3-bis(2-chloroethyl)-1-nitrosourea. (1999) Clin Cancer Res. 5: 3676-3681. [126] Kokkinakis DM, Moschel RC, Pegg AE, Schold SC. Potentiation of BCNU antitumor efficacy by 9-substituted O6-benzylguanines. Effect of metabolism. (2000) Cancer Chemother Pharmacol. 45: 69-77.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

67

[127] Cai Y, Wu MH, Xu-Welliver M, Pegg AE, Ludeman, SM, Dolan ME. Effect of O6benzylguanine on alkylating agent-induced toxicity and mutagenicity in Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases. (2000) Cancer Res. 60: 5464-5469. [128] Cai Y, Ludeman SM, Wilson LR, Chung AB, Dolan ME. Effect of O6-benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells. (2001) Mol Cancer Ther. 1: 21-28. [129] Shen DW, Pastan I, Gottesman MM. Cross-resistance to methotrexate and metals in human cisplatin-resistant cell lines results from a pleiotropic defect in accumulation of these compounds associated with reduced plasma membrane binding proteins. (1998) Cancer Res. 58: 268-275. [130] Reed E, Kohn KW. Cisplatin and platinum analogs. In: Chabner BA, Collins J. (ed), Cancer Chemotherapy Principles and Practice, pp. 465-490. Philadelphia: JB Lippincott, 1990. [131] Zamble DB, Lippard SJ. Cisplatin and DNA repair in cancer chemotherapy. (1995) Trends Biochem Sci. 20: 435-439. [132] Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A. HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease. (1994) Proc Natl Acad Sci U S A. 91: 10394-10398. [133] Kartalou M, Essigmann JM. Mechanisms of resistance to cisplatin. (2001) Mutat Res. 478: 23-43. [134] Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins, and hyperthermia. (1990) Biochem Pharmacol. 40: 2353-2362. [135] Chu G. Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. (1994) J Biol Chem. 269: 787 -790. [136] Ormerod MG, Orr RM, Peacock JH. The role of apoptosis in cell killing by cisplatin: a flow cytometric study. (1994) Br J Cancer. 69: 93-100. [137] Ikeguchi M, Tatebe S, Kaibara N, Ito H. Changes in levels of expression of p53 and the product of the bcl-2 in lines of gastric cancer cells during cisplatin-induced apoptosis. (1997) Eur Surg Res. 29: 396-402. [138] Ljungman M, Zhang F, Chen F, Rainbow AJ, McKay BC. Inhibition of RNA polymerase II as a trigger for the p53 response. (1999) Oncogene. 18: 583-592. [139] Auersperg N, Edelson MI, Mok SC, Johnson SW, Hamilton TC. The biology of ovarian cancer. (1998) Semin Oncol. 25: 281-304. [140] Pattanaik A, Bachowski G, Laib J, Lemkuil D, Shaw CF III, Petering DH, Hitchcock A, Saryan L. Properties of the reaction of cis-dichlorodiammineplatinum(II) with metallothionein. (1992) J Biol Chem. 267: 16121-16128. [141] Kelley SL, Basu A, Teicher BA, Hacker MP, Hamer DH, Lazo JS. Overexpression of metallothionein confers resistance to anticancer drugs. (1988) Science (Wash. DC). 241: 1813-1815. [142] De Vita V Jr, Hellman S, Rosenberg S. Cancer, 5th ed. (1997) Philadelphia: LippincottRaven.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

68

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

[143] Mack PC, Gandara DR, Lau AH, Lara PN, Edelman MJ, Gumerlock PH. Cell cycledependent potentiation of cisplatin by UCN-01 in non-small-cell lung carcinoma. (2003) Cancer Chemother Pharmacol. 51(4): 337-348. [144] Barret JM, Cadou M, Hill BT. Inhibition of nucleotide excision repair and sensitisation of cells to DNA cross-linking anticancer drugs by F11782, a novel fluorinated epipodophylloid. (2000) Biochem Pharmacol. 63: 251-258. [145] Gharehbaghi K, Szekeres T, Yalowitz JA, Fritzer-Szekeres M, Pommier YG, Jayaram HN. Sensitizing human colon carcinoma HT-29 cells to cisplatin by cyclopentenylcytosine, in vitro and in vivo. (2000) Life Sci. 68: 1-11. [146] Jiang H, Yang LY. Cell cycle checkpoint abrogator UCN-01 inhibits DNA repair: association with attenuation of the interaction of XPA and ERCC1 nucleotide excision repair proteins. (1999) Cancer Res. 59(18): 4529-4534. [147] Li QQ, Yunmbam MK, Zhong X, Yu JJ, Mimnaugh EG, Neckers L, Reed E. Lactacystin enhances cisplatin sensitivity in resistant human ovarian cancer cell lines via inhibition of DNA repair and ERCC-1 expression. (2001) Cell Mol Biol (Noisy-legrand). 47: Online Pub: OL61-OL72. [148] Yang LY, Li L, Keating MJ, Plunkett W. Arabinosyl-2-fluoroadenine augments cisplatin cytotoxicity and inhibits cisplatin-DNA cross-link repair. (1995) Mol Pharmacol. 47(5): 1072-1079. [149] Maki Y, Murakami J, Asaumi JI, Tsujigiwa H, Nagatsuka H, Kokeguchi S, Fukui K, Kawai N, Yanagi Y, Kuroda M, Tanaka N, Matsubara N, Kishi K. Role of O(6)methylguanine-DNA methyltransferase and effect of O(6)-benzylguanine on the antitumor activity of cis-diaminedichloroplatinum(II) in oral cancer cell lines. (2005) Oral Oncol. 41(10): 984-993. [150] Koul S, McKiernan JM, Narayan G, Houldsworth J, Bacik J, Dobrzynski DL, Assaad AM, Mansukhani M, Reuter VE, Bosl GJ, Chaganti RS, MurtyVV. Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors. (2004) Molecular Cancer. 3: 16. [151] Lee SM, Crowther D, Scarffe JH, Dougal M, Elder RH, Rafferty JA, Margison GP. Cyclophosphamide decreases O6-alkylguanine-DNA alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation. (1992) Cancer. 66: 331-336. [152] Wang L, Setlow RB. Inactivation of O6-alkylguanine-DNA alkyltransferase in HeLa cell by cisplatin. (1989) Carcinogenesis. 10: 1681-1684. [153] D'Atri S, Tentori L, Lacal PM, Graziani G, Pagani E, Benincasa E, Zambruno G, Bonmassar E, Jiricny J. Involvement of the mismatch repair system in temozolomideinduced apoptosis. (1998) Mol Pharmacol. 54: 334-341. [154] Balaña C, Ramirez JL, Taron M, Roussos Y, Ariza A, Ballester R, Sarries C, Mendez P, Sanchez JJ, Rosell R. O6-methyl-guanine-DNA methyltransferase Methylation in Serum and Tumor DNA Predicts Response to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea but not to Temozolamide Plus Cisplatin in Glioblastoma Multiforme. (2003) Clin Cancer Res. 9: 1461-1468.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of O6-Methyl Guanine-DNA Methyl Transferase …

69

[155] Kokkinakis DM, von Wronski MA, Vuong TH, Brent TP, Schold SC Jr. Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells. (1997) Br J Cancer. 75: 779-788. [156] Scanlon KJ, Newman EM, Lu Y, Priest DG. Biochemical basis for cisplatin and 5fluorouracil synergism in human ovarian carcinoma cells. (1986) Proc Natl Acad Sci USA. 83: 8923-2825. [157] Shionoya S, Lu Y, Scanlon KJ. Properties of amino acid transport systems in K562 cells sensitive and resistant to cis-diamminedichloroplatinum (II). (1986) Cancer Res. 46: 3445-3448. [158] Mineura K, Sasajima T, Sasajima H, Kowada M. Inhibition of methionine uptake by cis-diamminedichloroplatinum (II) in experimental brain tumors. (1996) Int J Cancer. 67: 681-683. [159] Scanlon KJ, Safirstein RL, Thies H, Gross RB, Waxman S, Guttenplan JB. Inhibition of amino acid transport by cis-diaminedichloroplatinum (II) derivatives in L1210 murine leukemia cells. (1983) Cancer Res. 43: 4211-4215. [160] Shirasaka T, Shimamoto Y, Ohshimo H, Saito H, FukushimaM. Metabolic basis of the synergistic antitumor activities of 5-fluorouracil and cisplatin in rodent tumor models in vivo. (1993) Cancer Chemother Pharmacol. 32: 167-172. [161] Araki H, Fukushima M, Kamiyama Y, Shirasaka T. fect of consecutive lower-dose cisplatin in enhancement of 5-fluorouracil cytotoxicity in experimental tumor cells in vivo. (2000) Cancer Lett. 60(2):185-191. [162] Fishel ML, Gamcsik MP, Delaney SM, Zuhowski EG, Maher VM, Karrison T, Moschel RC, Egorin MJ, Dolan ME. Role of glutathione and nucleotide excision repair in modulation of cisplatin activity with O 6-benzylguanine. (2005) Cancer Chemother Pharmacol. 55(4): 333-342. [163] Hoshiya Y, Kubota T, Matsuzaki S, Kitajima M, Hoffman RM. Methionine starvation modulates the efficacy of cisplatin on human breast cancer in nude mice. (1996) Anticancer Res. 16: 3515-3518. [164] Poirson-Bichat F, Gonçalves RA, Miccoli L, Dutrillaux B, Poupon MF. Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts. (2000) Clin Cancer Res. 6: 643-653. [165] Poirson-Bichat F, Lopez R, Bras Goncalves RA, Miccoli L, Bourgeois Y, Demerseman P, Poisson M, Dutrillaux B, Poupon MF. Methionine deprivation and methionine analogs inhibit cell proliferation and growth of human xenografted gliomas. (1997) Life Sci. 60: 919-931. [166] Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC, Anderson ME. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. (1992) Proc Natl Acad Sci USA. 89: 3070-3074. [167] Meister A, Anderson M. Glutathione. (1983) Biochem. 52: 711-760. [168] Meister A. In: Woolley PVIII, Tew KD. (ed), Mechanisms of Drug Resistance in Neoplastic Cells, pp.99-127. New York: Academic Press, 1988. [169] Ozols RF, Hamilton TC, Masuda H, Young RC. In: Woolley PVIII, Tew KD. (ed), Mechanisms of Drug Resistance in Neoplastic Cells. (1988) New York: Academic Press, pp.289-306..

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

70

Jun Murakami, Jun-ichi Asaumi, Hidetsugu Tsujigiwa et al.

[170] Tew KD. Glutathione-associated Enzymes in Anticancer Drug Resistance. (1994) Cancer Res. 54: 4313-4320. [171] Ozols RF, Louie KG, Plowman J, Behrens BC, Fine RL, Dykes D, Hamilton TC. Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumorbearing nude mice by buthionine sulfoximine depletion of glutathione. (1986) Biochem Pharmacol. 36: 147-153. [172] O'Dwyer PJ, Hamilton TC, Young RC, LaCreta FP, Carp N, Tew KD, Padavic K, Comis RL, Ozols RF. Depletion of glutathione in normal and malignant human cells in vivo by buthionine sulfoximine: clinical and biochemical results. (1992) J Natl Cancer Inst. 84: 264-267. [173] Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). (1979) J Biol Chem. 254: 7558-7560. [174] Griffith OW. Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. (1982) J Biol Chem. 257: 13704-13712. [175] Mader RM, Muller M, Steger GG. Resistance to 5-fluorouracil. (1998) Gen Pharmacol. 31: 661-666. [176] Langenback RJ, Danenberg PV. Heidelberger, C. Thymidylate synthethase: mechanism of inhibition by 5-fluoro-2'-deoxyuridylate. (1972) Biochem Biophys Res Commun. 48: 1565-1571. [177] Matsuoka H, Ueo H, Sugimachi K, Akiyoshi T. Preliminary evidence that incorporation of 5-fluorouracil into RNA correlates with antitumor response. (1992) Cancer Invest. 10: 265-269. [178] Parker WB, Cheng YC. Metabolism and mechanism of action of 5-fluorouracil, (1990) Pharmacol Ther. 381-395. [179] Major PP, Egan E, Herrick D, Kufe DW. 5-Fluorouracil incorporation in DNA of human breast carcinoma cells. (1982) Cancer Res. 42: 3005-3009. [180] Beck A, Etienne MC, Cheradame S, Fischel JL, Formento P, Renee N, Milano G.. A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil. (1994) Eur J Cancer. 30A(10): 1517-1522. [181] Salonga D, Danenberg KD, Johnson M, Metzger R, Groshen S, Tsao-Wei DD, Lenz HJ, Leichman CG, Leichman L, Diasio RB, Danenberg PV. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. (2000) Clin Cancer Res 6(4): 1322-1327. [182] Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, Christen RD, Boland CR, Koi M, Fishel R, Howell SB. Loss of DNA mismatch repair in acquired resistance to cisplatin. (1996) Cancer Res. 56: 3087-3090. [183] Jacob S, Aguado M, Fallik D, Praz F. The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells. (2001) Cancer Res. 61: 6555-6562.

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[184] de las Alas MM, Aebi S, Fink D, Howell SB, Los G. Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance. (1997) J Natl Cancer Inst. 89: 15371541. [185] Lin X, Ramamurthi K, Mishima M, Kondo A, Christen RD, Howell SB. P53 modulates the effect of loss of DNA mismatch repair on the sensitivity of human colon cancer cells to the cytotoxic and mutagenic effects of cisplatin. (2001) Cancer Res. 61: 15081516. [186] Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB, Boland CR. Mismatch repair proficiency and in vitro response to 5-fluorouracil. (1999) Gastroenterology. 117(1): 123-131. [187] Nagasaka T, Sharp GB, Notohara K, Kambara T, Sasamoto H, Isozaki H, MacPhee DG, Jass JR, Tanaka N, Matsubara N. Hypermethylation of O6-methylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases. (2003) Clin Cancer Res. 9(14): 5306-5312. [188] Day RSIII, Ziolkowski CHJ, Scudiero DA, Meyer SA, Mattern MR. Human tumor cell strains defective in the repair of alkylation damage. (1980) Carcinogenesis (Lond.). 1: 21-32. [189] Murakami J, Lee YJ, Kokeguchi S, Tsujigiwa H, Asaumi J, Nagatsuka H, Fukui K, Kuroda M, Tanaka N, Matsubara N. Depletion of O6-methylguanine-DNA methyltransferase by O6-benzylguanine enhances 5-FU cytotoxicity in colon and oral cancer cell lines. (2007) Oncol Rep. 17(6): 1461-1467. [190] Yoshioka T, Wada T, Uchida N, Maki H,Yoshida H, Ide N, Kasai H, Hojo K, Shono K, Maekawa R, Yagi S, Hoffman RM, Sugita K. Anticancer Efficacy in Vivo and in Vitro, Synergy with 5-Fluorouracil, and Safety of Recombinant Methioninase. (1998) Cancer Res. 58: 2583-2587. [191] Watts GS, Pieper RO, Costello JF, Peng YM, Dalton WS, Futscher BW. Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. (1997) Mol Cell Biol. 17(9): 5612-5619.

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In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter III

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy Drug Resistance: The Potential for Its Modulation to Achieve Therapeutic Gain A. Weickhardt∗ and M. Michael Division of Haematology and Medical Oncology Peter MacCallum Cancer Centre, Victoria, Australia

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Introduction Cancer cells grow within a host microenvironment that supports and nourishes their growth, enabling them to invade locally and metastasize to distant sites [1]. These supports can be either cellular in nature, comprising surrounding fibroblasts, pericytes and smooth muscles, or physical in nature due to the content and structure of the surrounding extracellular matrix with its associated interstitial hypoxia as well as high tumour interstitial fluid pressures (TIFP)—a direct consequence of abnormal tumoural vasculature. These components of the tumoural microenvironment can all contribute to difficulties in targeting and therapeutically inhibiting the growth of such tumours, quite distinct to the well-defined tumoural intracellular mechanisms of drug resistance [2]. Using drugs that target the cancer cells directly alone fails to account for this supporting tumoural microenvironment and can lead to therapeutic failure. The “seed and soil” hypothesis first proposed by Paget in 1889 suggests that both seed (the cancer cell) and soil (the tumour microenvironment) need to be targeted to achieve therapeutic success. The tumour microenvironment is therefore fertile ∗

Address for Correspondence: Dr. M. Michael, Consultant Medical Oncologist, Division of Haematology and Medical Oncology, Chair of GI Clinical Service, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Victoria, 8006, Australia. Telephone: +61-3-9656-1159; Facsimile: +61-3-9656-1408; [email protected]

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ground for drug discovery, with many different agents currently in pre-clinical, early phase 1 and 2 trials, and with some drugs already being used successfully in practice. This review will discuss the pathophysiology of the tumoural microenvironment and the current success and research that aim to take advantage of this for therapeutic gain.

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Tumour Interstitial Pressure Tumour cells are surrounded by a microenvironment with a high TIFP. This is a common feature of many different solid organ malignancies, and the pressures within these tumours can be in the range of 5–50 mmHg [3,4]. However, there may be substantial variation in intratumoural TIFP from region to region and potentially between the primary lesion and metastatic deposits, so reliability for in vivo assessment depends on multiple assessments within each tumour or site [5]. The intratumoural variation may be due to packing density of cells within those regions [6]. There is no current data analysing the differences for TIFP between the primary tumour and its metastases. There are different reasons for TIFP elevation. A possible cause is the growth of cancer cells within a confined anatomical space, i.e., tissue plane compartment. In one tumour xenograft study, treatment with paclitaxel and docetaxel reduced interstitial fluid pressure by direct cell apoptosis [7], TIFP may also be elevated because of disordered angiogenesis and small abnormal vessels, and the hence the resistance of the network correlates with interstitial pressure. In studies of three human melanoma xenograft lines (D-12, R-18, U-25), capillary diameter distribution was determined by stereological analysis of histological sections and IFP was measured using the wick-in-needle technique [8]. An inverse linear correlation was observed between tumour IFP and mean capillary diameter were found for each of the melanoma lines (P < 0.05). IFP also decreased during tumour growth whereas mean capillary diameter increased with increasing tumour volume (P < 0.001). These data suggest that the diameter distribution and thus the geometric resistance of the capillary network can exert significant influence on the IFP of tumours [8]. The lack of functional lymphatic circulation within tumours combined with leaky tumour vessels [9] means that there is difficulty in sustaining pressure gradients across the vessel wall [10], resulting in sustained elevated microvascular pressures which also contribute to high TIFP [10, 11]. Several molecules also seem to play important role within the surrounding host tissue contributing to TIFP. These include transforming growth factor (TGF)-β-1 and -β-3 [12]. Thyroid tumour xenografts that have been exposed to a recombinant TGF-β receptor type II murine antibody had significantly lowered TIFP in a time and concentration dependent manner [12]. TGF-β-1 is thought to mediate these actions through platelet derived growth factor (PDGF) and its receptor the platelet derived growth factor receptor (PDGFR), both of which are also important in TIFP [13]. PDGF, homo or heterodimers, composed of A and B 100 amino acid disulfide bonded polypeptides, act as ligands signalling through the alpha- and beta- tyrosine kinase receptors (i.e., PDGFR-α and PDGFR-β). Only PDGF BB can signal through PDGFR-β [14]. The receptors are mostly found on smooth muscle and fibroblasts. They are also on pericytes, suggesting a role in angiogenesis [15]. There is overexpression of PDGF and PDGFR in

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 75 many different tumours, such as ovarian, prostate, and colorectal cancer [16-18]. PDGFR-β in these tumours is thought to be important in causing conditions favourable to survival of the tumour cell such as raised IFP [19]. Fibroblasts, smooth muscle cells and pericytes within the tumour that express the PDGFR-β are likely to be the effector cells of this change in IFP [12]. This role in tumour development and growth is different to other tumours such as Dermatofibrosarcoma Protuberans which use PDGF and PDGFR in an autocrine loop as a central stimulus to growth, and are very sensitive to the tyrosine kinase inhibitor, imatinib [20].

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Clinical Sequelae: Prognostic Significance of These Changes; Drug Activity The delivery of drugs within tumour vasculature to their targets depends on penetration through tumour microvascular walls and into the interstitial compartment [21]. The mechanisms of transport can be with diffusion, using a concentration gradient of the drugs, or convection, relying on a pressure gradient. The consequences of the high TIFP may be decreased delivery of drugs to the cancer cells [7]. Fluid movement across vessel walls changes depending on dynamics between the interstitial hydrostatic and interstitial and intravascular oncotic pressures, hence drugs will only move out of blood vessels towards their targets according to favourable pressure gradients [9]. Tumours not only have high microvascular pressures, but also may have high interstitial oncotic pressures (i.e., protein, albumin concentrations), which may vary subject to tumour size [22]. Where intratumoural oncotic pressures are not elevated, especially in larger tumours, there is less convective pressure gradient to allow for delivery of drug [23]. Drug dosing therefore is affected by the variability in local and perhaps unpredictable factors such as vascular permeability and high IFP [10]. Larger molecules such as antibodies may also be prevented from penetrating tumours given the heterogeneous blood supply as well as elevated IFP and large transport distances within the interstitium [24]. High IFP has clinical prognostic significance, and is an independent predictor of survival in patients with cervix cancer [4]. High IFP can also predict rates of metastatic disease in melanoma xenografts [25]. High IFP correlates with hypoxia within cervical cancer and may provide an explanation for resistance to not only chemotherapy but also to radiotherapy as well [26]. Interestingly, it is probable that radiotherapy does not change IFP, with one experiment in mice showing no significant change in IFP in tumour xenografts over the duration of radiotherapy [27]. Therefore in tumours with high IFP that require radiotherapy, there may be a requirement for concurrent treatment aimed at modulation and reduction of IFP to achieve optimum results. IFP can be assessed by direct or indirect methods. A wick-in needle technique can be used to directly measure IFP, but is limited to tumours that are easily accessible [5], but radiology may also be important. CT may play a role in assessing permeability of capillaries within tumours using patlak analysis, and can track changes associated with chemotherapy [28]. An advantage of using CT for this assessment is that it can be incorporated with routine CT examinations. Although CT may have less sensitivity than MRI in analysis of these

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changes, images may be simpler and less time consuming to interpret, aiding application to general day to day practice where there are obvious time constraints. A recent study looking at the blood flow patterns of primary adenocarcinomas in 37 patients showed a high correlation between the amount of tumour blood flow and the subsequent risk post surgery of developing metastases [29]. Perfusion CT can be used to analyse the changes to liver metastases in response to therapy and also offers prognostic information [30]. Further work has been done in lung cancer correlating perfusion CT parameters with tumour size and rates of distant metastases [31], suggestive also of correlation with prognosis. Although there has been success in the use of CT in assessing tumour perfusion and correlation with prognosis there are two areas of concern that limit its day to day applicability. Firstly in clinical studies, there has not been a demonstration that tumoural perfusion changes induced by angiogenic agents can be measured by CT. Therefore there has not as yet been validation in clinical trials of this technique to show that it is not only prognostic, but also predictive of response to such agents. Secondly, to attempt to track clinical changes in perfusion, there are multiple imaging assessments of the tumour required, increasing exposure to radiation [32]. Finally tumour perfusion as assessed by CT scan itself only provides a very indirect measure of TIFP and has not been directly validated. MRI has an evolving role for functional tumour imaging. Dynamic contrast-enhanced MRI (DCE-MRI) can provide details of tumour perfusion that correlate with prognosis, and monitor changes that may occur with therapy [32]. The basic principle of DCE-MRI is obtaining sequential images before, during and following administration of contrast. Pharmacokinetic compartmental methodology has been utilised to derive several functional mathematical parameters from the imaging data. The volume transfer constant (Ktrans), which is the transfer rate constant between the blood plasma and the extravascular extracellular space per unit volume of tissue, is one of the more important parameters [33]. Although earlier studies by Lyng et al. [34] suggested that MRI was not useful in assessing IFP within melanoma xenografts, more recently Ktrans analysis using DCE-MRI has been shown to provide in vivo analysis of tumour permeability and vascularity [35]. In a study performed of cervical tumours in 32 patients prior to therapy: there was a significant negative correlation between Ktrans and IFP (r = -0.47, P = 0.008) [35]. Many other studies have demonstrated the utility of DCE-MRI in predicting or monitoring response to therapy [36, 37]. There are also studies correlating DCE-MRI measurements with prognostic factors in breast cancer [38], as well as other tumours [39]. DCE-MRI is hampered in clinical utilisation currently by differing imaging protocols which lack standardization across centres, as well as the long times taken to analyse images [33].

Therapeutic Agents Being Evaluated in the Clinic: Highlights and Promising Results Recent studies support the notion that lowering IFP and improving the transcapillary pressure gradient increases the uptake of tumour targeting antibodies, as well as low molecular weight compounds [40]. The most promising therapeutic target for changing the microenvironment in regard to the reduction of IFP is PDGFR inhibition. There are other

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The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 77 targets such as TGF-β1, -β3 [12], and prostaglandin E1 [41], but these have been studied in preclinical models and not yet tested in humans. Other drugs such as hydralazine injected directly into tumour xenografts will also change IFP [42], but the method of action and its clinical utility are uncertain. Although hydralazine is a vasodilator and may modulate IFP in this manner, there are other actions of hydralazine such as increasing Hypoxia Inducible Factor 1 alpha (HIF-1α) and Vascular Endothelial Growth Factor levels in endothelial and smooth muscle cells that may also affect change. Furthermore, hydralazine has DNA demethylating activity and possesses the ability to reactivate tumour suppressor gene expression, which is silenced by promoter hypermethylation in vitro and in vivo [43]. This latter indication is the major reason why hydralazine has been investigated in oral dosing in early phase clinical trials [44,45], but there has been little enthusiasm for the investigation of direct intratumoural injection in vivo to assess its ability to change IFP. Imatinib is a small molecule tyrosine kinase inhibitor of PDGFR-β, and lowers IFP as a consequence of that inhibition [13,46]. Preclinical work in prostate cancer [47], colorectal cancer [48] and ovarian cancer [49]has been encouraging in demonstrating that imatinib inhibits the growth of ovarian cancer cells in a PDGFR-specific manner, at clinically relevant concentrations which has led to the use of the drug in clinical trials. However, multiple clinical trials of single agent imatinib in breast [50], small cell lung cancer [51], pancreatic cancer [52] and ovarian cancer [53,54] have all failed to show significant activity. It thus appears that targeting microenvironment alone lacks therapeutic effect and hence it therefore may have a role in potentially enhance cytotoxic drug penetration and hence activity. Studies have been performed in mesothelioma xenografts, showing synergy of imatinib with gemcitabine [55]. Imatinib was combined with docetaxel in heavily pretreated ovarian cancer platinum-resistant patients that overexpressed c-kit or PDGFR-α. The combination resulted in 21.7% overall radiological response rates, with fatigue, nausea, diarrhoea and edema the most common side effects [56]. Imatinib has also been combined with fluorouracil and leucovorin in patients with gastric, colon and pancreatic cancer patients in a phase 1 trial, with dose-limiting side effects being mostly nausea and fluid accumulation: response rates were not reported [57]. Imatinib, at a dose of 400mg, was also combined with three-weekly docetaxel and estramustine in the treatment of androgen independent prostate cancer, but there was a high incidence of thrombosis [58]. A more promising and less toxic regimen combined imatinib, 600mg daily, with weekly docetaxel in the treatment of patients with androgen independent prostate cancer, with a 67% response rate versus 7% when imatinib was used alone [59]. This supports the notion that targeting microenvironment can potentially enhance cytotoxic drug penetration and hence activity. Another agent is CDP860, a humanised di-Fab which can block the activity of PDGFRβ [60]. CDP860 is an engineered Fab' fragment-polyethylene glycol conjugate, which binds to and blocks the activity of PDGFRβ. In a phase I trial, patients with advanced ovarian or colorectal cancer received intravenous infusions of CDP860 on days 0 and 28. Patients had serial DCE MRI studies to measure changes in tumour vascular parameters. Three of eight patients developed significant ascites, and seven of eight showed evidence of fluid retention. In some patients, the ratio of vascular volume to total tumour volume increased significantly (P < .001) within 24 hours following CDP860 administration, an effect suggestive of

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recruitment of previously non-functioning vessels. PDGFRβ blockade did not lead to any change in vascular permeability as measured by Ktrans, so changes in vascular permeability may not be the reason for fluid accumulation [60]. As alluded to above, PDGRB blockade alone is unlikely to be therapeutic without the use of cytotoxic chemotherapy, and may alone cause side effects such as fluid accumulation (ascites, etc.) [60].

Hypoxia Hypoxia: Definition and Its Causes Hypoxia, an inadequate supply of oxygen to cells, is a hallmark of solid tumours [61]. It is a direct result of abnormal tumour vasculature, both structurally and functionally, the increased diffusion distance from the tumour cells to capillaries and their dysregulated growth [62]. Intratumoural hypoxia is also exacerbated by anaemia, either as a paraneoplastic phenomenon or induced by treatment [63]. Individual tumour cells show variable sensitivity to hypoxia based on the distance from their blood supply and also their variable metabolic consumption of oxygen [62].

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The Tumour Cells’ Response to Hypoxia Cells take on different survival mechanisms to cope with the hypoxic surrounds [64]. Cancer cells become more reliant on glycolysis than the more efficient oxidative pathways, as a consequence of inadequate nutrients, and hence produce more carbon dioxide and carbonic acid [65]. This Warburg effect [66] takes place with low oxygen and high glucose requirements, and becomes the predominant pathway for ATP generation. In hypoxic regions within tumours, neoplastic cells generate lactic acid by glycolysis, with the subsequent hydrolysis of ATP: this then leads to a decreased pH within the tumour [67]. The acidic environment can lead to treatment resistance to some cytotoxics and selective growth advantage [68]. Hypoxia also induces or up-regulates hypoxia responsive genes, with the most important being HIF-1. This is composed of two protein subunits, constitutively expressed HIF-1β, and the active subunit HIF-1α [69]. Stimulation of the Phosphoinositide 3-kinase-protein kinase B (Akt) – mammalian target of rapamycin (mTor) pathway by Human Epidermal Growth Factor Receptor 2 (HER2), Insulin-like Growth Factor (IGF), Epidermal Growth Factor (EGF) and Steroid Receptor Coactivator-1 (SRC-1) receptors in malignant cells leads to increased translation of HIF-1α microRNA into protein [70]. This is usually undetectable in normoxic tissue due to the proteolytic destruction of HIF1α by the ubiquitin-proteasome system [71]. The von Hippel-Lindau (VHL) tumour suppressor gene product plays a critical role in regulating this process [72] In the presence of oxygen, there is hydroxylation of a portion of HIF-1α, and this is then bound to the VHL protein, which transports the molecule to the ubiquitin system for proteasomal destruction [73]. In hypoxia, HIF-1α is not broken

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 79 down and binds to the constitutively expressed other protein subunit HIF-1β to form the active HIF complex. The HIF-1α subunit is the active unit, that after dimerizing with beta subunits and translocating to the nucleus, leads to subsequent down stream activation of subsequent genes [74]. The overwhelming majority of genes upregulated by hypoxia are through the HIF-1 pathway [75]. These genes include angiogenic factors SUCH AS VEGF and thymidine phosphorylase (TP), survival and growth factors such as PDGF-β, TGF-β, IGF, glucose transporter 1 and 3 (GLUT1 and GLUT3) and glycolytic enzymes aldolases A and C, enolase 1 [76]. This leads to tumour cells evolving invasive characteristics and metastatic potential including increased angiogenesis as well as greater capacity for glycolytic metabolism. The tumour suppressor gene p53 is one of the most common genes mutated in cancer and confers a survival advantage to cells. Although some preclinical work [77,78] suggests that p53 expression is augmented by HIF, further studies analysing the correlation of HIF and p53 in vitro have showed no such correlation between the two in tumour specimens [79]. However, the combination of mutated p53 may lead to a lack of stress response within tumour cells and a failure of apoptosis that would otherwise normally occur in the presence of hypoxia. This may therefore leads to a clonal selection of cells capable of tolerating hypoxia due to HIF expression, becoming a potent stimulus for tumour survival, growth and metastases.

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The Evaluation of Hypoxia and Its Prognostic Importance Thus, as described above, in tumour cells there is the potent expression of a multitude of pro-survival elements via hypoxic response elements within certain genes. It is therefore not surprising that hypoxia, either measured via HIF expression, or via direct measurement of tumour pH or oxygenation, or by indirect imaging techniques, has been shown to correlate with adverse clinical outcomes in a number of cancers [63]. Nordsmark et al. used oxygen electrodes to measure tumour partial oxygen pressures (p02) in a large group of 397 patients with radically treated head and neck cancer and demonstrated a significant association with poor prognosis in this group [80]. In this study, pO2 values ≤ 2.5 mmHg in a multivariate Cox Proportional Hazards analysis was by far the most statistically significant factor in explaining the variability in survival. This prognostic model shows that the 5-year survival is in the range from 0 to 20% for pO2 values ≤ 2.5 mmHg, whereas the 5-year survival approaches 0% in the most hypoxic tumours [80]. A similar technique using microelectrodes to assess hypoxia in prostate cancer patients demonstrated similar poor prognosis amongst those with tumour hypoxia [81]. HIF expression as determined by immunohistochemistry correlated with a poor prognosis in many cancers, such as soft tissue sarcomas [82], head and neck cancer [83], esophageal cancer [79], gastric cancer [84,85], pancreatic cancer [86], cervical cancer [87,88] and bladder cancer [89]. Other immunohistochemical surrogate markers that have been used to assess tissue hypoxia and shown to correlate with prognosis have included Lactate dehydrogenase 5 in non small cell lung cancer [90] and carbonic anhydrase 9 in non small

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cell lung cancer [91] and cervical cancer [92]. The advantage of the use of these methods of determining hypoxia are the relative ease in which these additional immunohistochemical tests can be added to standard pathology performed on tumour samples, and this may lead to their uptake in clinical practice before the invasive methods detailed below. In contrast to invasive or tissue-based measures of hypoxia requiring microelectrodes or biopsies, in vivo analysis of hypoxia using functional imaging has many obvious attractions, and which can be repeated during treatment [93]. Initial work performed in head and neck cancer patients using 18F-misonidazole positron emission tomography (FMISO PET) to assess hypoxia [94], and validated by Gagel et al. [95,96], demonstrated a moderate correlation of needle electrode assessed hypoxia with indirectly measured hypoxia using FMISO-PET. Use of the imaging depends on skilled interpretation given there is heterogeneity of hypoxic assessment within the tumour primary [97]. Hypoxia on FMISO-PET in tumours and also been shown to be not only useful in assessing prognosis in head and neck cancer patients [98] but also may in future affect therapy choices for patients [99]. In 45 patients with head and neck cancer, 32 patients (71%) had detectable hypoxia in either or both primary and nodal disease by FMISO-PET. In patients who received standard chemoradiotherapy, one of 10 patients without hypoxia had local failure compared with eight of 13 patients with hypoxia; the risk of local failure was significantly higher in hypoxic patients (exact log-rank, P = 0.038; hazard ratio [HR] = 7.1). By contrast, in patients who received the radiotherapy with tirapazamine (a hypoxic activated cytotoxic) plus cisplatin and radiotherapy, only one of 19 patients with hypoxic tumours had local failure compared to standard therapy (P = 0.001; HR = 15) [99]. A more recent alternative PET technique, using (18F) fluoroazomycin-arabinoside (FAZA), may supplant the role FMISO-PET because of less background tracer uptake and significantly improved image interpretation [100]. FAZA-PET has more lately shown also to correlate to hypoxia in head and neck cancer patients [101] and this may make it a preferable technique in head and neck cancer patients [93]. Availability limits the use of FMISO and FAZA-PET currently to research protocols, and requires commercialisation for use in the clinical setting. Another promising imaging modality for hypoxia in-situ is with dynamic MRI using the fluorinated 2-nitroimidazole hypoxia probe (SR 4554) [102]. The use of dynamic MRI has been validated in vivo models showing correlation with tissue level hypoxia [103]. Use of such imaging in vivo may allow changes during therapy to redistribute radiotherapy doses to encompass areas of heterogeneous hypoxia [104]. Given the potential prognostic and predictive utility of these imaging modalities, once validated, the greatest challenge will be their incorporation within trial protocols to serve as additional tools to modify therapy and predict response to newer agents.

Hypoxia and Resistance to Therapy The consequences of hypoxia with subsequent cellular and stromal changes are reduced sensitivity to radiotherapy, as well as chemotherapy. Many years have passed since it was first demonstrated in 1953 that tumour cells are less sensitive to radiotherapy when there is

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 81 hypoxia [105]. Subsequent investigations have demonstrated chemotherapy is less cytotoxic in hypoxic conditions [106], through several mechanisms. Transient hypoxia causes an increase in the frequency of dihydrofolate reductase gene amplification and subsequent reduced response to folate antagonists [107]. Hypoxic conditions also enhance expression of P-glycoprotein expression and subsequent multidrug resistance [108]. Other drugs such as bleomycin, procarbazine and vincristine are all dependent on maximum cellular oxygenation for activity [109]. The changes to pH within the tumour also affects the cytotoxicity of many chemotherapeutic agents, either by decreased cellular uptake of weakly basic drugs such as doxorubicin [110], or by decreased active transport of drugs, such as methotrexate [111]. Hypoxia induced acidity may also cause resistance to mitoxantrone due to pH dependent topoisomerase type II activity [112]. Hypoxia may also explain difference between in vivo and in vitro sensitivity to chemotherapeutics, given in vitro cells are often grown in a monolayer with ample supply of oxygen, relative to the three dimensional tumour in vivo with a heterogeneous oxygen supply. Depriving pancreatic cancer cells of oxygen in vivo can induce resistance of the cells to gemcitabine compared to its activity under optimum oxygenation [113]. Data suggestive of the role of HIF-1 in chemoresistance has also been reported by Song in 2006, showing that silencing of the HIF-1 gene using viral vector mediated RNA interference can reverse the resistance of non small cell lung cancer cells to cisplatin and doxorubicin [114].

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Therapies Directed to Hypoxia Not surprisingly given the poor prognosis of tumours growing under hypoxic conditions there has been considerable attention to developing cytotoxic drugs to overcome and even take advantage of the hypoxic environment. Although there are many agents in the preclinical environment that have been investigated, from the relatively simple: glyceryl trinitrate (GTN) reversing resistance to doxorubicin in prostate cancer cells [115], to the relatively complex such as gene therapy targeted to hypoxic cells [116], there are relatively few agents that have proved useful in human studies. The latter include hypoxic cell cytotoxics and hypoxic cell sensitizers. Tirapazamine, alluded to above, is an example of a hypoxic cell cytotoxic that is active only under limited oxygen concentrations. It is a benzotriazine compound that is activated by intracellular reductases to a cytotoxic radical. In the presence of oxygen there is back oxidation of the radical to the non-toxic compound [117]. Tirapazamine was originally trialled in non small cell lung cancer (NSCLC) patients. In the phase III CATAPULT-1 study, the addition of tirapazamine to cisplatin in patients with advanced NSCLC had a significantly better overall survival compared to patients treated with cisplatin alone (34.6 weeks vs 27.7 weeks; P = 0.0078), and higher response rates (27.5% vs 13.7%, P < 0.001) [118]. Although smaller phase II trials suggested the addition of tirapazamine to platinum doublets was feasible [119.120], a subsequent South Western Oncology Group phase III study showed no additional benefit in overall survival when combined with carboplatin and paclitaxel in this patient group [121]. An equivalent phase III study, the National Cancer Institute i3T trial, randomised patients with NSCLC to receive tirapazamine with cisplatin

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and vinorelbine versus cisplatin and vinorelbine alone but although completed has still yet to be published. There have been studies of tirapazamine with chemoradiotherapy in head and neck cancer, but again the overall survival in the largest trial of 861 patients between the two treatment arms was equivalent, as was the relapse free survival [122]. However a previous analysis, described above, had shown in a 45 patient subgroup that the benefit of tirapazamine was mostly confined to those 32 patients with patients with hypoxia on FMISO PET [99]. One patient out of 19 with hypoxia who received tirapazamine with chemoradiotherapy had loco-regional failure, as compared to 8 out of 13 in those that received standard chemoradiotherapy. The results highlight the importance of using predictive biomarkers and/or imaging to assess for the presence of hypoxia when using hypoxia-directed cytotoxics. Further clinical trials should include these requirements to better enrich their target population. Other drugs in active development have included PR-104, which is in vivo converted to PR-104A then reduced selectively in hypoxic cells to a DNA cross-linking nitrogen mustard alkylating agent [123]. A phase 1 trial of PR-104 has been carried out with acceptable toxicities [124], and four current trials, some with FMISO-PET hypoxia imaging and biomarker analysis are currently recruiting. Other agents include a 2-nitroimidazole containing lead compound 3b (TH-302) which has up to 400 fold enhanced cytotoxicity in vitro in hypoxic conditions versus aerobic conditions, and is now currently the focus of a phase 1 trial. HIF-1 alpha is also a potential target that is obviously attractive, but there have not yet been great advances in the pharmacological inhibition of this agent. So far therapeutic agents have been developed towards molecules such as VEGF and PDGF-beta that are induced by hypoxia and subsequent act as master regulators of HIF-1 alpha expression. These “low hanging” fruit on the tree have shown considerable promise, but perhaps HIF-1 can be modulated by some therapeutic agents. There may be difficulties targeting HIF-1 because the drugs themselves would need to penetrate the nucleus and interact with a HIF-1, which does not have obvious sites for inhibition. However there are several small molecules and indirect mechanisms that are potential agents to achieve this. A small molecule inhibitor of interest is Geldanamycin, which is an inhibitor of Heat shock protein 90 (HSP-90), which is involved in the folding of HIF-1. Inhibition of the HSP90 in vivo in common cancer cell lines leads to degradation of HIF-1 [125], and the molecule has been trialled in a phase 1 trial [126]. The PI3K/AKT/mTor can lead to elevated levels of HIF-1 alpha: although the mechanism is unclear [61], this presents an opportunity to influence the expression of the transcription factor. In a prostate cancer model, HIF-1 activity was reversed with mTor inhibition [127]. However mTor inhibitors have many other cellular effects, so the efficacy in cancer treatment cannot be ascribed alone to HIF-1 modulation. Similarly topotecan, a topoisomerase 1 inhibitor that produces single strand breaks in DNA, has additional properties that lead to lowered levels of HIF-1 [128], but again this is probably much less important to its DNA damaging abilities. Another drug that has been demonstrated to inhibit the transcriptional activity of HIF-1 alpha is Bortezomib [129], but it does not seem to have single agent effectiveness in solid organ tumours. Although a phase 1 trial of bortezomib has established disease limiting

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 83 toxicity when combining the agent with oxaliplatin and 5FU [74], in colorectal cancer, a phase II trial has shown a lack of single agent activity in patients with metastatic colorectal cancer [130]. Furthermore the treatment of colorectal cancer with irinotecan and bortezomib also failed to show significant additional activity compared to irinotecan alone [131]. This suggests that cytotoxic therapy in the clinical setting can not provide synergistic or even additive benefit to Bortezomib despites its potential anti-hypoxic effect. Hypoxia is a common occurrence and adverse factor for tumours. The mechanisms that lead to hypoxia and its consequences are emerging. Studies so far point to the utility of imaging and biomarkers to select patients that may best benefit from drugs that utilise tumour hypoxia to their advantage. These drugs share similarities to other agents that may modulate microenvironmental factors, such as imatinib, as they lack single agent activity and require other cytotoxic agents to have maximum potential. The challenge is to combine the different classes of agents safely, and to design studies in such a way to best enrich the treatment population.

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Drug Penetration Tumour cells grow in a three dimensional manner within a complex structure governed by tissue planes and surrounding stromal support. A disorganised vascular network, a reduced density, in addition to areas of hypoxia, tumoural necrosis, pH gradients and often extracellular mucin, all ensure that tumour cells are mostly distant to the blood vessels [132]. Drugs need to travel from blood vessels through an extracellular matrix to reach the tumour cells. The diffusion and penetration of drugs is determined by their physiochemical properties (for example molecular weight, ionisation, solubility, etc.) but also limited by the above factors, and may lead to lack of exposure of many tumour cells to adequate concentrations of cytotoxic drugs or cytostatic drugs [133]. The penetration of these drugs can be assessed using different techniques, and these have led to a better understanding of the heterogeneous and often poor distribution of drugs in vitro. Cellular in-vivo models, using multilayered cell cultures (MCLs), and multicellular spheroids are two of the better studied techniques. Whereas as drug testing in cell suspensions may show dispersed cells are affected by cytotoxics, MCLs and multicellular spheroids presumably more closely mimicking the in vitro state. These studies have demonstrated that drug efficacy is limited, especially the more central cells relative to the more peripheral cells consistent with limited penetration of the drug [62]. Further experiments suggestive of the importance of solid tumour environment were conducted with cancer cells in multicellular layers grown on collagen coated Teflon membranes [134]. The penetration of drugs through the multicellular layers was evaluated and shown to be much slower than through the Teflon membrane alone [134]. Similar work with the multicellular layers had shown limited diffusion of doxorubicin, mitoxantrone, and to a lesser extent methotrexate and 5FU through these layers [135]. Taxanes have also been shown to have dramatically different cellular penetration through multilayered cell cultures, with little drug reaching more than 100μm into the tissue [136].

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More sophisticated cellular models, using multicellular spheroids have been conducted showing the limited penetration of doxorubicin in these models [137]. Alkylating agents and cisplatin may have demonstrated cytotoxicity to disaggregated cells but lack this ability with multidimensional tumours in other animals or spheroids [138]. The response of cells in spheroids may be different not only because of limited drug penetration, but also because of hypoxia and the cell contact effect [133]. Cells in contact with each other influence the expression of some genes, and may also contribute to changes in response to therapeutic agents by proposed mechanisms such as gap junctional ‘reciprocity’, cell shape mediated changes in (repair-related) gene expression, and alterations in chromatin packaging which influence DNA repair [139]. Although there is little data at present on the ability of newer agents of targeted monoclonal antibodies to penetrate tumour tissues in vitro, there are studies showing limited penetration of spheroids by a monoclonal antibody to CEA [140], and little distribution of the same antibody from blood vessels in two-dimensional chambers [141]. A method for quantifying the depth of penetration of anticancer agents within tissue is immunofluorence, which can provide information on the distances drugs travel from blood vessels into tumours. Doxorubicin is the best studied drug in this regard given its autofluorescence in the UV wavelength. With doxorubicin, work suggests that the there are many tumour cells not exposed to detectable concentrations of the drugs [142-143]. In the latter study, 10 patients with locally advanced breast cancer were given intravenous doxorubicin and an incision biopsy of the tumour was subsequently taken. The specimens were examined using computerized laser scanning microscopy to detect the doxorubicin autofluorescence and the microvessels were immunostained in the same sections. Overlays of both pictures revealed doxorubicin gradients existed in the tumour islets with high concentrations in the periphery and low concentrations in the centre of the tumour islets. Gradients were most pronounced shortly after the injection, but were still be detected 24 hours later. No gradients were observed in connective tissue [143]. Hence the limited drug penetration through multicellular tumoural masses may lead to failure of eradication of all tumour cells, and subsequent tumour progression The stromal network and its interaction with tumour cells may be another reason why there may be limited penetration of drug to its target. Tumours with a densely organised collagen network have lower penetration by high-molecular weight agents such as IgG antibodies than those with poorly organised collagen networks. Collagenase treatment of the more penetration-resistant tumours significantly increased the IgG interstitial diffusion rate. It appears that collagen by binding and stabilizing the glycosaminoglycan component of the extra-cellular matrix may hence retard the penetration and transport of macormolcules [144]. Thus, the extracellular matrix together with collagen and the interaction of tumoural cells with these structures may act as a sieve to reduce penetration. This has been demonstrated by reversal of these cell adhesion mediated forms of drug resistance by treating tissue with hyaluronidase, which increases penetration of drugs through multicellular spheroids [145,146]. Cell lines lacking the cellular adhesion molecule alpha-E catenin have better penetration of cytotoxic drugs, also supporting the importance of the extracellular matrix [147]. A possible problem translating this to the direct application in humans is the theoretical risk of increasing metastatic spread of the tumour62. Consistent with this concern

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 85 is the use of E-cadherin expression, a molecule involved in cell to cell adhesion as a prognostic marker. When E-cadherin levels are low, outcomes are worse in many different tumour types [148-150], suggestive of the powerful role cell to cell adhesion has on outcome in tumour growth and metastatic potential.

Summary The treatment of cancer is undergoing dramatic change as the biology is better understood. The stromal supports for tumours and the physical tumour microenvironment provide both challenges and opportunities for ongoing improvement in tumour-related outcomes. Although some of these mechanisms have been recognised for many years as adverse factors and possible targets, only small increments have so far been made that have translated through to mainstream clinical practice in this area. The challenges regarding drug resistance secondary to the tumour microenvironment will continue to be identification of suitable targets whose lack of target toxicity troubles many targeted agents, while identifying the best combination of cytotoxic medication to take with the newer agents. Over the next few years this may be achieved, allowing for concurrent treatment not only of the tumour, but also the permissive microenviroment that surrounds it.

References [1]

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[2] [3] [4]

[5]

[6] [7]

[8] [9]

De Wever O, Mareel M: Role of tissue stroma in cancer cell invasion. J. Pathol. 200:429-47, 2003. Fidler IJ: The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat. Rev. Cancer 3:453-8, 2003. Less JR, Posner MC, Boucher Y, et al: Interstitial hypertension in human breast and colorectal tumours. Cancer Res. 52:6371-4, 1992. Milosevic M, Fyles A, Hedley D, et al: Interstitial fluid pressure predicts survival in patients with cervix cancer independent of clinical prognostic factors and tumour oxygen measurements. Cancer Res. 61:6400-5, 2001. Milosevic MF, Fyles AW, Wong R, et al: Interstitial fluid pressure in cervical carcinoma: within tumour heterogeneity, and relation to oxygen tension. Cancer 82:2418-26, 1998. Tufto I, Rofstad EK: Interstitial fluid pressure, fraction of necrotic tumour tissue, and tumour cell density in human melanoma xenografts. Acta. Oncol. 37:291-7, 1998. Griffon-Etienne G, Boucher Y, Brekken C, et al: Taxane-induced apoptosis decompresses blood vessels and lowers interstitial fluid pressure in solid tumours: clinical implications. Cancer Res. 59:3776-82, 1999. Tufto I, Rofstad EK: Interstitial fluid pressure and capillary diameter distribution in human melanoma xenografts. Microvasc. Res. 58:205-14, 1999. Jain RK: Transport of molecules in the tumour interstitium: a review. Cancer Res. 47:3039-51, 1987.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

86

A. Weickhardt and M. Michael

[10] Jain RK, Tong RT, Munn LL: Effect of vascular normalization by antiangiogenic therapy on interstitial hypertension, peritumour edema, and lymphatic metastasis: insights from a mathematical model. Cancer Res. 67:2729-35, 2007. [11] Boucher Y, Jain RK: Microvascular pressure is the principal driving force for interstitial hypertension in solid tumours: implications for vascular collapse. Cancer Res. 52:5110-4, 1992. [12] Lammerts E, Roswall P, Sundberg C, et al: Interference with TGF-beta1 and -beta3 in tumour stroma lowers tumour interstitial fluid pressure independently of growth in experimental carcinoma. Int. J. Cancer 102:453-62, 2002. [13] Pietras K, Ostman A, Sjoquist M, et al: Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumours. Cancer Res. 61:2929-34, 2001. [14] Heldin CH, Westermark B: Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev. 79:1283-316, 1999. [15] Bergers G, Song S, Meyer-Morse N, et al: Benefits of targeting both pericytes and endothelial cells in the tumour vasculature with kinase inhibitors. J. Clin. Invest 111:1287-95, 2003. [16] Dabrow MB, Francesco MR, McBrearty FX, et al: The effects of platelet-derived growth factor and receptor on normal and neoplastic human ovarian surface epithelium. Gynecol Oncol 71:29-37, 1998. [17] Craven RJ, Xu LH, Weiner TM, et al: Receptor tyrosine kinases expressed in metastatic colon cancer. Int. J. Cancer 60:791-7, 1995. [18] Lindmark G, Sundberg C, Glimelius B, et al: Stromal expression of platelet-derived growth factor beta-receptor and platelet-derived growth factor B-chain in colorectal cancer. Lab. Invest. 69:682-9, 1993. [19] Kimura Y, Inoue K, Abe M, et al: PDGFRbeta and HIF-1alpha inhibition with imatinib and radioimmunotherapy of experimental prostate cancer. Cancer Biol. Ther. 6:176372, 2007. [20] McArthur GA: Dermatofibrosarcoma protuberans: a surgical disease with a molecular savior. Curr Opin Oncol 18:341-6, 2006. [21] Wijeratne NS, Hoo KA: Understanding the role of the tumour vasculature in the transport of drugs to solid cancer tumours. Cell Prolif. 40:283-301, 2007. [22] Stohrer M, Boucher Y, Stangassinger M, et al: Oncotic pressure in solid tumours is elevated. Cancer Res. 60:4251-5, 2000. [23] Jain RK: The next frontier of molecular medicine: delivery of therapeutics. Nat. Med. 4:655-7, 1998. [24] Jain RK: Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumours. Cancer Res. 50:814s-819s, 1990. [25] Rofstad EK, Tunheim SH, Mathiesen B, et al: Pulmonary and lymph node metastasis is associated with primary tumour interstitial fluid pressure in human melanoma xenografts. Cancer Res. 62:661-4, 2002. [26] Roh HD, Boucher Y, Kalnicki S, et al: Interstitial hypertension in carcinoma of uterine cervix in patients: possible correlation with tumour oxygenation and radiation response. Cancer Res. 51:6695-8, 1991.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 87 [27] Baumann M, Appold S, Zimmer J, et al: Radiobiological hypoxia, oxygen tension, interstitial fluid pressure and relative viable tumour area in two human squamous cell carcinomas in nude mice during fractionated radiotherapy. Acta. Oncol. 40:519-28, 2001. [28] Harvey C, Dooher A, Morgan J, et al: Imaging of tumour therapy responses by dynamic CT. Eur. J. Radiol. 30:221-6, 1999. [29] Goh V, Halligan S, Wellsted DM, et al: Can perfusion CT assessment of primary colorectal adenocarcinoma blood flow at staging predict for subsequent metastatic disease? A pilot study. Eur. Radiol., 2008. [30] Miles KA, Leggett DA, Kelley BB, et al: In vivo assessment of neovascularization of liver metastases using perfusion CT. Br. J. Radiol. 71:276-81, 1998. [31] Li Y, Yang ZG, Chen TW, et al: Whole tumour perfusion of peripheral lung carcinoma: evaluation with first-pass CT perfusion imaging at 64-detector row CT. Clin. Radiol. 63:629-35, 2008. [32] Atri M: New technologies and directed agents for applications of cancer imaging. J. Clin. Oncol 24:3299-308, 2006. [33] Hylton N: Dynamic contrast-enhanced magnetic resonance imaging as an imaging biomarker. J. Clin. Oncol 24:3293-8, 2006. [34] Lyng H, Tufto I, Skretting A, et al: Proton relaxation times and interstitial fluid pressure in human melanoma xenografts. Br. J. Cancer 75:180-3, 1997. [35] Haider MA, Sitartchouk I, Roberts TP, et al: Correlations between dynamic contrastenhanced magnetic resonance imaging-derived measures of tumour microvasculature and interstitial fluid pressure in patients with cervical cancer. J. Magn Reson Imaging 25:153-9, 2007. [36] Wedam SB, Low JA, Yang SX, et al: Antiangiogenic and antitumour effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J. Clin. Oncol 24:769-77, 2006. [37] Liu G, Rugo HS, Wilding G, et al: Dynamic contrast-enhanced magnetic resonance imaging as a pharmacodynamic measure of response after acute dosing of AG-013736, an oral angiogenesis inhibitor, in patients with advanced solid tumours: results from a phase I study. J. Clin. Oncol 23:5464-73, 2005. [38] Nagashima T, Suzuki M, Yagata H, et al: Dynamic-enhanced MRI predicts metastatic potential of invasive ductal breast cancer. Breast Cancer 9:226-30, 2002. [39] Lu JP, Wang J, Wang T, et al: Microvessel density of malignant and benign hepatic lesions and MRI evaluation. World J. Gastroenterol. 10:1730-4, 2004. [40] Pietras K, Rubin K, Sjoblom T, et al: Inhibition of PDGF receptor signaling in tumour stroma enhances antitumour effect of chemotherapy. Cancer Res. 62:5476-84, 2002. [41] Rubin K, Sjoquist M, Gustafsson AM, et al: Lowering of tumoural interstitial fluid pressure by prostaglandin E(1) is paralleled by an increased uptake of (51)Cr-EDTA. Int. J. Cancer 86:636-43, 2000. [42] Podobnik B, Sersa G, Miklavcic D: Effect of hydralazine on interstitial fluid pressure in experimental tumours and in normal tissue. In Vivo 15:417-24, 2001. [43] Arce C, Segura-Pacheco B, Perez-Cardenas E, et al: Hydralazine target: from blood vessels to the epigenome. J. Transl. Med. 4:10, 2006.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

88

A. Weickhardt and M. Michael

[44] Candelaria M, Gallardo-Rincon D, Arce C, et al: A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumours. Ann. Oncol. 18:1529-38, 2007. [45] Zambrano P, Segura-Pacheco B, Perez-Cardenas E, et al: A phase I study of hydralazine to demethylate and reactivate the expression of tumour suppressor genes. BMC Cancer 5:44, 2005. [46] Grinnell F, Ho CH, Lin YC, et al: Differences in the regulation of fibroblast contraction of floating versus stressed collagen matrices. J. Biol. Chem. 274:918-23, 1999. [47] Buchdunger E, Cioffi CL, Law N, et al: Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295:139-45, 2000. [48] Stahtea XN, Roussidis AE, Kanakis I, et al: Imatinib inhibits colorectal cancer cell growth and suppresses stromal-induced growth stimulation, MT1-MMP expression and pro-MMP2 activation. Int. J. Cancer 121:2808-14, 2007. [49] Matei D, Chang DD, Jeng MH: Imatinib mesylate (Gleevec) inhibits ovarian cancer cell growth through a mechanism dependent on platelet-derived growth factor receptor alpha and Akt inactivation. Clin. Cancer Res. 10:681-90, 2004. [50] Modi S, Seidman AD, Dickler M, et al: A phase II trial of imatinib mesylate monotherapy in patients with metastatic breast cancer. Breast Cancer Res. Treat 90:157-63, 2005. [51] Soria JC, Johnson BE, Chevalier TL: Imatinib in small cell lung cancer. Lung Cancer 41 Suppl 1:S49-53, 2003. [52] Gharibo M, Patrick-Miller L, Zheng L, et al: A phase II trial of imatinib mesylate in patients with metastatic pancreatic cancer. Pancreas 36:341-5, 2008. [53] lberts DS, Liu PY, Wilczynski SP, et al: Phase II trial of imatinib mesylate in recurrent, biomarker positive, ovarian cancer (Southwest Oncology Group Protocol S0211). Int. J. Gynecol. Cancer 17:784-8, 2007. [54] Schilder RJ, Sill MW, Lee RB, et al: Phase II evaluation of imatinib mesylate in the treatment of recurrent or persistent epithelial ovarian or primary peritoneal carcinoma: a Gynecologic Oncology Group Study. J. Clin. Oncol 26:3418-25, 2008. [55] Bertino P, Piccardi F, Porta C, et al: Imatinib mesylate enhances therapeutic effects of gemcitabine in human malignant mesothelioma xenografts. Clin. Cancer Res. 14:541-8, 2008. [56] Matei D, Emerson RE, Schilder J, et al: Imatinib mesylate in combination with docetaxel for the treatment of patients with advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis: a Hoosier Oncology Group trial. Cancer 113:723-32, 2008. [57] Al-Batran SE, Atmaca A, Schleyer E, et al: Imatinib mesylate for targeting the plateletderived growth factor beta receptor in combination with fluorouracil and leucovorin in patients with refractory pancreatic, bile duct, colorectal, or gastric cancer—a doseescalation Phase I trial. Cancer 109:1897-904, 2007. [58] Lin AM, Rini BI, Derynck MK, et al: A phase I trial of docetaxel/estramustine/imatinib in patients with hormone-refractory prostate cancer. Clin. Genitourin Cancer 5:323-8, 2007.

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The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 89 [59] Mathew P, Thall PF, Jones D, et al: Platelet-derived growth factor receptor inhibitor imatinib mesylate and docetaxel: a modular phase I trial in androgen-independent prostate cancer. J. Clin. Oncol 22:3323-9, 2004. [60] Jayson GC, Parker GJ, Mullamitha S, et al: Blockade of platelet-derived growth factor receptor-beta by CDP860, a humanized, PEGylated di-Fab', leads to fluid accumulation and is associated with increased tumour vascularized volume. J. Clin. Oncol 23:973-81, 2005. [61] Shannon AM, Bouchier-Hayes DJ, Condron CM, et al: Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies. Cancer Treat. Rev. 29:297307, 2003. [62] Tredan O, Galmarini CM, Patel K, et al: Drug resistance and the solid tumour microenvironment. J. Natl. Cancer Inst. 99:1441-54, 2007. [63] Vaupel P, Thews O, Hoeckel M: Treatment resistance of solid tumours: role of hypoxia and anemia. Med. Oncol. 18:243-59, 2001. [64] O'Donnell JL, Joyce MR, Shannon AM, et al: Oncological implications of hypoxia inducible factor-1alpha (HIF-1alpha) expression. Cancer Treat. Rev. 32:407-16, 2006 [65] Dang CV, Semenza GL: Oncogenic alterations of metabolism. Trends Biochem. Sci. 24:68-72, 1999. [66] Warburg O: On the origin of cancer cells. Science 123:309-14, 1956. [67] Tannock IF, Rotin D: Acid pH in tumours and its potential for therapeutic exploitation. Cancer Res. 49:4373-84, 1989. [68] Schmaltz C, Hardenbergh PH, Wells A, et al: Regulation of proliferation-survival decisions during tumour cell hypoxia. Mol. Cell Biol. 18:2845-54, 1998. [69] Wang GL, Semenza GL: Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270:1230-7, 1995. [70] Semenza GL: HIF-1 and tumour progression: pathophysiology and therapeutics. Trends Mol. Med. 8:S62-7, 2002. [71] Salceda S, Caro J: Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. 272:226427, 1997. [72] Maxwell PH, Wiesener MS, Chang GW, et al: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271-5, 1999. [73] Mazure NM, Brahimi-Horn MC, Berta MA, et al: HIF-1: master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs. Biochem. Pharmacol. 68:971-80, 2004. [74] Fedele AO, Whitelaw ML, Peet DJ: Regulation of gene expression by the hypoxiainducible factors. Mol. Interv. 2:229-43, 2002. [75] Greijer AE, van der Groep P, Kemming D, et al: Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J. Pathol. 206:291-304, 2005. [76] Kunz M, Ibrahim SM: Molecular responses to hypoxia in tumour cells. Mol. Cancer 2:23, 2003.

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90

A. Weickhardt and M. Michael

[77] Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumourigenesis. Cell 86:353-64, 1996 [78] Yuan XL, Yu SY, Xia S, et al: [Effects of hypoxia on expression of HIF-1alpha,P53, and cyclin D1 in human lung adenocarcinoma cell line A549]. Ai Zheng 23:1031-5, 2004. [79] Sohda M, Ishikawa H, Masuda N, et al: Pretreatment evaluation of combined HIF1alpha, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer. Int. J. Cancer 110:838-44, 2004. [80] Nordsmark M, Bentzen SM, Rudat V, et al: Prognostic value of tumour oxygenation in 397 head and neck tumours after primary radiation therapy. An international multicenter study. Radiother Oncol. 77:18-24, 2005. [81] Movsas B, Chapman JD, Greenberg RE, et al: Increasing levels of hypoxia in prostate carcinoma correlate significantly with increasing clinical stage and patient age: an Eppendorf pO(2) study. Cancer 89:2018-24, 2000. [82] Shintani K, Matsumine A, Kusuzaki K, et al: Expression of hypoxia-inducible factor (HIF)-1alpha as a biomarker of outcome in soft-tissue sarcomas. Virchows Arch. 449:673-81, 2006. [83] Winter SC, Shah KA, Han C, et al: The relation between hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression with anemia and outcome in surgically treated head and neck cancer. Cancer 107:757-66, 2006. [84] Ma J, Zhang L, Ru GQ, et al: Upregulation of hypoxia inducible factor 1alpha mRNA is associated with elevated vascular endothelial growth factor expression and excessive angiogenesis and predicts a poor prognosis in gastric carcinoma. World J. Gastroenterol 13:1680-6, 2007. [85] Sumiyoshi Y, Kakeji Y, Egashira A, et al: Overexpression of hypoxia-inducible factor 1alpha and p53 is a marker for an unfavorable prognosis in gastric cancer. Clin. Cancer Res. 12:5112-7, 2006. [86] Sun HC, Qiu ZJ, Liu J, et al: Expression of hypoxia-inducible factor-1 alpha and associated proteins in pancreatic ductal adenocarcinoma and their impact on prognosis. Int. J. Oncol. 30:1359-67, 2007. [87] Bachtiary B, Schindl M, Potter R, et al: Overexpression of hypoxia-inducible factor 1alpha indicates diminished response to radiotherapy and unfavorable prognosis in patients receiving radical radiotherapy for cervical cancer. Clin. Cancer Res. 9:223440, 2003. [88] Birner P, Schindl M, Obermair A, et al: Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res. 60:4693-6, 2000. [89] Theodoropoulos VE, Lazaris A, Sofras F, et al: Hypoxia-inducible factor 1 alpha expression correlates with angiogenesis and unfavorable prognosis in bladder cancer. Eur. Urol. 46:200-8, 2004. [90] Koukourakis MI, Giatromanolaki A, Sivridis E, et al: Lactate dehydrogenase-5 (LDH5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis. Br. J. Cancer 89:877-85, 2003.

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The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 91 [91] Swinson DE, Jones JL, Richardson D, et al: Carbonic anhydrase IX expression, a novel surrogate marker of tumour hypoxia, is associated with a poor prognosis in non-smallcell lung cancer. J. Clin. Oncol 21:473-82, 2003. [92] Loncaster JA, Harris AL, Davidson SE, et al: Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumour oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res. 61:6394-9, 2001. [93] Rischin D, Fisher R, Peters L, et al: Hypoxia in head and neck cancer: studies with hypoxic positron emission tomography imaging and hypoxic cytotoxins. Int. J. Radiat. Oncol. Biol. Phys. 69:S61-3, 2007. [94] Koh WJ, Bergman KS, Rasey JS, et al: Evaluation of oxygenation status during fractionated radiotherapy in human nonsmall cell lung cancers using [F18]fluoromisonidazole positron emission tomography. Int. J. Radiat. Oncol. Biol. Phys. 33:391-8, 1995. [95] Gagel B, Reinartz P, Dimartino E, et al: pO(2) Polarography versus positron emission tomography ([(18)F] fluoromisonidazole, [(18)F]-2-fluoro-2'-deoxyglucose). An appraisal of radiotherapeutically relevant hypoxia. Strahlenther Onkol. 180:616-22, 2004. [96] Gagel B, Piroth M, Pinkawa M, et al: pO polarography, contrast enhanced color duplex sonography (CDS), [18F] fluoromisonidazole and [18F] fluorodeoxyglucose positron emission tomography: validated methods for the evaluation of therapy-relevant tumour oxygenation or only bricks in the puzzle of tumour hypoxia? BMC Cancer 7:113, 2007 [97] Rasey JS, Koh WJ, Evans ML, et al: Quantifying regional hypoxia in human tumours with positron emission tomography of [18F]fluoromisonidazole: a pretherapy study of 37 patients. Int. J. Radiat. Oncol. Biol. Phys. 36:417-28, 1996. [98] Rajendran JG, Schwartz DL, O'Sullivan J, et al: Tumour hypoxia imaging with [F-18] fluoromisonidazole positron emission tomography in head and neck cancer. Clin. Cancer Res. 12:5435-41, 2006. [99] Rischin D, Hicks RJ, Fisher R, et al: Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumour hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J. Clin. Oncol 24:2098-104, 2006. [100] Reischl G, Dorow DS, Cullinane C, et al: Imaging of tumour hypoxia with [124I]IAZA in comparison with [18F]FMISO and [18F]FAZA--first small animal PET results. J. Pharm. Pharm. Sci. 10:203-11, 2007. [101] Beck R, Roper B, Carlsen JM, et al: Pretreatment 18F-FAZA PET predicts success of hypoxia-directed radiochemotherapy using tirapazamine. J. Nucl. Med. 48:973-80, 2007. [102] Aboagye EO, Kelson AB, Tracy M, et al: Preclinical development and current status of the fluorinated 2-nitroimidazole hypoxia probe N-(2-hydroxy-3,3,3-trifluoropropyl)-2(2-nitro-1-imidazolyl) acetamide (SR 4554, CRC 94/17): a non-invasive diagnostic probe for the measurement of tumour hypoxia by magnetic resonance spectroscopy and

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imaging, and by positron emission tomography. Anticancer Drug Des. 13:703-30, 1998. [103] Zhao D, Ran S, Constantinescu A, et al: Tumour oxygen dynamics: correlation of in vivo MRI with histological findings. Neoplasia 5:308-18, 2003. [104] Sovik A, Malinen E, Skogmo HK, et al: Radiotherapy adapted to spatial and temporal variability in tumour hypoxia. Int. J. Radiat. Oncol. Biol. Phys. 68:1496-504, 2007. [105] Gray LH, Conger AD, Ebert M, et al: The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br. J. Radiol. 26:638-48, 1953. [106] Brown JM: The hypoxic cell: a target for selective cancer therapy--eighteenth Bruce F. Cain Memorial Award lecture. Cancer Res. 59:5863-70, 1999. [107] Rice GC, Hoy C, Schimke RT: Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. U S A 83:5978-82, 1986. [108] Ledoux S, Yang R, Friedlander G, et al: Glucose depletion enhances P-glycoprotein expression in hepatoma cells: role of endoplasmic reticulum stress response. Cancer Res. 63:7284-90, 2003. [109] Teicher BA, Lazo JS, Sartorelli AC: Classification of antineoplastic agents by their selective toxicities toward oxygenated and hypoxic tumour cells. Cancer Res. 41:7381, 1981. [110] Gerweck LE, Vijayappa S, Kozin S: Tumour pH controls the in vivo efficacy of weak acid and base chemotherapeutics. Mol. Cancer Ther. 5:1275-9, 2006. [111] Cowan DS, Tannock IF: Factors that influence the penetration of methotrexate through solid tissue. Int. J. Cancer 91:120-5, 2001. [112] Greijer AE, de Jong MC, Scheffer GL, et al: Hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells. Cell Oncol. 27:43-9, 2005. [113] Yokoi K, Fidler IJ: Hypoxia increases resistance of human pancreatic cancer cells to apoptosis induced by gemcitabine. Clin Cancer Res. 10:2299-306, 2004. [114] Song X, Liu X, Chi W, et al: Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1alpha gene. Cancer Chemother Pharmacol. 58:776-84, 2006. [115] Frederiksen LJ, Siemens DR, Heaton JP, et al: Hypoxia induced resistance to doxorubicin in prostate cancer cells is inhibited by low concentrations of glyceryl trinitrate. J. Urol. 170:1003-7, 2003. [116] Dachs GU, Coralli C, Hart SL, et al: Gene delivery to hypoxic cells in vitro. Br. J. Cancer 83:662-7, 2000. [117] Brown JM: SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours. Br. J. Cancer 67:1163-70, 1993. [118] von Pawel J, von Roemeling R, Gatzemeier U, et al: Tirapazamine plus cisplatin versus cisplatin in advanced non-small-cell lung cancer: A report of the international CATAPULT I study group. Cisplatin and Tirapazamine in Subjects with Advanced Previously Untreated Non-Small-Cell Lung Tumours. J. Clin. Oncol 18:1351-9, 2000.

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The Role of Tumoural Microenvironment and Its Vasculature in Chemotherapy… 93 [119] Gatineau M, Rixe O, Chevalier TL: Tirapazamine with cisplatin and vinorelbine in patients with advanced non-small-cell lung cancer: a phase I/II study. Clin. Lung Cancer 6:293-8, 2005. [120] Reck M, von Pawel J, Nimmermann C, et al: [Phase II-trial of tirapazamine in combination with cisplatin and gemcitabine in patients with advanced non-small-celllung-cancer (NSCLC)]. Pneumologie 58:845-9, 2004. [121] Williamson SK, Crowley JJ, Lara PN, Jr., et al: Phase III trial of paclitaxel plus carboplatin with or without tirapazamine in advanced non-small-cell lung cancer: Southwest Oncology Group Trial S0003. J. Clin. Oncol 23:9097-104, 2005. [122] Rischin D, Peters L, O'Sullivan B, et al: Phase III study of tirapazamine, cisplatin and radiation versus cisplatin and radiation for advanced squamous cell carcinoma of the head and neck. J. Clin. Oncol 26 (May 20 suppl):abstr LBA 6008, 2008. [123] Patterson AV, Ferry DM, Edmunds SJ, et al: Mechanism of action and preclinical antitumour activity of the novel hypoxia-activated DNA cross-linking agent PR-104. Clin. Cancer Res. 13:3922-32, 2007. [124] Jameson M, Rischin D, Pegram M, et al: A phase I pharmacokinetic study of PR-104, a hypoxia-activated nitrogen mustard prodrug, in patients with solid tumour. J. Clin. Oncol 26: (May 20 suppl): Abstr 2562, 2008. [125] Mabjeesh NJ, Post DE, Willard MT, et al: Geldanamycin induces degradation of hypoxia-inducible factor 1alpha protein via the proteosome pathway in prostate cancer cells. Cancer Res. 62:2478-82, 2002. [126] Nowakowski GS, McCollum AK, Ames MM, et al: A phase I trial of twice-weekly 17allylamino-demethoxy-geldanamycin in patients with advanced cancer. Clin. Cancer Res. 12:6087-93, 2006. [127] Majumder PK, Febbo PG, Bikoff R, et al: mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat. Med. 10:594-601, 2004. [128] Puppo M, Battaglia F, Ottaviano C, et al: Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1alpha and -2alpha. Mol. Cancer Ther. 7:1974-84, 2008. [129] Shin DH, Chun YS, Lee DS, et al: Bortezomib inhibits tumour adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1. Blood 111:3131-6, 2008. [130] Mackay H, Hedley D, Major P, et al: A phase II trial with pharmacodynamic endpoints of the proteasome inhibitor bortezomib in patients with metastatic colorectal cancer. Clin. Cancer Res. 11:5526-33, 2005. [131] Kozuch PS, Rocha-Lima CM, Dragovich T, et al: Bortezomib with or without irinotecan in relapsed or refractory colorectal cancer: results from a randomized phase II study. J. Clin. Oncol 26:2320-6, 2008. [132] Thomlinson RH, Gray LH: The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9:539-49, 1955. [133] Minchinton AI, Tannock IF: Drug penetration in solid tumours. Nat. Rev. Cancer 6:583-92, 2006.

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[134] Tannock IF: Tumour physiology and drug resistance. Cancer Metastasis Rev. 20:12332, 2001. [135] Tunggal JK, Cowan DS, Shaikh H, et al: Penetration of anticancer drugs through solid tissue: a factor that limits the effectiveness of chemotherapy for solid tumours. Clin. Cancer Res. 5:1583-6, 1999. [136] Kyle AH, Huxham LA, Yeoman DM, et al: Limited tissue penetration of taxanes: a mechanism for resistance in solid tumours. Clin. Cancer Res. 13:2804-10, 2007. [137] Sutherland RM, Eddy HA, Bareham B, et al: Resistance to adriamycin in multicellular spheroids. Int. J. Radiat. Oncol. Biol. Phys. 5:1225-30, 1979. [138] Teicher BA, Herman TS, Holden SA, et al: Tumour resistance to alkylating agents conferred by mechanisms operative only in vivo. Science 247:1457-61, 1990. [139] Olive PL, Durand RE: Drug and radiation resistance in spheroids: cell contact and kinetics. Cancer Metastasis Rev 13:121-38, 1994. [140] Sutherland R, Buchegger F, Schreyer M, et al: Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumour spheroids. Cancer Res. 47:1627-33, 1987. [141] Baxter LT, Zhu H, Mackensen DG, et al: Biodistribution of monoclonal antibodies: scale-up from mouse to human using a physiologically based pharmacokinetic model. Cancer Res. 55:4611-22, 1995. [142] Primeau AJ, Rendon A, Hedley D, et al: The distribution of the anticancer drug Doxorubicin in relation to blood vessels in solid tumours. Clin. Cancer Res. 11:8782-8, 2005. [143] Lankelma J, Dekker H, Luque FR, et al: Doxorubicin gradients in human breast cancer. Clin. Cancer Res. 5:1703-7, 1999. [144] Netti PA, Berk DA, Swartz MA, et al: Role of extracellular matrix assembly in interstitial transport in solid tumours. Cancer Res. 60:2497-503, 2000. [145] St Croix B, Man S, Kerbel RS: Reversal of intrinsic and acquired forms of drug resistance by hyaluronidase treatment of solid tumours. Cancer Lett. 131:35-44, 1998. [146] Jang SH, Wientjes MG, Au JL: Enhancement of paclitaxel delivery to solid tumours by apoptosis-inducing pretreatment: effect of treatment schedule. J. Pharmacol. Exp. Ther. 296:1035-42, 2001. [147] Grantab R, Sivananthan S, Tannock IF: The penetration of anticancer drugs through tumour tissue as a function of cellular adhesion and packing density of tumour cells. Cancer Res. 66:1033-9, 2006. [148] Bremnes RM, Veve R, Hirsch FR, et al: The E-cadherin cell-cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 36:115-24, 2002. [149] Inada S, Koto T, Futami K, et al: Evaluation of malignancy and the prognosis of esophageal cancer based on an immunohistochemical study (p53, E-cadherin, epidermal growth factor receptor). Surg Today 29:493-503, 1999. [150] Siitonen SM, Kononen JT, Helin HJ, et al: Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer. Am. J. Clin. Pathol 105:394-402, 1996.

In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter IV

Inherent and MicroenvironmentMediated Mechanisms of Drug Resistance Malathy P. V. Shekhar∗ Breast Cancer Program, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan

Abstract

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Current treatments are successful at debulking disease; however, development of resistance to treatment made evident by the recurrence of a primary tumor or distant metastasis is, sadly, a frequent occurrence. An important question is whether drug resistant neoplasms represent expansion(s) of residual primary tumor subpopulations that fail to respond to treatment, or represent tumor subpopulations that are activated or rejuvenated by the treatment. Eradication of primary and metastatic disease requires intervention strategies that will target both the tumor cells as well as its tumor microenvironment. In this chapter, we will focus on the roles of mechanisms that are inherent to tumor cells and the tumor microenvironment as contributors to the evolution of drug-resistant neoplasms. The rationale for targeting the DNA damage tolerance or postreplication repair pathway as a novel tool for overcoming drug resistance is discussed. The need for carrying out drug sensitivity evaluations in clinically relevant model systems that take into account the three-dimensional organization and in vivo relationship of a tumor with its microenvironment is also addressed. It is anticipated that such integrative efforts will yield a more global understanding of the tumor- and microenvironment-derived mechanisms involved in drug resistance as well as provide novel intervention targets that will abrogate interactions between a tumor’s cells and its microenvironment.

∗

Malathy PV Shekhar, Ph.D., Breast Cancer Program, Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, 2204, Prentis Building, 110 East Warren Avenue, Detroit, Michigan 48201. Tel: (313) 578-4326. E-mail: [email protected]

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Introduction Resistance to therapy has been recognized as a major obstacle to efficacious cancer treatment. Several factors contributing to cellular drug resistance have been identified and include decreased intracellular drug uptake, mutation of target genes, alterations in signal transduction pathways that result in reduced drug sensitivity, and DNA repair processes. In addition, changes in cell cycle, transcriptional and cell death regulators, growth factors, tumor suppressor genes, and oncogenes also affect cellular sensitivity to anticancer drugs. Since a variety of mechanisms can contribute to drug resistance, it is conceivable that a combination of alterations in several sites within the tumor cell may play a significant role in the development of drug resistance. Drug resistance involves multiple mechanisms inherent to the tumor cells as well as those governed by interactions of the tumor cells with its stromal microenvironment. Alterations in the microenvironment of solid tumors affect tumor progression and drug sensitivity. Thus, modulation of one factor is unlikely to provide a response in a clinical situation. In this chapter, we will focus on the role of mechanisms inherent to tumor cells and those of the tumor microenvironment as contributors to the evolution of drug-resistant neoplasms. Due to the modest tumor specificity of many anticancer drugs, normal tissues are also damaged, leading to adverse side effects. These cytotoxic side effects prevent administration of high doses of drugs that are necessary for eradication of the more resistant tumor subpopulations, and consequently promotes the expansion of drug-resistant tumor cells. A number of strategies have been developed to overcome drug resistance such as high-dose chemotherapy, modulators of multidrug resistance, and gene therapy. However, in order to have a major impact, strategies have to be developed that will broaden the therapeutic range by separating the effective dose and toxic dose to obtain maximal and durable clinical benefit. Multidrug resistance phenotype. Development of simultaneous resistance to multiple drugs, termed multidrug resistance (MDR) is a frequent phenomenon in cancer cells [1]. Cancer cell lines selected for resistance to a specific anticancer compound frequently demonstrate cross-resistance to a broad spectrum of structurally and functionally unrelated agents [2]. The failure to import anticancer drugs into the cells has been attributed as a major mechanism of drug resistance [3 and references within]. Limited drug uptake, enhanced rates of drug efflux, or changes in membrane lipids block apoptosis that is activated by most anticancer drugs [4], and influence response mechanisms that detoxify drugs and repair damage to DNA [5]. Amplification and overexpression of P-glycoprotein (P-gp), a membrane-bound efflux pump and a product of the ABCB1 (MDR1) gene, was found to correspond with conversion to multidrug resistant phenotype [6]. Tumor cells that overexpress P-gp do not accumulate therapeutically effective concentrations of the drug and are, therefore, resistant to the drug’s cytotoxicity. Treatment with verapamil, the competitive inhibitor of several MDR transporters, significantly increases the susceptibility of drugresistant human lung cancer cell lines [7]. Reversal of MDR has been easily achieved in vitro with a variety of inhibitors. However, MDR reversal with MDR inhibitors such as verapamil, quinidine and cyclosporine in the clinical setting has proven to be inefficacious, or with unacceptable toxicities and unexpected pharmacokinetic interactions. In ovarian cancer patients receiving verapamil at plasma levels sufficient to cause inhibition of MDR-mediated

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adriamycin resistance in vitro, there was no evidence of enhanced response of adriamycin coadministered with verapamil [8]. In a murine model of adriamycin resistance, continuous infusion of verapamil at the maximum tolerated dose failed to increase the sensitivity of resistant P388 lymphocytes cells to adriamycin despite a strong in vitro effect [9]. The limited efficacy highlights the presence of multiple and/or redundant mechanisms of resistance; thus targeting one transporter may leave the drug resistance intact [10]. Additional MDR transporters such as breast cancer resistance protein [BCRP; ref 11] have been found, suggesting involvement of multiple transporters that may account for multiple drug resistance. Today, ABCB1 and ABCG2 are recognized as belonging to a family of at least 48 human ABC transporters involved in a variety of essential cellular transport processes. Despite the huge efforts by pharmaceutical industry and academia, no P-gp inhibitors have reached the pharmaceutical market [12,13]. Special drug delivery vehicles are being tested to overcome MDR [14 and references within]. Our recent studies have shown that nanoparticles formulated with FDA-approved dioctylsodium sulfosuccinate and polysaccharide sodium alginate significantly enhances sustained cellular delivery of water soluble drugs such as adriamycin with resultant improved therapeutic efficacy at significantly lower doses of the drug without the use of P-gp inhibitors in drug resistant P-gp overexpressing mammary tumor cells [14]. Targeted molecular therapies and challenges. This strategy involves therapies targeted to gene products involved in proliferation, angiogenesis and apoptosis. Examples of targeted therapies include imatinib mesylate (Gleevec) that is targeted to the Bcr/Abl fusion protein derived from the translocation between chromosomes 9 and 22 in chronic myelogenous leukemia [15, 16], trastuzumab (Herceptin), a chimeric monoclonal antibody to Her2/neu used in breast cancer, rituximab (Rituxan), a monoclonal antibody to CD20 used in nonHodgkin’s lymphoma, and gefitinib (Iressa), a tyrosine kinase inhibitor of EGFR used in non small cell lung cancer [17]. Response to Gleevec has been attributed to specific targeting of Bcr/Abl tyrosine kinase. However, point mutations in the Bcr/Abl kinase domain that induce conformational changes in the protein and impair binding to the drug have been attributed to emergence of Gleevec-resistant leukemia [18, 19]. Monotherapy with Herceptin has shown a modest response rate of 10-20% even in patient groups with Her2/neu overexpression. Currently, Herceptin is combined with anthracyclines and taxanes to improve the response rate [20]. Thus, an important question that remains unanswered is whether the response rates, albeit modest, observed in molecular targeted therapies actually result from direct modulation of the target gene. Challenges in obtaining samples following targeted therapy have made it difficult to correlate clinical efficacy or lack thereof with modulation of the specific molecular target. Regardless of the treatment strategies and tumor type, cancers that initially respond to therapy often acquire drug resistance during the course of treatment. Acquired resistance occurs by adaptation to the drug(s). This probably results form alterations in expression of gene(s), mutational events, and/or selection of additional bypass/alternate pathways for survival that contribute to disease progression during the course of treatment. A welldocumented example of acquired resistance that often does not involve loss or mutation of its target receptor is acquired resistance to tamoxifen in estrogen receptor (ERα) positive breast cancers. Anti-hormone therapies such as tamoxifen are most effective in treatment of ERα+

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breast cancer because tamoxifen and other anti-estrogen therapies block estrogen-mediated growth promoting pathways in ERα+ breast cancer cells. However, half of these initially responsive breast cancers despite retention of a functional target ER become insensitive to tamoxifen because these tumor cells have discovered ways to grow in the absence of estrogen. In contrast to acquired resistance, de novo or intrinsic resistance allows cancer cells to tolerate anticancer drug-induced stress during initial exposure to the drug. It is not known whether intrinsic and acquired resistances emerge from similar alterations in gene expression and signaling pathways or gene mutations. Alternately, survival of minor tumor subpopulations from stress induced by the first exposure to anticancer drugs could be the first step that facilitates their preferential expansion and acquisition of drug resistance. These problems further highlight the complexities of targeted therapy, and underscore the need for identifying critical biomarker molecular panels that will allow rapid and objective assessments of tumor response to treatment. Whole genome sequencing for single nucleotide polymorphisms, mutations, or promoter methylation, and gene expression analyses are being employed to classify patients and their tumor types. This group classification is currently exploited to identify and target individual patients with the right drug therapy. Molecular signatures identified are being commercialized to predict outcomes for breast cancer patients [21]. Similarly, a radiosensitivity prediction assay modeled on radiosensitivity of 48 human cancer cell lines has been developed [22]. Since such classification criteria are often made based upon the use of highly sensitive technologies that seek to determine even small changes in gene expression that are generally not backed up data for the corresponding protein expression, cellular localization, and activity, the interpretation of response (or failure to respond) to the selected treatment regimen can be overstated. Consequently, treatment decisions based upon such predictions could result in patients receiving under treatment or over treatment. Another potential caveat is that gene expression or whole genome analyses are performed on needle core, core biopsy sections, or preselected laser micro-dissected sections that may not accurately reflect the complete heterogeneity of the tumor pathology and response. An important factor to keep in mind is that tumor response to therapy is often transient and is frequently accompanied by progressive alterations mostly induced by the treatment itself, which in turn may negatively influence the effectiveness of the treatment even after a successful initial round. The operation of common mechanisms of resistance across different cytotoxic agents and types of cancer would facilitate application of standard approaches for identification of relevant biomarker panels. Studies by Efferth et al. [23] suggest that sensitive or resistant tumors could be predicted by one single drug, adriamycin. The pleiotropic modes of action of adriamycin may explain why adriamycin is capable of predicting broad-spectrum resistance. Adriamycin is transported by P-gp and involved in MDR phenotype. It is a DNA topoisomerase II inhibitor that is necessary for cell division, and adriamycin generates ROS and free radical containing molecules. However, Riedel et al. [24] have shown that the biology associated with resistance is specific to an agent and is influenced by the context of the tumor cell lines tested. For identification of pathways associated with resistance, gene expression profiling was performed on NCI-60 cancer cell lines deemed to be sensitive and resistant to adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouracil, paclitaxel, and topotecan, and compared with data for a series of 40 lung cancer cell lines for which

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sensitivity to docetaxel and cisplatin was determined [24]. When the NCI-60 cell line data were analyzed for adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouarcil, paclitaxel, or topotecan induced alterations, no single biological pathway was found to be associated with all cytotoxic agents tested. Furthermore, the pathways associated with docetaxel resistance by NCI-60 data were found to be significantly different from those identified in the lung cancer cell line series [24]. Successful identification of clinically relevant molecular pathways requires stringent tagging of data sets with cell and disease context, culture conditions, drug context, drug dose, and time of treatment. DNA damage tolerance pathway. Chemoresistant cancer cells are characterized by altered gene expression and genomic instability because of mutation, recombination, and gene amplification events. Deregulation of DNA repair is a major part of this phenomenon. Genetic interactions contributing to damage-induced mutagenesis was obtained by epistasis analysis of a large array of genetic mutants in the model organism, Saccharomyces cerevisiae. Depending upon the mechanisms by which damaged DNA is excised, by-passed or ligated, processes dealing with DNA damage can be classified into four broad classes: nucleotide excision repair, homologous recombination, nonhomologous end joining (NHEJ), and translesion synthesis (TLS). These pathways are highly conserved evolutionarily and are used to repair modified nucleotides, strand breaks or both. The specificity and fidelity of these processes differ but may be mutually compensatory in certain contexts [25]. Several genes influencing chemosensitivity have been linked to DNA repair, and treatment with chemotherapeutic drugs enhances expression of genes involved in DNA repair. Recent study has shown that BRCA1 deficiency renders cancer cells sensitive to irofulven, a new class of anticancer agents that induces DNA double strand breaks [26]. Cells expressing wild type BRCA1 confer chemoresistance to irofulven by controlling S and G2/M checkpoints that is critical for repairing DNA double strand breaks through RAD51-dependent homologous recombination and maintenance of genomic integrity [26]. Thus, the damage response induced in cells can be counterintuitive since DNA damage induced by anticancer drugs can enhance expression and recruitment of DNA repair proteins to the sites of damage with resultant repair of damaged lesions and development of chemoresistance. Damage caused by chemotherapeutic drugs or other mutagens have to be accurately repaired for maintenance of genomic integrity. If the repair process is incomplete, the persistent lesions could stall the DNA replication machinery at the site of lesion causing a gap opposite the site of damage in the newly synthesized strand (Figure 1). To avoid such roadblocks, all organisms express postreplicative repair (PRR) enzymes that allow replication to bypass the damaged sites on DNA and allow completion of replication. This repair is also called as translesion bypass (TLS) or DNA damage tolerance pathway as it does not remove the lesion but allows tolerance to damage. In the yeast, genes belonging to the RAD6 epistasis group are responsible for the PRR pathway [27]. The major members of this pathway are RAD6, RAD18, RAD30, RAD5, REV3 and REV7. DNA gaps caused by replication stalling induced by mutagenic lesions are filled by translesion synthesis either in an error-free or error-prone manner depending upon the context of the DNA damage and the DNA polymerase recruited (Figure 1) [28]. Error-free and error-prone types of translesion synthesis are carried out by DNA polymerase η (RAD30) and DNA polymerase ζ (a heterodimer of REV3 and REV7), respectively [28-30].

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Figure 1. A model for translesion DNA synthesis. (a) High-fidelity DNA replication (replication machinery) is blocked at the site of DNA damage (triangle). Specialized DNA polymerases (pol η, pol ζ, pol δ), RAD6 and RAD18 are shown in the proximity of the arrested replication fork. (b) RAD6 and RAD18 are recruited to the damaged site and induces monoubiquitination of PCNA at lysine 164. The presence of monoubiquitinated PCNA triggers switching of polymerase: (c) pol ζ mediates error-prone translesion synthesis; (d) monoubiquitinated PCNA is further ubiquitinated by chain extension at lysine 164 (polyubiquitination) by Ubc13/Mms2 and RAD5 which triggers recruitment of pol δ and error-free translesion DNA synthesis by template switching.

RAD6 and RAD18 play a critical role in controlling PRR, and major phenotype of rad6 and rad18 loss is hypersensitivity to a variety of structurally and functionally unrelated DNA damaging agents [25, 31,32]. RAD18 forms a heterodimer with RAD6, an ubiquitin conjugating enzyme (E2) [33,34]. It has been proposed that RAD18 recruits RAD6 protein to the sites of damage or replication-stalled sites on DNA by binding to single stranded regions, and the RAD6 in turn ubiquitinates and degrades target chromosomal proteins in the PRR process [33,34]. The switch from the replicative polymerase η (error-free repair) to TLS polymerases (error prone repair) is regulated by differential posttranslational ubiquitin modification of PCNA. RAD6 collaborates with RAD18 to monoubiquitinate PCNA at lysine 164 in response to DNA damage [35]. Monoubiquitination of PCNA if followed by polyubiquitination by Ubc13/Mms2 (an E2) and RAD5 (an E3 ligase), leads to error-free repair pathway that involves template switching and recombination with the newly synthesized strand (Figure 1) [36]. Two closely related human homologues of yeast RAD6, HHR6A and HHR6B (referred as RAD6A and RAD6B), encode ubiquitin conjugating enzymes and complement the DNA repair and UV mutagenesis defects of the S. cerevisiae rad6 mutant [37]. RAD6A and RAD6B share 95% amino acid identity and are localized on human chromosomes Xq24-q25

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and 5q23-q31, respectively [38]. The requirement for at least one functional RAD6A or RAD6B allele in all somatic cell types is supported by the fact that male and female mice lacking both homologs are nonviable [39]. All functions performed by RAD6 result from ubiquitination since replacement of the conserved Cys88 with serine produces a null phenotype [40, 41]. Mutations in the catalytic site of RAD6 confer hypersensitivity to lethal effects of a variety of DNA damaging agents, and defects in damage-induced mutagenesis [42]. RAD6B is overexpressed in human breast cancer cell lines and tumors, and constitutive overexpression of RAD6B in nontransformed MCF10A breast epithelial cells induces severe aneuploidy, resistance to adriamycin, cisplatin, and ionizing radiation, and a high-risk hyperplastic phenotype [43-45]. The ability to tolerate DNA damage induced by adriamycin and cisplatin correlates with RAD6B expression levels [44]. MCF10A cells stably transfected with antisense RAD6B were PRR-compromised and hypersensitive to the drugs as opposed to RAD6B overexpressing cells that were PRR-proficient and displayed chemoresistance [44]. These findings suggest an important role for RAD66B in normal breast cells, and imply that RAD6B overexpression, frequently found in human breast cancers, can impact the fidelity of PRR pathways, with resultant increase in error-prone repair and chemoresistance. Mammalian TLS DNA polymerases such as Pol η, Pol ζ and REV1 have been discovered and are proposed to function similarly as their yeast counterparts [46]. Most likely, DNA damage induced by chemotherapeutic drugs and ionizing radiation (IR) and bypassed by TLS DNA polymerases results in many point mutations. In patients with a variant form of the hereditary photosensitive and cancer prone xeroderma pigmentosum (XPV), the polymerase η, a homolog of yeast PRR protein RAD30, is mutated [47, 48]. XP-V cells have defective PRR in that the size of the newly replicated DNA is shorter than that in normal cells after UV irradiation [48]. Recently, the human RAD5 homolog, SHPRH was discovered which shares error-free template switching activity of RAD5 (Figure 1) [49]. Inactivation of SHPRH in human cells resulted in increased sensitivity to DNA damaging agents and enhanced chromosomal breakage [49]. Four different mutations of SHPRH have been discovered in several cancer cells including breast cancer and melanoma cells [50]. Mice and chicken DT40 cells deficient in RAD6/RAD18 exhibit hypersensitivity to various DNA damaging agents and enhanced genomic instability visualized by increase in number of sister chromatid exchanges [51]. These observations further underscore the importance of PRR pathways in maintenance of genomic integrity. Imbalances in expression levels/activity or mutation in PRR genes could seriously impact the fidelity of repair process, sensitivity to damaging agents, and the genomic integrity of cells. The incorporation of IR into multimodality cancer treatment regimens has significantly improved survival of patients, particularly in patients with solid tumors receiving curative therapy [52,53]. The tumoricidal effect of IR is derived from induction of DNA damage that is beyond the capacity of the cell’s capacity for repair [52,53]. However, sublethal doses of IR-induced DNA damage lead to elevated mutagenic accumulation (54). In normal tissues, IR-induced mutagenesis is associated with enhanced risk for malignancy [55-57]. Whereas in tumor cells, elevated mutagenesis enhances the risk of adaptive mutation acquisition, which in turn leads to therapeutic resistance [58,59]. Disruption of RAD6 caused significant disruption of IR-induced mutagenesis whereas deletion of the entire ORF of RAD18 had no effect [60]. The translesion synthesis or DNA damage tolerance pathway has received very

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little appreciation outside yeast genetics. Since this highly conserved RAD6 epistasis pathway accounts for majority of drug resistance and damage-induced mutagenesis in the yeast, targeting key genes such as RAD6 that service this pathway could provide a novel therapeutic approach for overcoming resistance to a broad spectrum of drugs. Further, considering the role of RAD6 in both error-free and error-prone PRR pathways, inhibition of the RAD6 ubiquitinating catalytic activity could sensitize tumors to lower doses of chemotherapy and IR as well as block damage-induced mutagenesis that is associated with error-prone PRR pathway. Proteasome inhibitors are currently used to overcoming drug resistance. However, achieving a desirable drug response with proteasome inhibitors in vivo could be difficult since multiple cell survival signaling networks are controlled by ubiquitin modification of antiapoptotic and cell cycle regulatory proteins. Therapeutic targeting of key ubiquitin conjugating E2 enzymes such as RAD6 or specific ubiquitin E3 ligases could provide greater flexibility and selectivity than proteasome inhibition per se. Microenvironmental influences on drug sensitivity. It is becoming increasingly clear that microenvironmental stromal factors can significantly affect the success of therapy. The tumor microenvironment not only actively participates in carcinogenesis but also influences the drug response and survival of cancer cells during treatment. The tumor microenvironment consists of the stroma, a supportive platform for epithelial cells, which is composed of fibroblasts, endothelial cells, immune cells, adipocytes, inflammatory cells, nerve cells, and a macromolecular network of proteoglycans and glycoproteins collectively termed as extracellular matrix (ECM). Factors required for premalignant progression, growth of primary cancer, as well as invasion and metastasis are all altered by tumor-tumor cell, tumorstromal, and tumor-ECM interactions. This stromal-mediated effect is related to the ability of the microenvironment to influence processes or pathways that are important for cancer cell survival, proliferation, and response of cells to anticancer drugs. Soluble factors such as cytokines and growth factors originating from the stromal microenvironment provide paracrine signals for cancer proliferation and survival. Thus, drug resistance of tumor could be regarded as aggregate of resistance arising from cell-cell and cell-ECM interactions, and from stromal-derived soluble factors (Figure 2) [61]. Evidence for drug resistance arising from direct cell-cell contacts or cell-cell adhesion-mediated mechanism was obtained when tumor cells grown as three dimensional masses or multicellular spheroids were found to be more resistant to anticancer drugs than the corresponding monolayers. Drug sensitivity profiles deduced from 3-D cultures are more representative as they more closely resemble their in vivo organization and hence more closely recapitulate the drug resistance properties of in vivo tumor cells as compared to the typical 2-D or monolayer cultures used in most laboratories [62]. These differences in drug sensitivities were not due to compromised ability of the drugs to penetrate the 3-D structures [63]; disruption of cell-cell interaction was shown to sensitize cancer cells to 5-fluorouracil, paclitaxel, and etoposide [64]. The MDR arising from direct cell contact with the ECM or other cells is referred to as cell adhesion-mediated drug resistance. Extracellular matrix (ECM). ECM is a complex assembly of collagens, proteoglycans and glycoproteins, and is an important component of normal tissue that provides essential signals for cell development, proliferation, migration, and survival [65].

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Figure 2. A model for mechanism of microenvironment mediated drug resistance. Cancer cells develop drug resistance through direct cell-cell (homotypic or heterotypic) interactions, cell-ECM interactions, and cancer cell interactions with soluble molecules released by stromal and cancer cells. Heterotypic interactions refer to interactions between cancer cells and stromal (fibroblasts, immune cells, endothelial, etc.) cells.

The tumor cells are surrounded by ECM, which is produced by the neighboring stromal cells or the tumor cells themselves. Thus, the ECM composition of tumors can be different which can affect their sensitivity to drugs. ECM components typically interact with integrins, a family of α and β-transmembrane proteins, which associate to form heterodimeric receptors [66]. The roles of integrins in cell survival have been established in studies that showed loss of ECM-integrin interactions lead to anoikis [67]. The same types of mechanisms may also promote resistance to drug-mediated apoptosis [68]. Adhesion to fibronectin has been shown to prevent drug-induced apoptosis [69-74]. Involvement of ECM proteins such as collagen IV, fibronectin and tenascin that are elevated in lung cancer were shown to protect small cell lung cancer cells from chemotherapy-induced induced apoptosis via signaling mediated through the integrin β1 receptor [73]. Breast cancer cells placed in reconstituted basement membrane matrix was shown to resist drug-induced apoptosis [74,75]. This induction of chemoresistance was accompanied by activation of NFκB, a transcription factor that is involved in mediating survival signals [76]. The tumor vasculature is a target for conventional and targeted therapy and the efficacy of these drugs depends on their ability to destroy tumor vasculature [77]. Fibronectin can inhibit drug-induced apoptosis in tumorderived endothelial cells (69), making this interaction a new target for tumor therapy [78].

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These observations provide further evidence on the role of ECM proteins and ECM-mediated interactions with tumor cells in providing survival signals to the tumor vasculature and protecting tumor from chemotherapy-induced apoptosis. An important point to keep in mind is that tumors produce their favorable ECM, and that some ECM constituents may be upregulated in response to exposure to certain drugs that could enhance their resistance to these compounds. Collagen VI is overexpressed in many ovarian tumors but is absent in normal ovarian epithelial cells [79]. Culturing ovarian cancer cells on ECM composed of collagen VI resulted in increased survival when exposed to cisplatin [79]. In addition to their role in influencing tumor cell signaling, the ECM could also influence the ability of the drugs from reaching the tumor cells. The composition of ECM can influence physical properties such as mechanical stiffness in the tumor and consequently act as a barrier that affects diffusion of drugs [80]. The limited ability of drugs to reach tumor cells particularly those that are distant from blood vessels creates a gradient with cells closest to drug delivering vessel to be most sensitive as compared to cells that are distant [81]. Soluble factors. The tumor microenvironment is a rich source of soluble factors that are secreted by immune cells and fibroblasts, and influences organ-specific metastasis as evidenced by the involvement of stromal-derived cytokines and growth factors such as TGFβ, RANKL, SDF-1, and IL-6 in breast and prostate cancer, and multiple myeloma metastasis to the bone. IL-6 is secreted by bone marrow derived stromal cells and has been reported to protect against chemotherapy-induced apoptosis in myeloma, prostate and breast cancer cells [82-84]. Stromal-derived soluble growth factors such as EGF, bFGF, IGF-1 and HGF have been shown to influence the survival of various cancers in the presence of chemotherapeutic drugs [85-89]. Evidence for tumor fibroblasts as a direct inducer of drug resistance was obtained from our studies with breast cancer cells [90]. Contact dependent 3-D cocultures on reconstituted basement membrane matrix of ERα positive/tamoxifen sensitive breast cancer cells with tumor fibroblasts derived from ERα-negative breast tumors resulted in loss of tamoxifen sensitivity, whereas similar cocultures of the breast cancer cells with tumor fibroblasts derived from ERα+ breast tumors preserved the cancer cells’ tamoxifen sensitivity [90]. Tumor fibroblast-mediated acquisition of tamoxifen resistance was accompanied by hyperactivation of MAPK and Akt pathways and implicates involvement of stromal-derived soluble growth factors [90]. Although the identity of the stromal-derived soluble growth factor(s) modulating tamoxifen sensitivity remains to be determined, our data have excluded EGF and IGF-1 as potential players [90]. Interestingly, the intrinsic tamoxifen resistance of breast cancer cells was unaffected by the tumor fibroblasts regardless of whether the fibroblasts were derived from ER+ or ER-negative breast tumors. Although it remains to be determined whether development of intrinsic and acquired resistance involves common pathways, hyperactivation of MAP kinase and Akt was observed in both [90]. Models for studying drug resistance. Synergistic interactions between cancer cells, cancer cells and ECM, and cancer cell-stromal cells determine the net drug sensitivity of tumor. Thus, models that more closely mimic these interactions are predicted to provide better insights into the tumor’s sensitivity to anticancer drugs (Figure 3). Model 1, the spheroid model, is generated by forcing tumor cells to form conglomerates by preventing adhesion of tumor cells to the tissue culture dish (Figure 3A) [63]. Model 2 focuses on 3-D interactions between tumor cell and ECM (Figure 3B).

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Figure 3. Models for screening drug sensitivity. Model 1 (A) is the spheroid model that encourages homotypic cancer cell-cell interactions. Model 2 (B) focuses on interactions between tumor cells and ECM, and results in activation of ECM-mediated signaling pathways in tumor cells. Model 3 (C and D) is the stromal model, which facilitates interactions between cancer cells and stroma, cancer cells and ECM as well as between cancer cells, stroma and ECM. Interactions with the stroma results in secretion of soluble factors by stromal cells and cancer cells that induce activation of signaling pathways that contributes to drug resistance.

The most favorable ECM constituent of the specific tumor type is used individually to permit establishment of cancer cell-ECM-mediated signaling pathways. Application of individual ECM components will allow establishment of specific and selective signaling pathways. However, it must be borne in mind that the tumor ECM is not a composed of a single component but is rather a heterogeneous mixture of variant composition that can influence the physical rigidity of the microenvironment, the type and activity of signaling pathways, and consequently drug sensitivity. Model 3 focuses on 3-D interactions between tumor cells, stromal cells and ECM (Figure 3C and D). This model facilitates tumor cellstromal interactions, tumor cell-ECM interactions, and tumor cell-stromal-ECM interactions. This model will enable both homotypic and heterotypic interactions, as well as establishment of soluble factor-mediated paracrine and autocrine mechanisms of tumor cell and stromal cell activation [91]. Using this model system, we have observed that epithelial differentiation and morphogenetic effects induced by stroma are apparent only when breast cancer cells are cocultured with organ-specific fibroblasts even when cultured on reconstituted basement membrane matrix [91]. These findings indicate a requirement for specific ECM molecules

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that are assembled or synthesized de novo by reciprocal epithelial-mesenchymal interactions and necessary for modeling of mammary gland architecture. These findings also underscore the specific demands that must be met for establishment of productive stromal-epithelialECM interactions both by contact-dependent and soluble factor-mediated mechanisms [91]. Reciprocal interactions between tumor cells and stroma provide signals for survival by divergent pathways via upregulation of growth, upregulation of antiapoptotic molecules, downregulation of antiapoptotic molecules, attenuation of DNA damage response, increase of DNA repair activity, etc. Screenings performed using these model systems should permit identification of drugs that effectively and simultaneously target both tumor cells and its tumor microenvironment and help eliminate or minimize acquisition of drug resistance.

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Conclusion Cancer cell subpopulations with variant phenotypes and genotypes that coexist with mixed stromal elements contribute to tumor heterogeneity. Alternatively, is the stromal microenvironment serving as a platform for driving tumor heterogeneity and heterogeneity in drug sensitivities? ECM components elaborated during stromal-cancer cell interactions are likely to be tumor specific. Different cancers (including multifocal and multicentric cancers of the same organ) and cancer subtypes are likely to mount different stromal reactions that enable them to synthesize/assemble variant ECM composition. Consequently, different cancers will display different requirements for optimal ECM-mediated protection from the insults. This necessitates the use of carefully selected, clinically relevant experimental systems for identification of specific signaling pathways that are established during tumorstroma-ECM interactions and necessary for the survival of cancer cells and stromal cells. Utilization of model systems that take into account the three-dimensional organization and in vivo relationship of a tumor with its tumor microenvironment will facilitate the identification of molecular pathways that are associated with resistance to specific anticancer drugs and specific disease states. It is expected that intervention strategies designed to target the identified signaling pathways will reduce interactions between the microenvironment and the tumor cells and consequently enhance the efficiency of cytotoxic therapy. Identification of critical survival pathways that simultaneously target both the cancer cells as well as the tumor’s unique stromal participants is crucial for development of therapies that will not only overcome drug resistance but could also prevent acquisition of drug resistance. Since most treatment regimens employ combination treatment modalities, strategies that target pathways such as the DNA damage tolerance or the PRR pathway, which confers resistance to a wide spectrum of anticancer drugs rather than to a specific single agent, should be exploited in the future for overcoming or reversing drug resistance. It is likely that such integrated efforts will yield a more global understanding of the mechanisms involved in drug resistance and provide novel intervention targets for a specific drug or combination of drugs.

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References [1]

[2]

[3] [4] [5] [6] [7]

[8] [9]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[10]

[11]

[12] [13] [14] [15] [16] [17]

Stein WD, Bates SE, Fojo T. Intractable cancers: The many faces of multidrug resistance and the many targets it presents for therapeutic attack. Curr. Drug Targets 2004, 5:333-346. Beidler JL, Riehm H. Cellular resistance to actinomycin D in Chinese hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. Cancer Res. 1970, 30:1174-1184. Gottesman MM. Mechanisms of cancer drug resistance. Ann. Rev. Med. 2002, 53:61527. Lowe SW, Ruley HE, Jacks T, Housman DE. p53 dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993, 74:957-67. Synold TW, Dussault I, Forman BM. The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. 2001, 7:584-90. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discovery 2006, 5:219-234. Merry S, Courtney ER, Fetherston CA, Kaye SB, Freshney RI. Circumvention of drug resistance in human non-small lung cancer in vitro by verapamil. Br. J. Cancer 1987, 56:401-405. Fojo A, Hamilton TC, Young RC, Ozols RF. Multidrug resistance in ovarian cancer. Cancer 1987, 60 (Suppl 8): 2075-2080. Rustum YM, Radel S, Campbell J, Mayhew E. Approaches to overcome in vivo anticancer drug resistance. Prog. Clin. Biol. Res. 1986, 223: 187-202. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med. 2001, 7:1028-1034. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, Ross DD. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 1998, 95:15665-15670. Nobilit S, Landini I, Giglioni B, Mini E. Pharmacological strategies for overcoming multidrug resistance. Curr. Drug Targets 2006, 7:861-79. Takara K, Sakaeda T, Okumura K. An update on overcoming MDR1-mediated multidrug resistance in cancer chemotherapy. Curr. Pharm. Des. 2006, 12:273-286. Chavanpatil MD, Khdair A, Gerard B, Bachmeier C, Miller DW, Shekhar MP, Panyam J. Surfactant-polymer nanoparticles overcome P-glycoprotein-mediated drug efflux. Mol. Pharm. 2007, 4: 730-738. Goldman JM, Melo JV. Targeting the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med. 2001, 344:1804-1806. Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach DR, Miller WT, Clarkson B, Kuriyan J. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res. 2002, 62:42364243.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

108

Malathy P. V. Shekhar

[18] Kim R, Toge T. Changes in therapy for solid tumors: potential for overcoming drug resistance in vivo with molecular targeting agents. Surg. Today 2004, 34:293-303. [19] von Bubnoff N, Schneller F, Peschel C, Duyster J. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukemia to ST1571: a prospective study. Lancet 2002, 359:487-491. [20] Nimmanapalli R, Bhalla K. Mechanisms of resistance to imatinib mesylate in Bcr-Ablpositive leukemias. Curr. Opin. Oncol. 2002, 14:616-620. [21] Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2. N. Engl. J. Med. 2001, 344: 783-792. [22] Paik S, Shak S, Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T, Hiller W, Fisher ER, Wickerham DL, Bryant J, Wolmark N. A multigene assay to predict recurrence of tamoxifen-treated, nodenegative breast cancer. N. Engl. J. Med. 2004, 351:2817-26. [23] Yeatman TJ, Mule J, Dalton WS, Sullivan D. On the eve of personalized medicine in oncology. Cancer Res. 2008, 68:7250-7252. [24] Efferth T, Konkimalla B, Wang Yi-F, Sauerbrey A, Meinhardt S, Zintl F, Mattern J, Volm M. Prediction of broad spectrum resistance of tumors towards anticancer drugs. Clin. Cancer Res. 2008, 14:2405-2412. [25] Riedel RF, Porrello A, Pontzer E, Chenette EJ, Hsu DS, Balakumaran B, Potti A, Nevins J, Febbo PG. A genomic approach to identify molecular pathways associated with chemotherapy resistance. Mol. Cancer Ther. 2008, 7:3141-3149. [26] Friedberg E, Graham C, Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington D.C., 1995. [27] Wiltshire T, Senft J, Wang Y, Konat GW, Wenger SL, Reed E, Wang W. BRCA1 contributes to cell cycle arrest and chemoresistance in response to anticancer agent, irofulven. Mol. Pharmacol. 2007, 71:1051-1060. [28] Broomfield S, Hryciw T, Xiao W. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat Res 2001, 486:167-184. [29] Kunz BA, Straffon AF, Vonarx EJ. DNA damage-induced mutation: tolerance via translesion synthesis. Mutat Res 2000, 451:169-185. [30] McDonald JP, Levine AS, Woodgate R. The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism. Genetics 1997, 147:1557-1568. [31] Nelson JR, Lawrence CW, Hinkle DC. Thymine-thymine dimer bypass by yeast DNA polymerase zeta. Science 1996, 272:1646-1649. [32] Lawrence CW, Christensen R. UV mutagenesis in radiation-sensitive strains of yeast. Genetics 1976, 82:207-232. [33] Prakash L. Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol. Gen. Genet. 1981, 184:471-478. [34] Bailly V, Lamb J, Sung P, Prakash S, Prakash L. Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev. 1994, 8:811-820.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Inherent and Microenvironment-Mediated Mechanisms…

109

[35] Bailly V, Prakash P, Domains required for dimerization of yeast Rad6 ubiquitinconjugating enzyme and Rad18 DNA binding protein. Mol. Cell Biol. 1997, 17:45364543. [36] Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 2002, 419:135-141. [37] Friedberg EC, Lehmann AR, Fuchs RPP. Trading places: How do DNA polymerases switch during translesion DNA synthesis. Mol. Cell 2005, 18:499-505. [38] Koken MH, Reynolds P, Jaspers-Dekker I, Prakash L, Prakash S, Bootsma D, Hoeijmakers JH. Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. Proc. Natl. Acad. Sci. USA 1991, 88:8865-9. [39] Koken MH, Smit EM, Jaspers-Dekker I, Oostra BA, Hagemeijer A, Bootsma D, Hoeijmakers JH. Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31. Genomics 1992, 12:447-53. [40] Roest HP, Baarends WM, de Wit J, van Klaveren JW, Wassenaar E, Hoogerbrugge JW, van Cappellen WA, Hoeijmakers JH, Grootegoed JA. The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice. Mol. Cell Biol. 2004, 24:5485-95. [41] Sung P, Prakash S, Prakash L. Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions. Proc. Natl. Acad. Sci. USA 1990, 87:2695-2699. [42] Sung P, Prakash S, Prakash L. Stable ester conjugate between the Saccharomyces cerevisiae RAD6 protein and ubiquitin has no biological activity. J. Mol. Biol. 1991, 221:745-749. [43] Prakash S, Sung P, Prakash L. Structure and function of RAD3, RAD6, and other DNA repair genes of Saccharomyces cerevisiae. In: Strauss PR, Wilson SH, editors. The Eukaryotic nucleus. Caldwell (NJ): Telford Press: 1990. Vol 1, pp 275-292. [44] Shekhar MP, Lyakhovich A, Visscher DW, Heng H, Kondrat N. Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation. Cancer Res. 2002, 62:2115-2124. [45] Lyakhovich A, Shekhar MP. RAD6B overexpression confers chemoresistance: RAD6 expression during cell cycle and its redistribution to chromatin during DNA damageinduced response. Oncogene 2004, 23:3097-3106. [46] Shekhar MP, Tait L, Gerard B. Essential role of T-cell factor/beta-catenin in regulation of Rad6B: a potential mechanism for Rad6B overexpression in breast cancer cells. Mol. Cancer Res. 2006, 4: 729-745. [47] Myung K, Smith S. The RAD5-dependent postreplication repair pathway is important to suppress gross chromosomal rearrangements. JNCI Monogr 2008, 39:12-15. [48] Boyer JC, Kaufman WK, Brylawski BP, Cordeiro-Stone M. Defective postreplication repair in xeroderma pigmentosum variant fibroblasts. Cancer Res. 1990, 50:2593-2598. [49] Masutani C, Kusumoto R, Yamada A, Yuasa M, Araki M, Nogimori T, Yokoi M, Eki T, Iwai S, Hanaoka F. Xeroderma pigmentosum variant: from a human genetic disorder to a novel DNA polymerase. Cold Spring Harb. Symp. Quant. Biol. 2000, 65:71-80.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

110

Malathy P. V. Shekhar

[50] Motegi A, Sood R, Helen M, Markowitz SD, Liu PP, Myung K. Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination. J. Cell Biol. 2006, 175:703-708. [51] Sood R, Makalowska I, Galdzicki M, Hu P, Eddings E, Robbins CM, Moses T, Namkoong J, Chen S, Trent JM. Cloning and characterization of a novel gene, SHPRH, encoding a conserved putative protein with SNF2/helicase and PHD-finger domains from the 6q24 region. Genomics 2003, 82:153-161. [52] Tateishi S, Niwa H, Miyazaki J, Fujimoto S, Inoue H, Yamaizum M. Enhanced genomic instability and defective postreplication repair in Rad18 knockout mouse embryonic stem cells. Mol. Cell Biol. 2003, 23:474-481. [53] Senan S, De Ruysscher De D, Giraud P, Mirimanoff R, Budach V. Literature-based recommendations for treatment planning and execution in high-dose radiotherapy for lung cancer. Radiother Oncol. 2004, 71:139-146. [54] Soffietti R, Ruda R, Mutani R. Management of brain metastases. J Neurol 2002, 249:1357-1369. [55] Beir V. Health effects of exposure to low levels of ionizing radiation. National Academy Press, Washington D.C., 1990. [56] Brada M, Ford D, Ashley S, Bliss JM, Crowley S, Mason M, Rajan B, Traish D. BMJ 1992, 304:1343-1346. [57] Kimball Dalton VM, Gelber RD, Li F, Donnelly MJ, Tarbell NJ, Sallan SE. Second malignancies in patients treated for childhood acute lymphoblastic leukemia. J. Clin. Oncol. 1998, 16:2848-2853. [58] Sadetzki S, Flint-Richter P, Ben-Tal T, Nass D. Radiation-induced meningioma: a descriptive study of 253 cases. J. Neurosurg. 2002, 97:1078-1082. [59] Lin X, Okuda T, Trang J, Howell SB. hREV1 modulates the cytotoxicity and mutagenicity of cisplatin in human ovarian carcinoma cells. Mol. Pharmacol. 2006, 69:1748-1754. [60] Okuda T, Lin X, Trang J, Howell SB. Suppression of hREV1 expression reduces the rate at which human ovarian carcinoma cells acquire resistance to cisplatin. Mol. Pharmacol. 2005, 67:1852-1860. [61] Chen CC, Motegi A, Hasegawa Y, Myung K, Kolodner R, D’Andrea A. Genetic analysis of ionizing radiation-induced mutagenesis in Saccharomyces cerevisiae reveals translesion synthesis (TLS) independent of PCNA K164 SUMOylation and ubiquitination. DNA repair 2006, 5:1475-1488. [62] Li Z-W, Dalton WS. Tumor microenvironment and drug resistance in hematologic malignancies. Blood Reviews 2006, 20:333-342. [63] Croix St. B, Kerbel RS. Cell adhesion and drug resistance in cancer. Curr. Opin. Oncol. 1997, 9:549-556. [64] Durand RE, Sutherland RM. Effects of intercellular contact on repair of radiation damage. Exp. Cell Res. 1972, 71:75-80. [65] Green SK, Francia G, Isidoro C, Kerbel RS. Antiadhesive antibodies targeting Ecadherin-sensitize multicellular tumor spheroids to chemotherapy in vitro. Mol. Cancer Ther. 2004, 3:149-159.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Inherent and Microenvironment-Mediated Mechanisms…

111

[66] Dwayne G. Stupack and David A. Cheresh. Get a ligand, get a life: integrins, signaling and cell survival. J. Cell Sci. 2002, 115:3729-3738. [67] Jin H, Varner J. Integrins: roles in cancer development and as treatment targets. Br. J. Cancer 2004, 90:561-565. [68] Frisch SM, Screaton RA. Anoikis mechanisms. Curr. Opin. Cell Biol. 2001, 13:555562. [69] Shain KH, Dalton WS. Cell adhesion is a key determinant in de novo multidrug resistance (MDR): new targets for the prevention of acquired MDR. Mol. Cancer Ther. 2001, 1:69-78. [70] Hoyt DG, Rusnak JM, Mannix RJ, Modzelewski RA, Johnson CS, Lazo JS. Integrin activation suppresses etoposide-induced DNA strand breakage in cultured murine tumor-derived endothelial cells. Cancer Res. 1996, 56:4146-4149. [71] Ferber I, Bachmann SO, Specht H, Wimmel A, Gross MW, Schlegel J, Suske G, Schuermann M. In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways. Oncogene 2002, 21:8683-8695. [72] Kouniavsky G, Khaikin M, Zvibel I, Zippel D, Brill S, Halpern Z, Papa M. Stromal extracellular matrix reduces chemotherapy-induced apoptosis in colon cancer cell lines. Clin. Exp. Metastasis 2002, 19:55-60. [73] Maubant S, Cruet-Hennequart S, Poulain L, Carreiras F, Sichel F, Luis J, Staedel C, Gauduchon P. Altered adhesion properties and alphav integrin expression in a cisplatinresistant human ovarian carcinoma cell line. Int. J. Cancer 2002, 97:186-194. [74] Sethi T, Rintoul RC, Moore SM, MacKinnon AC, Salter D, Choo C, Chilvers ER, Dransfield I, Donnelly SC, Strieter R, Haslett C. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat. Med. 1999, 5:662-668. [75] Weaver VM, Lelièvre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F, Werb Z, Bissell MJ. beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2002, 2:205-216. [76] Zahir N, Weaver VM. Death in the third dimension: apoptosis regulation and tissuearchitecture. Curr. Opin. Genet Dev. 2004, 14:71-80. [77] Wang CY, Mayo MW, Baldwin AS Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996, 274:784-787. [78] Miller KD, Sweeney CJ, Sledge GW Jr. Redefining the target: chemotherapeutics as antiangiogenics. J. Clin. Oncol. 2001, 19:1195-1206. [79] Kalluri R. Basement membranes: structure, assembly, and role in tumor angiogenesis. Nat. Rev. Cancer 2003, 3:422-433. [80] Sherman-Baust CA, Weeraratna AT, Rangel LB, Pizer ES, Cho KR, Schwartz DR, Shock T, Morin PJ. Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells. Cancer Cell 2003, 3:377-386. [81] Jain RK. Vascular and interstitial barriers to delivery of therapeutic agents in tumors. Cancer Metastasis Rev. 1990, 9:253-266.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

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Malathy P. V. Shekhar

[82] Tannock IF, Lee CM, Tunggal JK, Cowan DS, Egorin MJ. Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy. Clin. Cancer Res. 2002, 8:878-884. [83] Frassanito MA, Cusmai A, Iodice G, Dammacco F. Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis. Blood 2001, 97:483-489. [84] Borsellino N, Belldegrun A, Bonavida B. Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of human prostate carcinoma cell lines. Cancer Res. 1995, 55:4633-4639. [85] Conze D, Weiss L, Regen PS, Bhushan A, Weaver D, Johnson P, Rincón M. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Res. 2001, 61:8851-8858. [86] Jankowski K, Kucia M, Wysoczynski M, Reca R, Zhao D, Trzyna E, Trent J, Peiper S, Zembala M, Ratajczak J, Houghton P, Janowska-Wieczorek A, Ratajczak MZ. Both hepatocyte growth factor (HGF) and stromal-derived factor-1 regulate the metastatic behavior of human rhabdomyosarcoma cells, but only HGF enhances their resistance to radiochemotherapy. Cancer Res. 2003, 63:7926-7935. [87] Guo YS, Jin GF, Houston CW, Thompson JC, Townsend CM Jr. Insulin-like growth factor-I promotes multidrug resistance in MCLM colon cancer cells. J. Cell Physiol. 1998, 175:141-148. [88] Miyake H, Hara I, Gohji K, Yoshimura K, Arakawa S, Kamidono S. Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human bladder cancer cell line. Cancer Lett. 1998, 123:121-126. [89] Navolanic PM, Steelman LS, McCubrey JA. EGFR family signaling and its association with breast cancer development and resistance to chemotherapy. Int. J. Oncol. 2003, 22:237-252. [90] Alexia C, Fallot G, Lasfer M, Schweizer-Groyer G, Groyer A. An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against druginduced apoptosis. Biochem. Pharmacol. 2004, 68:1003-1015. [91] Shekhar MP, Santner S, Carolin KA, Tait L. Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity. Am. J. Pathol. 2007, 170:15461560. [92] Shekhar MP, Werdell J, Santner SJ, Pauley RJ, Tait L. Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer Res. 2001, 61:1320-1326.

In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter V

Studies on the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitor Gefitinib in NSCLC Cell Lines: Evidence that Ligand-Induced Endocytosis of EGFR via the Early/Late Endocytic Pathway is Associated with Gefitinib Sensitivity of NSCLC Cell Line Yukio Nishimura∗

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Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Abstract The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, I examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, I provide novel evidence that efficient endocytosis of EGF-EGFR occurs via the ∗

E-mail: [email protected]. Tel and Fax: +81-92-642-6616

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Yukio Nishimura endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because, even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, I demonstrate that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines.

Keywords: Gefitinib; EGF receptor (EGFR); phosphorylated EGF receptor (pEGFR); Endosome/Lysosomes; Endocytosis; Non-small cell lung cancer cell lines; cathepsin.

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Introduction The epidermal growth factor (EGF) and its receptor (EGFR) play an important role in the pathogenesis of different tumors; therefore, therapies directed at inhibiting EGFR function are potential anti-cancer treatments (de Bono and Rowinsky, 2002; Mendelsohn and Baselga, 2000). Each EGFR is composed of an extracellular binding domain and a cytoplasmic domain with tyrosine kinase activity, and it can bind to the EGF. Following ligand binding, the EGFR is dimerized and the intracellular tyrosine kinase region is activated, causing receptor tyrosine autophosphorylation and transphosphorylation of another receptor monomer. These events lead to the recruitment and phosphorylation of several intracellular substrates (Ullrich and Schlessinger, 1990; Yarden, 2001). The endocytosis of EGFR has served as a model for studying ligand-induced receptormediated endocytosis for many years. In receptor-mediated endocytosis, ligand-receptor complexes are internalized and transported via clathrin-coated vesicles to the early endosomes. EGF is a secreted peptide that stimulates cell growth and cell division by binding to a receptor tyrosine kinase at the plasma membrane. EGFR then recruits and phosphorylates signaling molecules, leading to the activation of a MAPK-signal transduction cascade—an important mechanism for regulating cell growth (Schlessinger, 2004). EGF-EGFR complexes are finally degraded on delivery to the lysosomes via endocytosis, a process known as receptor down-regulation. Therefore, intracellular lysosomal dysfunction may reflect carcinomatous malignant transformation (Kroemer and Jaattela, 2005; Mohamed and Sloane, 2006). Recently, gefitinib (Iressa, ZD1839), a new EGFR inhibitor, was developed and used in clinical trials for cancer (Arteaga and Johnson, 2001; Barker et al., 2001; Baselga and

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Averbuch, 2000; Woodburn, 1999). Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor that functions by competing with ATP in binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells. It has exhibited significant in vivo antitumor activity against a broad range of mouse tumor xenograft models (Sirotnak et al., 2000) and tumor cell lines in vitro (Hirata et al., 2004). A recent in vitro study demonstrated that of the nine NSCLC cell lines examined, PC9 was the most sensitive to the effect of gefitinib when assayed under basal growth conditions for EGFR phosphorylation and activation of EGFR downstream effectors such as AKT and those in the ERK1/2 pathway for its survival and proliferation. This result suggests that the mechanism underlining the sensitivity of the EGFR pathway could be useful in predicting the potential effectiveness of gefitinib in NSCLC patients. Inefficient EGFR down-regulation was observed in the gefitinib-resistant cell line QG56, whereas rapid down-regulation occurred in the gefitinib-sensitive cell line PC9 wherein the cells were in the exponential phase of growth, thereby suggesting that a different unknown down-regulation mechanism operates in each cell types (Ono et al., 2004). The mechanism of drug sensitivity to gefitinib has not been fully understood at present. Furthermore, the detailed molecular mechanisms underlying the sorting and trafficking of EGFR via the endocytic pathway have not yet been demonstrated, although the outline of the EGFR endocytic pathway appears to be established. The identification and characterization of each regulatory element involved in EGFR trafficking are of importance for the further clarification of its machinery. In the present study, to further substantiate the effect of gefitinib on the EGFR downregulation pathway and to understand the detailed internalization mechanism for gefitinibsensitive PC9 cells or gefitinib-resistant QG56 cells, I examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. Confocal immunofluorescence microscopy was then used to assess the distribution of internalized Texas red-EGF, early endosomes stained with an anti-transferrin receptor antibody, and late endosomes/lysosomes stained with an anti-LIMPII antibody. Here, I found novel evidence that an aberration in some steps of the EGF-EGFR traffic out of the early endosomes to the late endosomes/lysosomes does occur in the gefitinib-resistant human cancer cell line derived from NSCLC, whereas endocytosis of EGFR is normal in the gefitinib-sensitive PC9 cells.

Results Intracellular Distribution of EGFR and pEGFR between the GefitinibSensitive or Gefitinib-Resistant NSCLC Cell Lines Firstly, in order to examine the intracellular distribution of EGFR in the gefitinibsensitive NSCLC cell line PC9 or the gefitinib-resistant cell line QG56, each cell lines were double-labeled with specific antibodies to EGFR or with specific antibodies to cathepsin D and LIMPII, respectively (Figure 1). I determined the intracellular distribution of late endosomes/lysosomes by using specific antibodies to lysosomal aspartic protease cathepsin D or LIMPII/LGP85.

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Figure 1. Intracellular distribution of EGFR and pEGFR in the NSCLC cell lines. To examine the intracellular localization of EGFR, the gefitinib-sensitive NSCLC cell line, PC9 (A), or the gefitinibresistant cell line, QG56 (C) was fixed and double-stained for cathepsin D (green in a, c) or LIMPII (green in d, f) and EGFR (red). Superimposed images of cathepsin D or LIMPII and EGFR are shown in c and f. In A, it is notable that EGFR is mostly colocalized with LIMPII-positive swollen vacuolar structures in PC9 cells, reminiscence of late endosmes, in the perinuclear region, and also some EGFR stainings are seen on the plasma membrane at the cell-cell contact sites. However, no colocalization of EGFR with cathepsin D was seen (A). In contrast, EGFR is mostly localized on the plasma membranes, and is not associated with late endosomal vesicular structures in QG56 (C). Next, to investigate the localization of pEGFR, PC9 cell (B) or QG56 cells (D) was double-stained for LIMPII (red in b, h) or cathepsin D (red in e, k) and pEGFR (green). Superimposed images of cathepsin D or LIMPII and pEGFR are shown in c, f, i, l. In both PC9 and QG56 cells, it is notable that pEGFR-positive small punctate vesicles are clearly seen in the nucleus of both PC9 and QG56 cells. Bar, 10μm. (Figure 1 is the synthesis of data previously published by Nishimura et al. in 2007 and 2008).

These proteins are distributed within the endocytic organelles at the highest concentration in late endosome/lysosomes as observed for the other lysosomal glycoproteins,

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LAMP-1 and LAMP-2 (Nishimura et al. 2003; Nishimura et al. 2004; Nishimura et al. 2006; Nishimura et al. 2007; Nishimura et al. 2008). In PC9 cell, it is notable that most EGFR is localized within large swollen vacuoles in the perinuclear region of cells, and some EGFR stainings were found to be associated with plasma membrane (Figure 1A). Thus observed large numbers of EGFR-positive large swollen vesicles were costained with LIMPII antibody, however, the localization of these vacuoles appears to be distinct from that of cathepsin D, lysosomal marker protease. These results indicate that major distribution of EGFR would be LIMPII-positive late endosomes in PC 9 cells where complete maturation of lysosomes by fusing with late endosomes does not take place. In contrast to PC9 cells, most EGFR were localized in plasma membrane and intracellular EGFR- and LIMPII-double positive swollen vacuoles as seen in PC9 cells were not observed in QG56 cells (Figure 1C). These findings implicate that intracellular endocytic trafficking of EGFR from plasma membrane through early endosomes toward late endsomes/lysosomes may be impaired in gefitinib-resistant cell line QG56. Secondly, to examine the intracellular distribution of pEGFR in the gefitinib-sensitive NSCLC cell line PC9 (Figure 1B) or the gefitinib-resistant cell line QG56 (Figure 1D), in the absence of EGF stimulation, each cell line was double-labeled either with antibodies specific to pEGFR or with those specific to cathepsin D and LIMPII In PC9 cells (B), it is notable that most pEGFRs were localized within small vesicular structures distributed throughout the cytoplasm, and it is clear that some punctate signals were found in the nucleus (a, d). The immunostaining pattern of pEGFR in QG56 cells (D) was similar to that in PC9 cells. Moreover, pEGFR staining was distributed in the cytosol and in the nucleus: some cytosolic pEGFR was stained diffusely, while no staining was observed in the plasma membrane (g, j). In both PC9 and QG56 cells, the small pEGFR-positive vesicular structures observed in large numbers were not costained with LIMPII or the cathepsin D antibody (c, f, i, l). These results indicate that pEGFR would be mainly distributed in the cytoplasmic vesicles and possibly in early endosomes; however, its distribution was also indicated in the nucleus at the steadystate level without EGF stimulation.

Aggregated Vesicular Structures of Early Endocytic Compartments Are Found to be Overlapped With Sorting Nexin 1 (SNX1) Localized in the Perinuclear Region of Gefitinib-Resistant NSCLC Cell Line It was reported previously that sorting nexin 1 (SNX1), originally identified as a protein that interacts with EGFR (Kurten et al., 1996), is preferentially localized to early endosomes through its phospholipid-binding motif termed the phox homology (PX) domain (Worby and Dixon, 2002). It was also shown that overexpression of SNX1 caused enhanced EGFR degradation and that a deletion mutant of SNX1 blocked EGFR degradation but failed to inhibit receptor endocytosis (Kurten et al., 1996; Zhong et al., 2002). Therefore, it is suggested that SNX1 plays a role in endosome-lysosome trafficking. In the present study, to investigate the intracellular distribution of SNX1 with endocytosed transferrin—a marker of early endosomes in NSCLC cell lines—PC9 or QG56 cells were allowed to internalize Texas red-labeled transferrin for 20 min. After transferrin

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binds to its receptor on the cell surface, it is internalized via clathrin-coated vesicles and is subsequently delivered to the early endosomes. Confocal immunofluorescence microscopy studies revealed that endogenous SNX1 was distributed primarily to punctate vesicles and that it showed considerable overlap with endocytosed transferrin in the cytoplasm of PC9 cells (Figure 2A, h). However, it was also shown that SNX1 staining did not overlap with late endosomes/lysosomes labeled by the LIMPII antibody (Figure 2A, D). In contrast, SNX1 was distributed in the aggregated vesicular structures in the perinuclear region of QG56 cells, and SNX1 staining overlapped with Texas red-transferrin-positive early endosomes (Figure 2B, p). Interestingly, a part of SNX1-positive aggregated vesicles were also colocalized with late endosomes labeled with the LIMPII antibody (Figure 2B, l), therefore indicating that membrane trafficking of EGFR between early endosomes and late endosomes might be considerably suppressed in QG56 cells. However, it was revealed to be normal in PC9 cells. Furthermore, quantitative analysis was performed to determine the amounts of SNX1 that colocalized with LIMPII (Figure 2C) or with endocytosed Texas red-transferrin (data not shown). These results confirm the presence of an aberration in the early endosomes of QG56 cells.

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Ligand-Induced EGFR Endocytosis Operates Normally via the Early/Late Endocytic Pathway in the Gefitinib-Sensitive NSCLC Cell Line, however, Aberrant Endocytosis of EGFR Occurs in the Gefitinib-Resistant NSCLC Cell Line Receptor tyrosine kinases play important roles in cell growth, survival, migration and differentiation. Members of the EGFR play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of receptor tyrosine kinases leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways (Schlessinger, 2004). The ligand-activated receptor tyrosine kinases also undergo rapid endocytosis. The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. These receptors are targeted to the lysosomes for degradation, which terminates receptor signaling. We previously reported the efficient endocytosis of ligand-induced EGFR in human breast cancer cells by employing the double–immunolabeling confocal immunofluorescence microscopy (Nishimura et al., 2004). To follow the endocytic pathway and determine the intracellular fate of internalized labeled ligand, I studied the fate of the internalized labeled EGF in early endosomes or late endosomes/lysosomes in PC9 cells or QG56 cells by confocal immunofluorescence microscopy. To clarify EGFR internalization, I followed the uptake of Texas red-conjugated-EGF with time by each cell lines. The cells were incubated at 370C for 5, 15, and 30 min with Texas red-EGF. The distribution of internalized Texas redEGF, early endosomes stained with anti-transferrin receptor antibody, and late endosomes/lysosomes stained with anti-LIMPII antibody was then assessed by confocal immunofluorescence.

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Figure 2. Endocytosed Texas red-transferrin is downloaded into the SNX1-positive aggregated vesicular structure of early endosomes in the perinuclear region of gefitinib-resistant QG56 cells. The PC9 cells (A), or the QG56 cells (B) were fixed and double-stained for SNX1 (green in b, j) and LIMPII (red in c, k). Superimposed images of SNX1 and LIMPII are shown in d, l. The merged confocal images as yellow color were quantified and presented as the percentage of total amounts of SNX1-positive vesicles per cell in C. The error bar denotes SD. In PC9 cells (A), SNX1-positive small vesicles are not colocalized with LIMPII-positive vesicles (d), however, in QG56 cells (B), part of LIMPII-positive vesicles colocalized with SNX1-positive early endosomes is seen in the vicinity of nuclear region (l). Furthermore, the PC9 cells (A) or QG56 cells (B) were incubated for 20 min with Texas red-labeled transferrin (Tf), and the distribution of early endosomes stained with anti-SNX1 antibody (green in f, n) and the internalized Texas red-transferrin (red in g, o) was analyzed by confocal immunofluorescence microscopy after fixation of each cells. Superimposed images of SNX1 and Texas red-transferrin are shown in h and p. Each cell line was stained with DAPI (blue) to reveal nuclei. It appears that SNX1-positive early endosomes forms large aggregated vesicular structures around the perinuclear region (p) in QG56 cells, and that these newly formed aggregated structures are overlapped well with endocytosed Texas red-transferrin; however no aggregated early endosomal vesicles are not seen in PC9 cells (h). Moreover, part of LIMPII-positive late endosomes/lysosomes are also colocalized with SNX1-positive aggregates (l). (Figure 2 is the synthesis of data previously published by Nishimura et al. in 2008).

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In gefitinib-sensitive cell line PC9, an efficient internalization of Texas red-EGF was observed after 15 min internalization, and part of the internalized Texas red-EGF was already found to be overlapped with the LIMPII-positive late endosomes/lysosomes, and after 30 min internalization, the small punctate vesicles that positively stained for the internalized EGF were revealed to be colocalized well with the LIMPII-positive late endosomes/lysosomes in the perinuclear region (Figure 3A).

Figure 3. Evidence for an efficient endocytosis of ligand-induced EGFR in the gefitinib-sensitive PC9 cells, but for an aberration of EGF-EGFR endocytic traffic out of the early endosomes toward the late endosomes in the gefitinib-resisitant QG56 cells. In A and B, PC9 cells or QG56 cells were incubated at 37oC with Texas red-EGF for 5, 15, or 30 min. The distribution of the internalized Texas red-EGF in early endosomes (stained with anti-transferrin receptor antibody) and the late endosomes/lysosomes (stained with anti-LIMPII antibody) was studied by confocal immunofluuorescence microscopy after fixation of the cells, and superimposed images of EGF and transferrin receptor (TfR) or EGF and LIMPII are shown. The white arrows indicate the colocalization of the EGF- and transferrin receptorpositive structures or the EGF-positive and LIMPII-positive structures, respectively. Bar, 10μm. (Figure 3 is the synthesis of data previously published by Nishimura et al. in 2007).

By contrast, in the gefitinib-resistant cell line QG56, a suppressed internalization of Texas red-EGF was observed in the cells and even after 30 min internalization, the internalized Texas red-EGF-positive punctuate vesicles were found to be accumulated in transferrin receptor-positive early endosomes and no colocalization of Texas red-EGF with LIMPII-positive vesicular structures in the perinuclear region (Figure 3B). These data indicate that the endocytic traffic machinery of EGF-EGFR out of early endosomes toward late endosomes is apparently suppressed in QG56 cells.

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To further investigate EGFR internalization through the endocytic pathway, we followed the uptake of Texas red-conjugated-EGF with time periods up to 60 min by each cell lines. The cells were incubated at 370C for 5, 15, 30, and 60 min with Texas red-EGF. The distribution of internalized Texas red-EGF and early endosomes stained with anti-transferrin receptor antibody or EGFR-positive vacuolar structures (late endosomes) stained with antiEGFR antibody was then assessed by confocal immunofluorescence (Figure 4).

Figure 4. Efficient endocytosis and lysosomal degradation of ligand-induced EGFR via the endocytic pathway in the gefitinib-sensitive PC9 cells, however a suppressed endocytosis and considerable accumulation of EGFR in the early/late endosomal vesicles in the gefitinib-resisitant QG56 cells. In A, PC9 cells were incubated at 37oC with Texas red-EGF for 5, 15, 30, or 60 min. The distribution of the internalized Texas red-EGF in the early endosomes (stained with anti-transferrin receptor antibody) and the late endosomes (stained with anti-EGFR antibody) was studied by confocal immunofluuorescence microscopy after fixation of the cells. The superimposed images of EGF- and transferrin receptor (TfR)-positive structures or of EGF- and EGFR-positive structures are shown. In B, QG56 cells were treated as described above. The white arrow indicates the colocalization of the EGF- and transferrin receptor-positive structures or of the EGF-positive and EGFR-positive structures. Bar, 10μm. (Figure 4 is the synthesis of data previously published by Nishimura et al. in 2007).

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In gefitinib-sensitive cell line PC9, an efficient internalization of Texas red-EGF was observed after 5 min internalization because large amounts of EGF stainings were colocalized with transferrin receptor-positive early endosomes, and after 15 min internalization part of the internalized Texas red-EGF was already found to be overlapped with the EGFR-positive late endosomes. After 30 min internalization, the internalized EGF stainings were revealed to be colocalized well with the EGFR-positive late endosomes, and accumulated within these vesiclular structures in the perinuclear region (Figure 4A), indicating that efficient delivery of endocytosed EGF-EGFR complex from early endosomes to late endosomes. After 60 min, the EGF stainings accumulated within EGFR-positive vesicular structures as observed after 30 min internalization were completely disappeared from the cell, therefore, indicating that EGF-EGFR complex reached lysosomes where extensive degradation for EGF-EGF complex took place after 60 min internalization. These results further confirm that ligand-induced EGFR endocytosis operates normaly in PC9 cells. By contrast, in the gefitinib-resistant cell line QG56, a suppressed internalization of Texas red-EGF was observed in the cells. Even after 30-60 min internalization, Texas redEGF stainings revealed to be overlapped with EGFR-positive vesicular structures, and large amounts of EGF stainings seemed to be accumulated as an aggregated form within early and late endosomal vesicles (Figure 4B). Therefore, these data indicate that an aberration of EGFR endocytosis occurs through the early/late endocytic pathway, and that the delivery of EGFR from late endosomes to lysosomes is also considerably perturbed in QG56 cells. Quantitative analysis was carried out to determine the amounts of transferrin receptorpositive early endosomal marker or EGFR-positive late endosomal marker that colocalized with the endocytosed Texas red-EGF after 60 min internalization in each PC9 or QG56 cell (data not shown). These data indicate efficient endocytosis and delivery of EGFR into lysosomes in PC9 cells, by contrast, aberration of EGFR endocytosis occurs through the early/late endocytic pathway in QG56 cells.

Gefitinib Impedes Endocytosis of Ligand-Induced Phosphorylated EGFR via the Early Endocytic Pathway in NSCLC Cell Lines To further substantiate the detailed mechanisms for endocytosis of the ligand-induced activated form of EGFR, i.e., pEGFR, via the early/late endocytic pathway in both NSCLC cell lines, i.e., PC9 and QG56 cell lines, the cells were incubated with Texas red-EGF in the absence (A, B) or presence (C, D) of gefitinib at 37°C for 5, 10, and 15 min. Confocal immunofluorescence microscopy was then used to assess the distribution of internalized Texas red-EGF and endocytosed vesicles stained with the anti-pEGFR antibody (Figure 5). In the gefitinib-sensitive cell line PC9, efficient internalization of pEGFR was observed after 5 min of internalization, since large amounts of pEGFR staining was observed to have colocalized with Texas red-EGF-positive small endocytic vesicles, presumably early endosomes, in the vicinity of the plasma membrane (Figure 5A, a). Moreover, these vesicles costained with internalized pEGFR and Texas red-EGF were maturated and distributed in the periphery of the nucleus; they showed a gradual increase in size until 15 min of incubation (Figure 5A; b, c). These pEGFR- and EGFR-costained vesicles are considered to be late

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endosomes/lysosomes. This therefore indicates rapid delivery of the endocytosed EGF-EGFR complex from the early endosomes to the late endosomes, after EGF stimulation in PC9 cells (Nishimura et al., 2007).

Figure 5. Evidence for a rapid endocytosis of ligand-induced pEGFR in the gefitinib-sensitive PC9 cells, but for inefficient endocytic traffic of EGF-pEGFR in the gefitinib-resistant QG56 cells. The PC9 (in A and C) or QG56 (in B and D) cells were incubated in the absence (in A and B) or presence (in C and D) of gefitinib at 37oC with Texas red-EGF for 5, 10, or 15 min, and cells were fixed and doublestained for pEGFR (green). Superimposed images of pEGFR and Texas red-EGF are shown. The white arrowheads indicate the colocalization of the pEGFR- and Texas red-EGF-positive vesicular structures, respectively. It is notable that rapid endocytosis of EGF-EGFR occurs in PC9 cells, since large amounts of pEGFR- and Texas red-EGF-costained small vesicular structures appear in the cytoplasm after 5 min incubation (a) and these costained vesicles are increased at 15 min (b, c). By contrast in QG 56 cells, pEGFR stainings are mostly associated with plasma membrane at 10 min (h) and only small amounts of vesicular structures costained for Texas red-EGF and pEGFR appear in the vicinity of plasma membrane after 15 min incubation (i). Also, gefitinib significantly suppresses phosphorylation of EGFR in PC9 and QG 56 cells, and amount of pEGFR stainings are considerably reduced during the incubation (d, e, f, j, k, l). Further, in gefitinb-treated cells, ligand-induced endocytosis of EGFR is delayed via the early/late endocytic pathway and EGF-EGFR is not internalized even after 15 min incubation (d, e, f, j, k, l). Bar, 10μm. (Figure 5 is the synthesis of data previously published by Nishimura et al. in 2008).

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In contrast, in the gefitinib-resistant cell line QG56, internalization of Texas red-EGF was suppressed (Figure 5B). After 5 min of incubation, large amounts of pEGFR-positive vesicular structures were associated with the plasma membrane, and internalization of Texas red-EGF was not observed in the cell. Some pEGFR-positive vesicles overlapping with Texas red-EGF staining were revealed to have accumulated as aggregated structures in the vicinity of the plasma membrane at 15 min of incubation (i). These data indicate that EGF stimulation induces phosphorylation of EGFR in the plasma membrane of QG56 cells; however, internalization of the pEGFR-EGF complex from the plasma membrane to endocytic vacuoles is fairly suppressed in this cell line. This is consistent with our previously reported novel evidence that in QG56 cells, the endocytic machinery of EGFR is basically impaired at the level of the early endocytic pathway (Nishimura et al., 2007). I next examined the effect of gefitinib on the phosphorylation of EGFR and the subsequent internalization of pEGFR in the presence of gefitinib in each cell line for various time periods up to 15 min. Confocal immunofluorescence microscopy was used to assess the distribution of internalized Texas red-EGF and intracellular vesicles stained with the antipEGFR antibody. The results revealed that the gefitinib treatment strongly reduced the phosphorylation level of EGFR and that the endocytosis of EGFR was significantly suppressed in PC9 cells (Figure 5C). Ever after 15 min of internalization, most of the Texas red-EGF remained associated with the plasma membrane of gefitinib-treated PC9 cells instead of being trafficked to the early endosomes (Figure 5C; f). Similarly, the suppression of EGFR phosphorylation was observed in QG56 cells; in most of the Texas red-EGF-stained cells, no internalization of Texas red-EGF staining was observed even after 15 min of incubation, since it remained attached to the plasma membrane (Figure 5D; l). These results indicate that in PC9 cells, gefitinib significantly inhibits the efficient phosphorylation of EGFR and rapid internalization of pEGFR in the early stage of endocytosis, from the plasma membrane to the early endosomes. Further, the suppressive effect of gefitinib on the endocytosis of pEGFR proved to be much stronger in PC9 cells than in QG56 cells, since pEGFR trafficking via the early endocytic pathway is basically perturbed in QG56 cells; however, the pEGFR endocytic pathway is normal in PC9 cells.

Phosphorylated EGFR Is Efficiently Trafficked to Late Ensosmes via Early Endosmes in the Gefitinib-Sensitive NSCLC Cell Line, however an Accumulation of Phosphorylated EGFR Is Observed in the Early Endosomes instead of its Delivery to Late Endosomes in GefitinibResistant NSCLC Cell Line To clarify pEGFR internalization, confluent NSCLC cell lines were cultured in serumfree medium for 3 h, and then EGFR phosphorylation was induced by incubation with EGF (100 ng/ml) for 15 min on ice in RPMI medium. The cells were then rinsed with ice-cold phosphate-buffered saline (PBS), incubated in the presence of Texas red-transferrin in a prewarmed medium, and chased at 37°C for 3, 6, and 15 min. Using confocal immunofluorescence microscopy, I then assessed the intracellular distribution of pEGFR and internalized Texas red-transferrin, an endocytic marker (Figure 6).

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Figure 6. Evidence for an efficient phosphorylation of EGFR and rapid delivery of pEGFR into the early endosomes after EGF stimulation in PC9 cells. The PC9 (A) or QG56 (B) cells stimulated with EGF for 15 min on ice were further incubated at 37oC with Texas red-transferrin (red) for 3, 6, or 15 min, and cells were fixed and double-stained for pEGFR (green). Superimposed images of pEGFR and Texas red-transferrin are shown. Each cell line was stained with DAPI (blue) to reveal nuclei. The white arrows indicate the colocalized early endosomal vesicular structures positive for pEGFR and Texas red-transferrin. In PC9 cells (A), upon EGF stimulation, pEGFR-positive small vesicles colocalized with Texas red transferrin-positive early endosomes appear in the cytoplasm and number of these costained vesicles is gradually increased during incubation up to 15 min (c), indicating that ligand-induced EGFR phosphorylation occurs efficiently in early endosomes or in plasma membrane. By contrast, only small fraction of pEGFR staining associated with early endosomal vesicles is seen in the cytoplasm of QG56 cells even after 15 min incubation (f). Furthermore, the PC9 cells were preincubated with EGF followed by a chase experiment at 37oC for 3, 6, or 15 min as described above, and double-stained for pEGFR (green) or LIMPII (red), and superimposed images of pEGFR and LIMPII are shown in C. The white arrows indicate the colocalized LIMPII-positive late endosomes/lysosomes and pEGFR-positive cytoplasmic vesicular structures. The merged confocal images as yellow color as indicated by white arrows in PC9 cells (i) or in QG56 cells (data not shown) at 15 min incubation were quantified and presented as the percentage of total amounts of LIMPIIpositive vesicles per cell in D. In PC9 cells (A), pEGFR-positive vesicular structures colocalized with LIMPII-positive late endosomes/lysosomes are seen predominantly in the cytoplasm and number of these vesicles is gradually increased up to 15 min during incubation (i), indicating an efficient trafficking of pEGFR to late endosomes/lysosomes in PC9 cells, but not in QG56 cells. These data also support a role for membrane trafficking in pEGFR signaling through the degradative pathway. (Figure 6 is the synthesis of data previously published by Nishimura et al. in 2008).

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The gefitinib-sensitive cell line PC9 showed rapid internalization of pEGFR since pEGFR-positive vesicular structures were observed in the cell at 3 min of internalization; moreover, these pEGFR-positive vesicles were colocalized with transferrin receptor-positive early endosomes (Figure 6A; a), indicating rapid phosphorylation of EGFR and its efficient delivery to early endosomes after EGF stimulation. After 15 min of internalization, an increasing number of pEGFR-positive vesicular structures costained with endocytosed Texas red-transferrin were observed in the vicinity of the nucleus (Figure 6A; c). In contrast, in the gefitinib-resistant cell line QG56, internalization of pEGFR was suppressed (Figure 6B). Even after 15 min of internalization, only a small number of pEGFRpositive vesicles associated with the internalized Texas red-transferrin staining were observed in the perinuclear region of QG56 cells (Figure 6B, f). These data indicate that in QG56 cells, an aberration in pEGFR endocytosis occurs via the early/late endocytic pathway and the delivery of pEGFR from the early endosomes to late endosomes/lysosomes is also considerably perturbed. Quantitative analysis was performed to determine the amount of Texas red-transferrin-positive early endosomal markers (Figure 6D) that colocalized with the endocytosed pEGFR at 3 min of internalization; it was confirmed that rapid delivery of pEGFR to early endosomes is observed to a greater extent in PC9 cells than in QG56 cells. To further examine pEGFR internalization, the intracellular fate of pEGFR that colocalized with the late endosome/lysosome marker stained with the LIMPII antibody was monitored by confocal immunofluorescence microscopy for various time periods upto 15 min by using each cell line. As shown in Figure 6C, in PC9 cells, an increasing number of pEGFR-positive vesicles colocalized with LIMPII-positive late endosomes/lysosomes were observed in the cytoplasm at 15 min of internalization (Figure 6C, i); however, in QG56 cells, no pEGFR-positive vesicular structures overlapping with LIMPII-positive late endosomes/lysosomes were observed (data not shown). We have previously reported that the EGF-EGFR complex associated with the plasma membrane is efficiently endocytosed and translocated to LIMPII-positive late endosomes/lysosomes at 15 min after EGF stimulation in PC9 cells (Nishimura et al., 2006; Nishimura et al., 2007); therefore, the present data showing the efficient trafficking of pEGFR from the plasma membrane via early endosomes to late endosomes within 15 min of EGF stimulation is consistent with that reported previously. These results further confirm that ligand-induced pEGFR endocytosis operates normally in PC9 cells, but pEGFR trafficking is attenuated at the level of the early endocytic pathway in QG56 cells. Quantitative analysis for determining the amounts of LIMPII-positive late endosome/lysosome markers (Figure 6D) that colocalized with the endocytosed pEGFR at 15 min of internalization confirmed the efficient trafficking of pEGFR to late endosomes in PC9 cells.

Discussion In the present study, to investigate the suppression mechanism of gefitinib on EGFR down-regulation from the cell surface and to examine the distinct EGFR down-regulation mechanism between the cell lines most sensitive and resistant to gefitinib, the NSCLC cell line PC9 and the cell line QG56, respectively, I analyzed the ligand-induced endocytosis of

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EGFR and pEGFR with time via the endocytic pathway by confocal immunofluorescence microscopy, using each cell line. I provide evidence that in PC9 cells, the internalized Texas red-EGF-positive small punctate vesicles colocalized with transferrin-positive early endosomes after 5 min of internalization and then overlapped with the LIMPII-positive late endosomes/lysosomes in the perinuclear region after 15-30 min of internalization. This demonstrates that efficient endocytosis of EGFR occurs via the endocytic pathway within the PC9 cells. Moreover, I demonstrated that in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR occurred at 3 min after EGF stimulation, since large amounts of small pEGFR-positive punctate vesicles were colocalized with the internalized Texas redtransferrin-positive early endosomes in the vicinity of the plasma membrane. These internalized pEGFR-positive vesicles were maturated and subsequently trafficked to LIMPIIpositive late endosomes/lysosomes in the periphery of nucleus, along with gradual increases in its size at 15 min after EGF stimulation. These results indicate that efficient membrane trafficking of the EGF-pEGFR complex from early endosomes to late endosomes occurs in PC9 cells, and also suggest that ligand-induced EGFR signaling might operate in the early endosomes/late endosomes via the endocytic pathway. In contrast, an inefficient ligand-induced endocytosis of EGFR was observed in gefitinibresistant QG56 cells. The internalized EGF-EGFR was accumulated in the transferrin receptor-positive early endosomes and EGFR-positive late endosomes even after 60 min of internalization. It was not transported to the lysosomes in QG56 cells, thereby, suggesting that the endocytic traffic via the early/late endosomes/lysosomal pathway would be basically suppressed in QG56 cells. Also I found that in the gefitinib-resistant cell line QG56, internalization of pEGFR-positive vesicles was suppressed (Figure 5). After 5 min of incubation, large amounts of pEGFR-positive vesicular structures were associated with the plasma membrane, and internalization of Texas red-EGF was not observed in the cell. Some pEGFR-positive vesicles overlapping with Texas red-EGF staining were revealed to have accumulated as aggregated structures in the vicinity of the plasma membrane at 15 min of incubation (Figure 6). These data indicate that EGF stimulation induces phosphorylation of EGFR in the plasma membrane of QG56 cells; however, internalization of the pEGFR-EGF complex from the plasma membrane to endocytic vacuoles is fairly suppressed in this cell line. This is consistent with our recently reported novel evidence that in QG56 cells, the endocytic machinery of EGFR is basically impaired at the level of the early endocytic pathway (Nishimura et al., 2007; Nishimura et al., 2008). Regarding the suppressive effect of gefitinib on the endocytosis of EGFR and pEGFR in NSCLC cell lines, I found that gefitinib treatment on PC9 cells showed a strong inhibitory effect on the endocytosis of ligand-induced EGFR and the internalization rate of the EGFEGFR complex from the plasma membrane via the early endosomes to the late endosomes/lysosomes is considerably delayed. This confirms that gefitinib efficiently inhibits the internalization of EGFR via the early endocytic pathway in PC9 cells. The gefitinib treatment strongly reduced the phosphorylation level of EGFR and that the endocytosis of EGFR was significantly suppressed in PC9 cells, thereby endocytic trafficking of EGFR was significantly impaired and unphosphorylated EGFR remained associated with the plasma membrane. Therefore, the suppressive effect of gefitinib on the phosphorylation level of

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EGFR was demonstrated in PC9 cells. However, internalization of pEGFR was basically suppressed in QG56 cells; therefore, the inhibitory effect of gefitinib on pEGFR trafficking is limited. Collectively, my data indicate that an aberration in pEGFR endocytosis occurs via the early/late endocytic pathway and that the delivery of pEGFR from the early endosomes to the late endosomes/ysosomes is considerably perturbed in QG56 cells. Based on these findings, I postulate that efficient endocytosis of ligand-induced EGFR is closely related to EGFR-tyrosine kinase inhibitor sensitivity of human lung cancer cell lines. With regard to the endocytosis of EGFR via the early endocytic pathway in gefitinibresistant cell lines, for QG56 cells, we reported novel evidence regarding the accumulation of internalized EGF-EGFR in the early endosomes, instead of its trafficking to the lysosomes; this evidence suggests that the endocytic machinery of EGFR might be considerably impaired at the level of the early/late endosomes (Nishimura et al., 2007). To further substantiate this, I examined the intracellular distribution of SNX1 along with endocytosed transferrin in NSCLC cell lines. SNX1 is a mammalian homologue of yeast Vps5p, which recognizes the lysosomal targeting code of EGFR and participates in lysosomal trafficking of the receptor (Kurten et al., 1996; Chin et al., 2001); SNX1 is preferentially localized to early endosomes through its phospholipid-binding motif termed the PX domain (Worby and Dixon, 2002). In the present study, using confocal immunofluorescence microscopy, we demonstrated novel evidence that in QG56 cells, early endosomes labeled with endocytosed Texas red-transferrin formed an aggregated vesicular structure distributed in the perinuclear region and SNX1 distribution overlapped with these aggregated early endosomal vesicles. Surprisingly, I found that a part of SNX1-positive aggregates was also colocalized with late endosomes labeled with the LIMPII antibody, implying that membrane trafficking of EGFR from the early endosomes to late endosomes might be significantly impaired in QG56 cells; however, no such accumulation was noted in PC9 cells. Therefore, I speculate that impairment of SNX1 trafficking might cause the perturbation of EGFR endocytosis, which then leads to the acquisition of gefitinib-resistance in NSCLC cell lines. I further detected clear nuclear staining of pEGFR in PC9 and QG56 cells, although considerable amounts of pEGFR were distributed in small cytoplasmic punctate vesicles. Since we recently demonstrated that in PC9 cells, most EGFR is localized in the plasma membrane and in LIMPII-positive swollen vacuoles, i.e., late endosomes (Nishimura et al., 2007), it is interesting to note that a part of pEGFR is already localized in the nucleus even in the absence of EGF stimulation. These results imply that pEGFR might be trafficked into the nucleus through the conventional nuclear importing system associated with the nuclear pore complex (that is, the Ran/importin pathway), and it might then operate as a transcription factor. In fact, it has recently been reported that nuclear localization of EGFR is detected in the highly proliferating state of human cancer tissues in vivo and human breast cancer cell lines in vitro, supporting the close correlation between nuclear EGFR and tumor tissues with high proliferation (Hoshino et al., 2007; Lin et al., 2001; Lo et al., 2006). It was also shown that nuclear EGFR levels were increased on treatment with EGF and that the EGFR which accumulated in the nucleus was highly tyrosine phosphorylated; it was further demonstrated that nuclear EGFR acts as a transcription factor for activating gene expression of cyclin D1, a well-known cell growth-promoting factor (Lin et al., 2001). Therefore, further studies with

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respect to the functions of these cell growth nuclear receptors should be conducted to create new avenues in the field of receptor signaling. It has been previously reported that gefitinib-sensitive lung tumors were found to express EGFR variants with a higher sensitivity for the drug than the wild-type receptor (Lynch et al., 2004; Paez et al., 2004). Since it has been known that PC9 cells express EGFR mutant variants and QG56 cells express wild-type EGFR (Ono et al., 2004), it can be assumed that the drug suppresses the endocytosis of EGFR more strongly in gefitinib-sensitive PC9 cells than in gefitinib-resistant QG56 cells. Recently, cellular targets of gefitinib were identified in HeLa cells by a proteomic method in which gefitinib could interact with more than 20 previously unknown kinase targets (Brehmer et al., 2005). It was further found that gefitinib could interact with the serine/threonine kinase, cyclin G-associated kinase (GAK), which has been recently indicated to act as a negative regulator of EGFR signaling (Zhang et al., 2004), thereby proposing that the gefitinib-mediated inactivation of a negative regulator would antagonize the inhibitory effect of the drug on EGFR signaling. It is important to further analyze the mechanism by which the interaction of EGFR and other cellular proteins with gefitinib might regulate the process of EGFR down regulation in NSCLC cell lines. Further investigation of the endocytosis machinery in gefitinib-sensitive or gefitinib-resistant NSCLC cell lines could be useful for clarifying the effectiveness of gefitinib.

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Conclusion I found novel evidence for efficient EGFR phosphorylation and rapid endocytic delivery of pEGFR from plasma membrane to early endosomes/late endosomes/lysosomes in the gefitinib-sensitive cell line PC9 cells after EGF stimulation; however, pEGFR trafficking via the early endocytic pathway was basically impaired in the gefitinib-resistant cell line QG56 cells, since internalized pEGFR was accumulated in the aggregated vesicular structures of early endosomes associated with SNX1 instead of its trafficking to late endosomes/lysosomes. Therefore, I postulate that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib resistance in QG56 cells. Thus, impairment of protein function such as SNX1 regulating EGFR trafficking in the early endocytic pathway might cause the perturbation of EGFR endocytosis, which then leads to the acquisition of gefitinib resistance in NSCLC cell lines.

References Arteaga, C.L. and Johnson, D.H. (2001)Tyrosine kinase inhibitors-ZD1839 (Iressa). Curr. Opin. Oncol. 13, 491-498. Barker, A.J., Gibson, K.H., Grundy, W., Godfrey, A.A., Barlow, J.J., Healy, M.P., Woodburn, J.R., Ashton, S.E., Curry, B.J., Scarlett, L., Henthorn, L., and Richards, L. (2001) Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg. Med. Chem. Lett. 11, 1911-1914.

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Baselga, J. and Averbuch, S.D. (2000) ZD1839 (‘Iressa’) as an anticancer agent. Drugs 60, Suppl 1: 33-40; discussion 41-42. de Bono, J.S. and Rowinsky, E.K. (2002) The ErbB receptor family: a therapeutic target for cancer. Trends Mol. Med. 8, 19–26. Brehmer, D., Greff, Z., Godl, K., Blencke, S., Kurtenbach, A., Weber, M., Muller, S., Klebl, B., Cotten, M., Keri, G., Wissing, J., and Daub, H. (2005) Cellular targets of gefitinib. Cancer Res. 65, 379-382. Chin , L-S., Raynor, M. C., Wei, X., Chen, H-Q., and Li, L. (2001) Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J. Biol. Chem. 276, 7069-7078. Hirata, A., Uehara, H., Izumi, K., Naito, S., Kuwano, M., and Ono, M. (2004) Direct inhibition of EGF receptor activation in vascular endothelial cells by gefitinib ('Iressa', ZD1839). Cancer Sci. 95, 614-618. Hoshino, M., Fukui, H., Ono, Y., Sekikawa, A., Ichikawa, K., Tomita, S., Imai, Y., Imura, J., Hiraishi, H., and Fujimori, T. (2007) Nuclear expression of phosphorylated EGFR is associated with poor prognosis of patients with esophageal squamous cell carcinoma. Pathobiology 74, 15-21. Kroemer, G. and Jaattela, M. (2005) Lysosomes and autophagy in cell death control. Nature Rev.Cancer 5, 886-897. Kurten, R. C., Cadena, D. L., and Gill, G.N. (1996) Enhanced degradation of EGF receptors by a sorting nexin, SNX1. Science 272, 1008 –1010. Lin, S.Y., Makino, K., Xia, W., Matin, A., Wen, Y., Kwong, K.Y., Bourguignon, L., and Hung, M.C. (2001) Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat. Cell Biol. 3, 802-808. Lo, H.W. and Hung, M.C. (2006) Nuclear EGFR signaling network in cancer; linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br. J. Cancer 94, 184-188. Lynch, T.J., Bell, D.W., Sordella, R., Gurubhagavatula, S., Okimoto, R.A., Brannigan, B.W., Harris, P.L., Haserlat, S.M., Supko, J.G., Haluska, F.G., Louis, D.N., Christiani, D.C., Settleman, J., and Haber, D.A. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non small-cell lung cancer to gefitinib. N. Eng. J. Med. 350, 2129-2139. Mendelsohn, J. and Baserga, J. (2000) The EGF receptor family as targets for cancer therapy. Oncogene, 19, 6550–6565. Mohamed, M.M. and Sloane, B.F. (2006) Cysteine cathepsins: multifunctional enzyme in cancer. Nature Rev. Cancer 6, 764-775. Nishimura, Y., Itoh, K., Yoshioka, K., Tokuda, K., and Himeno, M. (2003) Overexpression of ROCK in human breast cancer cells. Evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. Pathol. Oncol. Res. 9: 83-95. Nishimura, Y., Yoshioka, K., Bernard, O., Himeno, M., and Itoh, K. (2004) LIM kinase 1: evidence for a role in the regulation of intracellular vesicle trafficking of lysosomes and endosomes in human breast cancer cells. Eur. J. Cell Biol. 34, 189-213.

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Nishimura, Y., Yoshioka, K., Bernard, O., Bereczky, B., and Itoh, K. (2006). A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells. Histochem. Cell Biol. 126, 627-638. Nishimura, Y., Bereczky, B., and Ono, M. (2007) The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines. Histochem. Cell Biol. 127, 541-553. Nishimura, Y., Yoshioka, K., Bereczky, B., and Itoh, K. (2008) Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line. Molecular Cancer 7:42, 1-13. Ono, M., Hirata, A., Kometani, T., Miyagawa, M., Ueda, S., Kinoshita, H., Fujii, T., and Kuwano, M. (2004) Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the EGF receptor/extracellular signalregulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol. Cancer Ther. 3, 465-472. Paez, J.G., Jänne, P.A., Lee, J.C., Tracy, S., Greulich, H., Gabriel, S., Herman, P., Kaye, F.J., Lindeman, N., Boggon, T.J., Naoki, K., Sasaki, H., Fujii, Y., Eck, M.J., Sellers, W.R., Johnson, B.E., Meyerson, M. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 1497-1500. Schlessinger, J. (2004) Common and distinct elements in cellular signaling via EGF and FGF receptors. Science 306, 1506-1507. Sirotnak, F.M., Zakowski, M.F., Miller, V.A., Scher, H.I., and Kris, M.G. (2000) Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin. Cancer Res. 6, 4885-4892. Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61. 203–212. Woodburn, J.R. (1999) The epidermal growth factor receptor and its inhibition in cancer therapy. Pharmacol. Ther. 82, 241-250. Worby, C. A. and Dixon, J. E. (2002) Sorting out the cellular function of sorting nexins. Nat. Rev. Mol. Cell Biol. 3, 919-931. Yarden, Y. (2001) The EGFR family and its ligands in human cancer signaling mechanisms and therapeutic opportunities. Eur. J.Cancer 37, 3-8. Zhang, L., Gjoerup, O., and Roberts, T.M. (2004) The serine/threonine kinase cyclin Gassociated kinase regulates epidermal growth factor. Proc.Natl.Acad.Sci.USA 101,1029610301. Zhong, Q., Lasar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S.D., and Gill, G. N. (2002) Endosomal localization and function of sorting nexin 1. Proc. Natl. Acad. Sci. USA 99, 67676772.

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In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter VI

Mechanisms of Resistance to EGF Receptor-Tyrosine Kinase Inhibitor in NSCLC Cell Lines: Gefitinib Sensitivity is Closely Correlated with LigandInduced Endocytosis of Phosphorylated EGF Receptor

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1

Yukio Nishimura*1, Kiyoko Yoshioka2 and Kazuyuki Itoh2 Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2 Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan

Abstract Gefitinib is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that functions by competing with ATP for binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells. It has exhibited significant anti-tumor activity against a broad range of mouse tumor xenograft models in vivo and non-small cell lung cancer (NSCLC) cell lines in vitro. We recently demonstrated that gefitinibsensitive NSCLC cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. In contrast, gefitinib-resistant NSCLC cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. * Corresponding author, E-mail: Yukio Nishimura* - [email protected], Tel & Fax: +81-92-6426616

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Yukio Nishimura, Kiyoko Yoshioka and Kazuyuki Itoh To further investigate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) using confocal immunofluorescence microscopy in the absence or presence of gefitinib. In the gefitinib-sensitive PC9 cells and the gefitinib-resistant QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures were not colocalized with late endosomes/lysosomes, but spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Moreover, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Taken together, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early/late endocytic pathway might confer gefitinib-resistance in QG56 cells.

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Introduction The epidermal growth factor receptor (EGFR) is a prototypical member of the ErbB family of tyrosine kinases and plays an important role in the pathogenesis of different tumors [1, 2]. Each EGFR comprises an extracellular binding domain and a cytoplasmic domain with tyrosine kinase activity [3]. After EGF stimulation, the EGFR is dimerized and the intracellular tyrosine kinase region is activated, causing receptor tyrosine autophosphorylation and transphosphorylation of another receptor monomer [4]. These events lead to the recruitment and phosphorylation of several intracellular substrates and the subsequent transmission of extracellular signals to the nucleus via an intracellular signaling network [4, 5]. Gefitinib (Iressa, ZD1839) is a selective EGFR tyrosine kinase inhibitor that functions by competing with ATP for binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells [6-9]. Gefitinib has been reported to show significant antitumor activity against a broad range of mouse tumor xenograft models in vivo [10] and tumor cell lines in vitro [11]. Moreover, recent study demonstrated that the PC9 cell line, one of the non-small cell lung cancer (NSCLC) cell lines, was most sensitive to the effect of gefitinib as compared with other NSCLC cell lines when assayed under basal growth conditions for EGFR phosphorylation and activation of EGFR downstream effectors such as AKT and those in the ERK1/2 pathway, which are required for its survival and proliferation [11]. This observation

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indicates that the mechanism underlying the sensitivity of the EGFR pathway could be useful in predicting the potential effectiveness of gefitinib in NSCLC patients. It has been established that the endocytosis of EGFR has served as a model for studying ligand-induced, receptor-mediated endocytosis. Upon EGF binding, EGF-EGFR complexes are internalized and transported via clathrin-coated vesicles to early endosomes. EGFR then recruits and phosphorylates signaling molecules, leading to the activation of a MAPK-signal transduction cascade—an important mechanism for regulating cell growth [12]. EGF-EGFR complexes are successively degraded upon delivery to the lysosomes to cease intracellular EGFR signaling via endocytosis; this process is known as receptor down-regulation. Thereby, endocytosis of EGF-pEGFR complex is closely related with attenuation of intracellular pEGFR signaling. Regarding the sensitivity of gefitinib on EGFR endocytosis, we have recently investigated the effect of gefitinib on the EGFR endocytosis via the endocytic pathway, and we have examined the endocytosis of Texas red-EGF in the absence or presence of gefitinib in both the gefitinib-sensitive NSCLC cell line PC9 and the gefitinibresistant cell line QG56, and then assessed the endocytic pathway of internalized Texas redEGF by using confocal immunofluorescence microscopy [13]. We demonstrated that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to the late endosomes/lysosomes does occur in the gefitinib-resistant QG56 cell line, whereas endocytosis of EGFR is normal in gefitinib-sensitive PC9 cells [13], suggesting that a different unknown down-regulation mechanism operates in each cell type. We therefore assume that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. In the present paper, to further investigate the relationship of EGFR endocytosis and EGFR signaling, we have used confocal immunofluorescence microscopy to substantiate the detailed mechanisms for endocytosis of the ligand-induced activated form of EGFR, i.e., phosphorylated EGFR (pEGFR), via the early endosome/late endosome/lysosome endocytic pathway in both NSCLC cell lines, namely, the PC9 and QG56. We herein report novel data regarding the occurrence of rapid EGFR phosphorylation and endocytic delivery of pEGFR from early endosomes to late endosomes/lysosomes in PC9 cells after EGF stimulation; however, endocytosis of pEGFR is significantly perturbed via the early/late endocytic pathway in QG56 cells.

Results Phosphorylated EGFR is Localized in the Cytosol and the Nucleus in Gefitinib-Sensitive or Gefitinib-Resistant NSCLC Cell Lines We firstly examined the intracellular distribution of pEGFR in the gefitinib-sensitive NSCLC cell line PC9 or the gefitinib-resistant cell line QG56, in the absence of EGF stimulation. Each cell line was double-labeled either with antibodies specific to pEGFR or with those specific to cathepsin D and lysosomal integral membrane protein(LIMPII) (Fig. 1). In order to determine the intracellular distribution of late endosomes/lysosomes, we used antibodies specific to lysosomal aspartic protease cathepsin D or LIMPII/lysosomal

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glycoprotein 85 (LGP85), since these proteins are distributed within endocytic organelles and are at the highest concentration in the late endosomes/lysosomes, as observed for other lysosomal glycoproteins, namely, LAMP-1 and LAMP-2 [14-17].

Figure 1. Intracellular distribution of pEGFR in the gefitinib-sensitive or gefitinib-resistant NSCLC cell lines. The gefitinib-sensitive cell line, PC9 or the gefitinib-resistant cell line, QG56 was fixed and double-stained for LIMPII (red in A) or cathepsin D (red in B) and pEGFR (green) as described in the Materials and methods. Also superimposed images of cathepsin D or LIMPII and pEGFR are shown. In both PC9 and QG56 cells, it is notable that pEGFR-positive small punctate vesicles are spreading in the cytoplasm and these vesicles are not colocalized with the LIMPII-, or cathepsin D-positive vesicular structures, and also pEGFR-positive punctate stainings are clearly seen in the nucleus. (Figure 1 is the syntheses of data published previously by Nishimura et al. in 2008)

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In PC9 cells, most pEGFRs were localized within small vesicular structures distributed throughout the cytoplasm, and it is interesting to note that many punctate signals were found in the nucleus. The immunostaining pattern of pEGFR in QG56 cells was similar to that in PC9 cells. Moreover, some cytosolic pEGFR was stained diffusely, while no staining was observed in the plasma membrane. In both PC9 and QG56 cells, the small pEGFR-positive vesicular structures observed in the cytosol were not overlapped with cathepsin D or LIMPII antibody. These results indicate that pEGFR is mainly distributed in the cytoplasmic vesicles, possibly in early endosomes/late endosomes; however, its localization was also indicated in the nucleus at the steady-state level without EGF stimulation.

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Early Endosomes Forms Aggregated Vesicular Structures which are Associated with Sorting Nexin 1 (SNX1) in Gefitinib-Resistant NSCLC Cell Line It was reported that SNX1, originally identified as a protein that interacts with EGFR [18], is preferentially distributed in the early endosomes through its phospholipid-binding motif termed the phox homology (PX) domain [19, 20]. Thereby, we next investigated the intracellular distribution of SNX1 with endocytosed transferrin—a marker of early endosomes in NSCLC cell lines—PC9 or QG56 cells. Each cell was allowed to internalize Texas red-labeled transferrin for 20 min. Transferrin binds to its receptor on the cell surface, then it is internalized via clathrin-coated vesicles and is subsequently delivered to the early endosomes. Each cell line was fixed, labeled with antibody specific to SNX1 as described in the Materials and methods, and analyzed the cellular distribution of SNX1 by confocal immunofluorescence microscopy. As shown in Fig. 2, it revealed that endogenous SNX1 was distributed primarily to punctate vesicles and that it showed considerable overlap with endocytosed transferrin in the cytoplasm of PC9 cells (Fig. 2A). On the other hand, SNX1 staining did not overlap with late endosomes/lysosomes labeled by the LIMPII antibody (Fig. 2A). In contrast, SNX1 was mainly distributed in the aggregated vesicles in the perinuclear region of QG56 cells, and SNX1 staining overlapped with Texas red-transferrin-positive early endosomes (Fig. 2B). Furthermore, a part of SNX1-positive aggregated vesicles were colocalized with LIMPIIpositive late endosomes (Fig. 2B). These results indicate that membrane trafficking of EGFR between early endosomes and late endosomes was considerably impaired in QG56 cells, while it was found to be normal in PC9 cells. Quantitative analysis was performed to determine the amounts of SNX1 that colocalized with LIMPII (Fig. 2C) or with endocytosed Texas red-transferrin (data not shown). These results further confirm an aberration of early endosomal vesicular trafficking in QG56 cells.

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Figure 2. Texas red-labeled transferrin is internalized and downloaded into the SNX1-positive aggregated vesicular structure of early endosomes in the perinuclear region of gefitinib-resistant QG56 cells. The PC9 cells (A), or the QG56 cells (B) were fixed and double-stained for SNX1 (green) and LIMPII (red) as described in the Materials and methods. Superimposed images of SNX1 and LIMPII are shown. Each cell line was stained with DAPI (blue) to reveal nuclei. The merged confocal images as yellow color were quantified and presented as the percentage of total amounts of SNX1-positive vesicles per cell in C. The error bar denotes SD. In PC9 cells (A), SNX1-positive small vesicles are not colocalized with LIMPII-positive vesicles, however, in QG56 cells (B), part of LIMPII-positive vesicles colocalized with SNX1-positive early endosomes is seen in the perinuclear region as indicated by white arrows. Furthermore, superimposed images of SNX1 and the internalized Texas red-transferrin in the PC9 cells and the QG56 cells show that the internalized Texas red-transferrin is overlapped well with the SNX1 staining as indicated by white arrowheads in (B). Note that SNX1-positive early endosomes form large aggregated vesicular structures in the perinuclear region in QG56 cells, and that these aggregated structures are overlapped with Texas red-transferrin (white arrowheads in B) or with LIMPII (white arrows in B) ; however no aggregated vesicles are seen in PC9 cells. (Figure 2 is the syntheses of data published previously by Nishimura et al. in 2008)

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Rapid Endocytosis of Phosphorylated EGFR via the Early/Late Endocytic Pathway in the Gefitinib-Sensitive NSCLC Cell Line Ligand-induced activation of receptor tyrosine kinases leads to the assembly of signaling protein complexes, followed by subsequent activation of downstream signaling pathways, and then the endocytosed receptors undergo a sorting process that determines the fate of the receptor and signal intensity [12]. These receptors are targeted to the lysosomes for degradation—a process that terminates receptor signaling [21]. Next, to clarify EGFR internalization for each cell line, we substantiated the detailed mechanisms for endocytosis of the ligand-induced activated form of EGFR, i.e., pEGFR, via the early/late endocytic pathway in both NSCLC cell lines, i.e., PC9 and QG56 cell lines. The cells were incubated with Texas red-EGF in the absence or presence of gefitinib at 37°C for 5, 10, and 15 min. Confocal immunofluorescence microscopy was then used to analyze the distribution of internalized Texas red-EGF and endocytosed vesicles stained with the antipEGFR antibody (Fig. 3). In the gefitinib-sensitive PC9 cells, rapid internalization of pEGFR was seen after 5 min of internalization, since large amounts of pEGFR staining was observed to have colocalized with Texas red-EGF-positive small endocytic vesicles, presumably early endosomes, in the periphery of the plasma membrane (Fig.3 A). These vesicles costained with internalized pEGFR and Texas red-EGF showed a gradual increase in size until 15 min of incubation and distributed in the perinuclear region. These pEGFR- and EGFR-positive vesicles are considered to be late endosomes/lysosomes. Thereby, this observation indicates EGFdependent rapid delivery of the endocytosed EGF-EGFR complex from the early endosomes to the late endosomes in gefitinib-sensitive cell line. In contrast, in the gefitinib-resistant cell line QG56, endocytosis of Texas red-EGF was suppressed (Fig. 3 B). After 5 min of incubation, considerable amounts of pEGFR-positive vesicular structures were associated with the plasma membrane, and internalization of Texas red-EGF was not observed in the cell. Some pEGFR-positive vesicles overlapping with Texas red-EGF staining were revealed to have accumulated as aggregated structures in the vicinity of the plasma membrane at 15 min of incubation. These data imply that EGF stimulation induces phosphorylation of EGFR in the plasma membrane of QG56 cells; however, internalization of the EGF-pEGFR complex from the plasma membrane to endocytic vacuoles is suppressed in this cell line. This is consistent with our previously reported novel evidence that in QG56 cells, the endocytosis of EGFR is basically impaired via the early endocytic pathway [13]. It has been reported that gefitinib is a selective EGFR tyrosine kinase inhibitor that functions by competing with ATP for binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells [6-9]. We have recently demonstrated by using confocal immunofluorescence microscopy that gefitinib significantly inhibited the efficient internalization rate of EGFR in the early stage of endocytosis, from the plasma membrane to the early endosomes in PC9 cells; furthermore, we also indicated that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger in PC9 cells than in QG56 cells [13].

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Figure 3. Evidence for a rapid endocytosis of ligand-induced pEGFR in the gefitinib-sensitive PC9 cells, but inefficient endocytic traffic of EGF-pEGFR in the gefitinib-resisitant QG56 cells. The PC9 (A) or QG56 (B) cells were incubated in the absence or presence of gefitinib at 37oC with Texas redEGF for 5, 10, or 15 min, and cells were fixed and double-stained for pEGFR (green) as described in the Materials and methods. Superimposed images of pEGFR and Texas red-EGF are shown. The white arrowheads indicate the colocalization of the pEGFR-positive vesicles and Texas red-EGF-positive vesicular structures. It is notable that rapid endocytosis of EGF-EGFR occurs in PC9 cells, since large amounts of pEGFR-positive small vesicles co-stained with Texas red-EGF appear in the cytoplasm after 5 min incubation and these co-stained vesicles are increased at 15 min. By contrast, in QG 56 cells, pEGFR stainings are mostly associated with plasma membrane even after 15 min incubation. Further, gefitinib significantly suppresses phosphorylation of EGFR in NSCLC cell lines and amount of pEGFR stainings are considerably reduced during the incubation. (Figure 3 is the syntheses of data published previously by Nishimura et al. in 2008)

To further substantiate the effect of gefitinib on the EGFR down-regulation pathway, we examined the effect of gefitinib on the phosphorylation of EGFR and the subsequent endocytosis of pEGFR in the presence of gefitinib in each cell line for various time periods up to 15 min. Confocal immunofluorescence microscopy was used to assess the distribution of internalized Texas red-EGF and intracellular vesicles stained with the anti-pEGFR antibody. We demonstrated that the gefitinib strongly reduced the phosphorylation level of

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EGFR and that the endocytosis of EGFR was significantly suppressed in PC9 cells (Fig. 3A). Most of the Texas red-EGF remained associated with the plasma membrane of gefitinibtreated PC9 cells instead of being trafficked to the early endosomes even after 15 min of internalization. Similarly, the suppression of EGFR phosphorylation was observed in QG56 cells; in most of the Texas red-EGF-stained cells, no internalization of Texas red-EGF staining was observed even after 15 min of incubation, and it remained attached to the plasma membrane (Fig. 3 B). These results indicate that gefitinib significantly inhibits the efficient phosphorylation of EGFR and rapid internalization of pEGFR in the early stage of endocytosis from the plasma membrane to the early endosomes in PC9 cells. It was also suggested that the suppressive effect of gefitinib on the endocytosis of pEGFR proved to be much stronger in PC9 cells than in QG56 cells, since pEGFR trafficking via the early/late endocytic pathway is basically impaired in QG56 cells; however, the pEGFR endocytic pathway is normal in PC9 cells.

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Phosphorylated EGFR is Efficiently Endocytosed and Trafficked to Late Endosomes/Lysosomes in the Gefitinib-Sensitive NSCLC Cell Line Next we investigated the intracellular distribution of pEGFR and internalized Texas redtransferrin, an endocytic marker, by using confocal immunofluorescence microscopy (Fig. 4). To clarify pEGFR internalization, confluent NSCLC cell lines were cultured in serum-free medium for 3 h, and then EGFR phosphorylation was induced by incubation with EGF (100 ng/ml) for 15 min on ice in a binding medium (1 mg/ml bovine serum albumin (BSA) in RPMI medium). The cells were then rinsed with ice-cold phosphate-buffered saline (PBS), incubated in the presence of Texas red-transferrin in a prewarmed medium, and chased at 37°C for 3, 6, and 15 min. The gefitinib-sensitive cell line PC9 showed rapid internalization of pEGFR since pEGFR-positive vesicular structures were observed in the cell at 3 min of internalization; moreover, these pEGFR-positive vesicles were colocalized with the internalized transferrinpositive early endosomes (data not shown), suggesting EGF-stimulated rapid phosphorylation of EGFR and its efficient delivery to early endosomes. After 15 min of internalization, an increasing number of pEGFR-positive vesicular structures costained with endocytosed Texas red-transferrin were observed in the vicinity of the nucleus (Fig. 4 A). In contrast, the internalization of pEGFR was suppressed in the gefitinib-resistant cell line QG56. Even after 15 min of internalization, only a small number of pEGFR-positive vesicles colocalized with the internalized Texas red-transferrin staining were seen in the perinuclear region of QG56 cells (Fig. 4 A). These data indicate that in the gefitinib-resistant QG56 cells, an aberration in pEGFR endocytosis occurs via the early/late endocytic pathway and the delivery of pEGFR from the early endosomes to late endosomes/lysosomes is considerably perturbed. Quantitative analysis was carried out to determine the amount of Texas red-transferrinpositive early endosomal markers that colocalized with the endocytosed pEGFR at 3 min of internalization (data not shown); we confirmed that efficient delivery of pEGFR to early endosomes is observed to a greater extent in the gefitinib-senseitive PC9 cells than in the gefitinib-resistant QG56 cells.

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Figure 4. Evidence for an efficient phosphorylation of EGFR and rapid delivery of pEGFR into the early endosomes after EGF stimulation in PC9 cells. In A, the PC9 or QG56 cells stimulated with EGF for 15 min on ice were further incubated at 37oC with Texas red-transferrin (red) for 15 min, and cells were fixed and double-stained for pEGFR (green) as described in the Materials and methods. Superimposed images of pEGFR and Texas red-transferrin are shown. In B, each cell line was incubated at 37oC for 15 min after EGF stimulation for 15 min on ice as described in the Materials and methods. The cells were then fixed and double-stained for pEGFR (green) or LIMPII (red). Superimposed images of pEGFR and LIMPII are shown. Each cell line was stained with DAPI (blue) to reveal nuclei. The white arrows indicate the colocalized early endosomal vesicular structures positive for pEGFR and Texas red-transferrin (A) or for pEGFR and LIMPII (B). The merged confocal images as yellow color as indicated by white arrows at 15 min incubation (B) were quantified and presented as the percentage of total amounts of LIMPII-positive vesicles per cell in C. It is notable that an efficient trafficking of pEGFR to late endosomes is seen in PC9 cells, but is not observed in QG56 cells.

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We further examined the internalization of pEGFR by using confocal immunofluorescence microscopy. The intracellular fate of pEGFR that colocalized with the late endosome/lysosome marker stained with the LIMPII antibody was monitored for various time periods up to 15 min by using each cell line. In the gefitinib-sensitive PC9 cells, an increasing number of pEGFR-positive vesicles that colocalized with LIMPII-positive late endosomes/lysosomes were observed in the cytoplasm after 15 min of internalization (Fig. 4 B). We previously reported that the EGFR associated with the plasma membrane is efficiently endocytosed and transported to LIMPII-positive late endosomes/lysosomes at 15 min after EGF stimulation in PC9 cells [13]; therefore, our data showing the efficient trafficking of pEGFR from the plasma membrane via early endosomes to late endosomes within 15 min of EGF stimulation is consistent with that reported previously. In contrast, in the gefitinib-resistant QG56 cells, no pEGFR-positive vesicular structures were colocalized with LIMPII-positive late endosomes/lysosomes (Fig. 4 B). Thereby, we confirm that ligandinduced pEGFR endocytosis operates normally in PC9 cells, but pEGFR trafficking is impaired via the early/late endocytic pathway in QG56 cells. Quantitative analysis was carried out to determine the amounts of LIMPII-positive late endosome/lysosome markers (Fig. 4 C) that colocalized with the endocytosed pEGFR at 15 min of internalization; we confirmed the efficient transport of pEGFR to late endosomes in PC9 cells and suppression of endocytic translocation of pEGFR in QG56 cells.

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Discussion We herein demonstrated that the efficient phosphorylation of EGFR and rapid internalization of pEGFR occurred after EGF stimulation in the gefitinib-sensitive cell line PC9, since large amounts of small pEGFR-positive punctate vesicles were colocalized with the internalized Texas red-transferrin-positive early endosomes in the vicinity of the plasma membrane at 3 min after EGF stimulation. These internalized pEGFR-positive vesicles were subsequently trafficked to LIMPII-positive late endosomes/lysosomes in the periphery of nucleus, along with gradual increases in its size at 15 min after EGF stimulation. These results indicate that efficient membrane trafficking of the EGF-pEGFR complex from early endosomes to late endosomes operates in PC9 cells, and also suggest that EGF-induced EGFR signaling might occur efficiently via the early/late endocytic pathway. Moreover, in the PC9 cells-treated with gefitinib, endocytic trafficking of EGFR was significantly impaired, and unphosphorylated EGFR remained associated with the plasma membrane. Thereby, we demonstrated the suppressive effect of gefitinib on the phosphorylation level of EGFR in PC9 cells. In contrast, in the gefitinib-resistant QG56 cells, internalization of pEGFR was basically suppressed; therefore, the inhibitory effect of gefitinib on pEGFR trafficking is limited. Taken together, we indicate that an aberration in pEGFR endocytosis occurs via the early/late endocytic pathway and that the delivery of pEGFR from the early endosomes to the late endosomes/ysosomes is considerably perturbed in QG56 cells. Based on these findings, we postulate that efficient endocytosis of ligand-induced EGFR is closely related to EGFR-tyrosine kinase inhibitor sensitivity of NSCLC cell lines.

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We further detected by confocal immunofluorescencemicroscopy nuclear staining of pEGFR in PC9 and QG56 cells, although considerable amounts of pEGFR were distributed in small cytoplasmic punctate vesicles. We recently demonstrated that in PC9 cells, most EGFR is localized in the plasma membrane and in LIMPII-positive swollen vacuoles, i.e., late endosomes [13], it is interesting to note that a part of pEGFR is localized in the nucleus even in the absence of EGF stimulation. These results imply that pEGFR might be translocated into the nucleus through the conventional nuclear importing system associated with the nuclear pore complex (that is, the Ran/importin pathway), and it might then operate as a transcription factor. It has recently been reported that nuclear localization of EGFR is detected in the highly proliferating state of human cancer tissues in vivo and human breast cancer cell lines in vitro, thereby, suggesting the close correlation between nuclear EGFR and tumor tissues with high proliferation [22-24]. It was also shown that nuclear EGFR levels were increased on treatment with EGF and that the EGFR which accumulated in the nucleus was highly tyrosine phosphorylated; it was further demonstrated that nuclear EGFR acts as a transcription factor for activating gene expression of cyclin D1, a well-known cell growthpromoting factor [23]. Regarding the functions of these cell growth nuclear receptors, further studies will be required to shed new light on the field of receptor signaling. Furthermore, we found novel evidence that in the gefitinib-resistant cell line QG56, the internalized pEGFR was not trafficked efficiently to late endosomes/lysosomes after EGF stimulation and accumulated in the aggregated vesicular structures of early endosomes which was associated with SNX1. Also, a part of SNX1-positive aggregates was colocalized with LIMPII-positive late endosomes, therefore, implying that vesicle trafficking of pEGFR from the early endosomes to late endosomes was significantly impaired in QG56 cells; however, no such accumulation was noted in PC9 cells. SNX1 is a mammalian homologue of yeast Vps5p, which recognizes the lysosomal targeting code of EGFR and participates in lysosomal trafficking of the receptor [18, 25]; SNX1 is preferentially localized to early endosomes through its phospholipid-binding motif termed the PX domain [19, 20]. Therefore, we speculate that impairment of SNX1 trafficking via the early/late endocytic pathway causes the perturbation of EGFR endocytosis, which then leads to the acquisition of gefitinibresistance in NSCLC cell lines. It is important to analyze the mechanism by which the interaction of EGFR and other cellular proteins might regulate the process of EGFR down regulation in NSCLC cell lines. Furthermore, an analysis of the pEGFR endocytosis machinery in gefitinib-sensitive or gefitinib-resistant NSCLC cell lines could be useful for clarifying the effectiveness of gefitinib.

Materials and Methods Materials Gefitinib was provided from AstraZeneca (Macclesfield, United Kingdom). Texas redlabeled transferrin, Texas red-labeled EGF, and SlowFade anti-fade reagent were purchased from Molecular Probes (Eugene, OR, USA). DAPI was obtained from Sigma (St. Louis, MO,

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USA). Recombinant human EGF was purchased from PeproTech (London, United Kingdom). Other chemicals were of reagent grade and were obtained from commercial sources.

Cell Culture Cell lines PC9 and QG56 (Kyushu Cancer Center, Fukuoka, Japan) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS). Cells were maintained under standard cell culture conditions at 37oC and 5% CO2 in a humid environment.

Antibodies Alexa 488-labeled goat anti-mouse and goat anti-rabbit secondary antibodies, Texas redlabeled human transferrin, and Texas red-labeled EGF were obtained from Molecular Probes (Eugene, OR, USA). Normal goat serum was purchased from Sigma (St. Louis, MO, USA). Antisera were raised in rabbits (New Zealand white male) against the mature form of rat liver lysosomal cathepsin D [26, 27] and the native form of LIMPII/LGP85 [15] as described previously. Anti-cathepsin D or anti-LIMPII IgG was affinity-purified by protein A Sepharose CL-4B (Sigma), followed by immunoaffinity chromatography using antigenconjugated Sepharose 4B. A mouse monoclonal anti-pEGFR was obtained from DakoCytomation (Denmark) and BD Biosciences (San Jose, CA, USA). Mouse monoclonal antibody to SNX1 was purchased from BD Biosciences (San Jose, CA, USA).

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Immunofluorescence Microscopy Immunofluorescence microscopy was described previously [13, 28-31]. In brief, cells were grown for 2 days on glass coverslips in 6-well plates in RPMI with 10% fetal bovine serum. Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), pH 7.4, permeabilized in PBS containing 0.1 % saponin. After washing with PBS, cells were blocked with PBS-10% normal goat serum. All subsequent antibody and wash solutions contained 0.1% saponin. The PC9 or QG56 cells were incubated with specific primary antibodies (rabbit anti-cathepsin D and anti-LIMPII IgGs, mouse anti-pEGFR mAb, or mouse antiSNX1 mAb), for 1 h, followed by washes with PBS containing 0.1 % saponin and incubation for 1 h with the secondary antibodies at 20 μg/ml. Each cell line was stained with DAPI to reveal nuclei. To label early endosomes, cells were incubated with RPMI without FBS for 3 h at 37oC followed by 20 min incubation in culture medium containing Texas red-conjugated transferrin, and then cells were fixed and stained for SNX1. Controls for antibody specificity were either preimmune serum (rabbit or mouse) or omission of the primary antibodies. To follow the endocytic pathway and determine the intracellular fate of internalized labeled ligand, the uptake of Texas red-conjugated-EGF by the cells was measured. The PC9 or QG56 cells were starved for 3 h with RPMI without FBS at 37oC. The serum-starved cells

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were preincubated for 3 h in the absence or presence of 0.1μM gefitinib before incubation with Texas red-EGF (100ng/ml) at 37oC for 5, 10, and 15 min, and then the cells were fixed and stained for pEGFR. The distribution of the labeled proteins was analyzed by confocal immunofluorescence microscopy of the fixed cells. In some cases, PC9 or QG56 cells were starved for 3 h with RPMI without FBS at 37oC and then the phosphorylation of the EGFR was induced with EGF (100ng/ml) for 15 min on ice in binding medium (1mg/ml BSA in RPMI medium). The cell were then rinsed with ice-cold PBS, incubated in the presence of Texas red-transferrin in prewarmed medium, and chased at 37oC for 3, 6, and 15 min. Slides were mounted with SlowFade anti-fade reagent and observed on a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany), equipped with krypton/argon laser sources. For quantification of colocalization between Texas red-EGF and pEGFR, between Texas red-transferrin and SNX1, between Texas red-transferrin and pEGFR, or between LIMPII and pEGFR, merged images as yellow color were quantified and presented as the percentage of total amounts of SNX1-, pEGFR- or LIMPII-positive vesicles per cell.

Abbreviations EGFR pEGFR NSCLC LIMPII SNX1

Epidermal growth factor receptor Phosphorylated epidermal growth factor receptor Non-small cell lung cancer cell lines; Lysosomal integral membrane protein II Sorting nexin 1

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References [1] [2] [3] [4] [5] [6] [7]

de Bono JS, Rowinsky EK: The ErbB receptor family: a therapeutic target for cancer. Trends Mol Med 2002, 8: 19–26. Mendelsohn J, Baserga J: The EGF receptor family as targets for cancer therapy. Oncogene 2000, 19: 6550–6565. Carpenter G, Cohen S: Epidermal growth factor. J Biol Chem 1990, 265: 7709-7712. Yarden Y: The EGFR family and its ligands in human cancer signaling mechanisms and therapeutic opportunities. Eur J Cancer 2001, 37: 3-8. Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 1990, 61: 203–212. Arteaga C L, Johnson DH : Tyrosine kinase inhibitors-ZD1839 (Iressa). Curr Opin Oncol 2001, 13: 491-498. Barker AJ, Gibson KH, Grundy W, Godfrey A A, Barlow JJ, Healy MP, Woodburn JR, Ashton SE, Curry BJ, Scarlett L, Henthorn L, Richards L: Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett 2001, 11: 1911-1914.

Mechanisms of Resistance to EGF Receptor-Tyrosine Kinase Inhibitor… [8] [9] [10]

[11]

[12] [13]

[14] [15]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[16]

[17]

[18] [19] [20]

[21]

[22]

147

Baselga J, Averbuch SD: ZD1839 (‘Iressa’) as an anticancer agent. Drugs 2000, 60, Suppl 1: 33-40; discussion 41-42. Woodburn JR: The epidermal growth factor receptor and its inhibition in cancer therapy. Pharmacol Ther 1999, 82: 241– 250. Sirotnak FM, Zakowski MF, Miller VA, Scher HI, Kris MG: Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000, 6: 48854892. Ono M, Hirata A, Kometani T, Miyagawa M, Ueda S, Kinoshita H, Fujii T, Kuwano, M: Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the EGF receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol Cancer Ther 2004, 3: 465472. Schlessinger J: Common and distinct elements in cellular signaling via EGF and FGF receptors. Science 2004, 306:1506-1507. Nishimura Y, Bereczky B, Ono M: The EGFR inhibitor gefitinib suppresses ligandstimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines. Histochem Cell Biol 2007, 127: 541-553. Kornfeld S, Mellman I: The biogenesis of lysosomes. Ann Rev Cell Biol 1989, 5:483525. Okazaki I, Himeno M, Ezaki J, Ishikawa T, Kato K: Purification and characterization of an 85 kDa sialoglycoprotein in rat liver. J Biochem 1992, 111: 763-769. Sandoval IV, Arredondo JJ, Alcalde J, Gonzalez-Noriega A, Vandekerckhove J, Jimenez MA, Rico M: The residues Leu (Ile) 475-Ile (Leu) 476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMPII to lysosomes. J Biol Chem 1994, 269: 6622-6631. Tabuchi N, Akasaki K, Tsuji H: Two acidic amino acid residues, Asp (470) and Glu (471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMPII, are important for its accumulation in secondary lysosomes. Biochem Biophys Res Commun 2000, 270: 557-563. Kurten R C, Cadena D L, Gill GN: Enhanced degradation of EGF receptors by a sorting nexin, SNX1. Science 1996, 272: 1008 –1010. Worby C A, Dixon J E: Sorting out the cellular function of sorting nexins. Nat Rev Mol Cell Biol 2002, 3: 919-931. Zhong Q, Lasar C S, Tronchere H, Sato T, Meerloo T, Yeo M, Songyang Z, Emr SD, Gill G N: Endosomal localization and function of sorting nexin 1. Proc Natl Acad Sci USA 2002, 99: 6767– 6772. Feugaing DDS, Tammi R, Echtermeyer FG, Stenmark H, Kresse H, Smollich M, Schönherr E, Kiesel L, Götte M: Endocytosis of the dermatan sulfate proteoglycan decorin utilizes multiple pathways and is modulated by epidermal growth factor receptor signaling. Biochimie 2007, 89: 637-657. Hoshino M, Fukui H, Ono Y, Sekikawa A, Ichikawa K, Tomita S, Imai Y, Imura J, Hiraishi H, Fujimori T: Nuclear expression of phosphorylated EGFR is associated with

148

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[31]

Yukio Nishimura, Kiyoko Yoshioka and Kazuyuki Itoh poor prognosis of patients with esophageal squamous cell carcinoma. Pathobiology 2007, 74: 15-21. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, Bourguignon L, Hung MC: Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 2001, 3: 802-808. Lo HW, Hung MC: Nuclear EGFR signaling network in cancer; linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer 2006, 94: 184-188. Chin L-S, Raynor MC, Wei X, Chen H-Q, Li L:Hrs Interacts with Sorting Nexin 1 and Regulates Degradation of Epidermal Growth Factor Receptor. J Biol Chem 2001, 276: 7069-7078. Nishimura Y, Higaki M, Kato K: Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Biochem Biophys Res Commun 1987, 148: 335-343. Nishimura Y, Kawabata T, Kato K: Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Arch Biochem Biophys 1988, 261: 64-71. Nishimura Y, Itoh K, Yoshioka K, Tokuda K, Himeno M: Overexpression of ROCK in human breast cancer cells. Evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. Pathol Oncol Res 2003, 9: 83-95. Nishimura Y, Yoshioka K, Bernard O, Himeno M, Itoh K: LIM kinase 1: evidence for a role in the regulation of intracellular vesicle trafficking of lysosomes and endosomes in human breast cancer cells. Eur J Cell Biol 2004, 34: 189-213. Nishimura Y, Yoshioka K, Bernard O, Bereczky B, Itoh K: A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells. Histochem Cell Biol 2006, 126: 627-638. Nishimura Y, Yoshioka K, Bereczky B, Itoh K: Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line. Molecular Cancer 2008, 7(42): 1-13.

In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter VII

Targeting Adverse Features of Hormone-Resistant Breast Cancer Stephen Hiscox∗, Liam Morgan, Nicola Jordan, Julia Gee and Robert I. Nicholson Welsh School of Pharmacy, Cardiff University, Cardiff, UK CF10 3NB

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Abstract Endocrine therapy is the treatment of choice in hormone receptor-positive breast cancer. However, the effectiveness of anti-estrogenic agents is limited by the development of drug resistance, ultimately leading to disease progression and patient mortality. Cell models of endocrine resistance have demonstrated a role for altered growth factor signalling in the development of an endocrine-insensitive phenotype. Significantly, recent studies have revealed that the acquisition of endocrine resistance in breast cancer is also accompanied by the development of an adverse cellular phenotype, with such resistant cells exhibiting altered adhesive interactions, enhanced migratory and invasive behaviour and the ability to promote angiogenic responses in vitro. Elucidation of the molecular mechanisms underlying these adverse tumour cell features and their subsequent targeting may provide a means of limiting tumour spread in vivo and may ultimately improve the outcome for breast cancer patients on endocrine therapy.

1. Introduction Endocrine therapy is the treatment of choice for hormone receptor-positive breast cancer, both early stage and metastatic, where these agents have proven efficacy at reducing breast cancer recurrence rates and improving patient survival. Unfortunately, however, while a substantial proportion of breast tumours will display an intrinsic resistance to hormone ∗

Address for correspondence: Dr. S. Hiscox, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF. Tel: 029 20 875226; Fax: 029 20 875152; Email: [email protected]

.

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therapies despite being hormone receptor positive (de novo resistance), more than a third of patients with endocrine-responsive, early stage breast cancer and almost all of those with metastatic disease will develop hormone resistance during the course of their disease (acquired resistance) [1-3]. Much research has been undertaken in order to understand the mechanisms that underlie the phenomenon of endocrine resistance and to reveal markers that predict response to, or early relapse on, such treatments, in addition to identifying potential therapeutic targets through which endocrine resistance may be delayed or prevented. Through these studies it is increasingly apparent that the tumour cells’ ability to harness a variety of growth factor signalling pathways to drive proliferation in the presence of endocrine agents plays a major role in promoting a resistant phenotype. Indeed, the inappropriate activation of growth factor signalling cascades is now regarded to play a significant role in the promotion of antihormone failure in breast cancer cells [4]. ItIndeed, it is also becoming clear that antihormones themselves can promote the expression of a number of growth factors and their receptors in the drug-responsive phase, which subsequently play key roles in the regulation of tumour growth during the drug-resistant phase [5, 6]. This phenomena of enhanced growth factor signalling have been described for a number of growth factors/growth factor receptors and include heregulins, acting through HER3 and HER4 ([7, 8]; epidermal growth factor (EGF) and transforming growth factor (TGF)-α, acting through the epidermal growth factor receptor (EGFR) [9] [6, 10]; and IGF-I and -II acting through the IGF-IR [11, 12]. Additionally, the HER2 receptor may also contribute to failure of anti-hormones either directly, via overexpression [13], or indirectly, through heterodimerisation with other members of the erbB receptor family [10]. Moreover, breast cancer models of endocrine (tamoxifen and fulvestrant) resistance consistently demonstrate overexpression of the EGFR gene and protein [9, 10, 14] as well as increases in HER2 expression compared with their endocrine-sensitive counterparts [15]. Importantly, tamoxifen-resistant MCF7 cells also express several EGFR ligands [10], supporting the hypothesis that such resistant cells possess EGFR-driven autocrine regulatory loop which can drive endocrine-resistant cellular growth. The importance of growth factor signalling in endocrine resistance is further revealed in that a high degree of interaction exists between intracellular signalling pathways downstream of the estrogen receptor and growth factor receptors, further contributing to the development of endocrine insensitive state. For example, both insulin-like growth factor (IGF-1) and oestradiol (E2)-induced MAPK activity in breast cancer cells are known to be mediated by the transactivation of the EGFR by IGF-1 receptor [16-18]. Such crosstalk between these pathways is further supported by studies which have demonstrated the ability of IGF-1R pathway blockade or inhibition of ER expression to abrogate E2 or IGF-1R response respectively [19]. Interestingly, the ER, IGF-1R and EGFR can be co-expressed in clinical breast cancers, further supporting interplay between these elements [20]. In addition to that mediated by the IGF-1R, much data also supports direct crosstalk between the ER and the EGFR. For example, induction of breast cancer cell proliferation following EGF stimulation of the EGFR can be inhibited by estrogen antagonists such as fulvestrant [21], while EGF can activate genes regulated by estrogen-responsive elements [22]. Moreover, induction of EGFR expression is observed in response to endocrine therapy [23, 24] which can subsequently provide an efficient mechanism to drive anti-hormone resistant growth. Such observations

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have been further supported by studies of clinical tissue, where increased expression and activity of EGFR and HER2 and enhanced MAPK is reported in sequential samples obtained from tamoxifen-treated, ER positive primary breast cancers [25]. In such cases as these mentioned above, the enhanced expression of growth factor signalling pathways and networks are likely to contribute to endocrine resistance through cross-talk with the ER resulting in a ligand-independent activation of the ER and sustained cellular growth [6, 26, 27]. Growth factor signalling pathways that promote the proliferation of breast cancer cells in an endocrine resistant context are also known also to play prominent roles as promoters of cellular migration and invasion in other cell systems [28-30]; it therefore follows that resistance to endocrine agents in breast cancer may result in the development of an adverse cellular phenotype. Indeed, our recent observations in multiple breast cancer cell models of acquired drug resistance have demonstrated this to be the case, with these cells demonstrating a highly invasive phenotype in vitro [31-33]. However, inhibition of the dominant growth regulatory pathways in these models results only in modest suppression of their invasive phenotype [32] suggesting that other, as yet unidentified, mechanisms must be present that control an adverse cellular behaviour associated with antihormone resistance. A number of key elements with pro-invasive/migratory roles are now known to be induced by a range of endocrine treatments and, if recapitulated in vivo, suggest that anti-hormones themselves may further augment the cells’ metastatic capacity and promote tumour progression. Several key molecular elements are described here that regulate these processes and which may present future targets through which endocrine resistance and an associated adverse cell phenotype may be prevented.

2. Endocrine Resistance Is Accompanied by an Adverse Cellular Phenotype Growth factor signalling pathways such as those mentioned above which promote the proliferation of breast cancer cells in an endocrine resistant context are also known also to play prominent roles as promoters of cellular migration and invasion in other cell systems [28-30] and it therefore follows that resistance to endocrine agents in breast cancer may result in the development of an adverse cellular phenotype. Indeed, through our studies of acquired resistance to anti-hormones or anti-growth factors, we have shown that, in many instances, drug resistant growth in cancer cells is associated with a gain of invasive behaviour. Thus, for example, in comparison with MCF-7 cells, our tamoxifen-resistant variants display a 6-fold increase in their capacity to invade through Matrigel, while cells that are dually resistant to tamoxifen and gefitinib (TAMR/TKIR) show over a 20-fold increase [32, 34, 35]. The resistant cells commonly show a more angular, dedifferentiated morphology than their parental cells with numerous lamellipodia and membrane ruffling and. They also appear to be undergoing an epithelial-to-mesenchymal transition (EMT) since they grow as loose, disorganised colonies in which cells have partially-dissociated cell-cell contacts promoting cell scattering [32, 36]. However, since inhibition of the dominant growth regulatory pathways in these models results only in modest suppression of their invasive phenotype [32]

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other, as yet unidentified, mechanisms must be present that control an adverse cellular behaviour. A number of key elements with pro-invasive roles are now known to be induced by a range of endocrine treatments which suggest that and, if recapitulated in vivo, suggest that anti-hormones themselves may have the ability to further augment the cells’ metastatic capacity and promote tumour progression in vivo. Several key molecular elements are described here that regulate these processes and which may present future targets through which endocrine resistance and an associated adverse cell phenotype may be prevented.

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2.1. Endocrine Resistant Breast Cancer Cells Overexpress Cell Surface Receptors That May Sensitize Them to the Tumour Microenvironment As mentioned above, certain signalling pathways are known to be overexpressed in endocrine resistance (e.g., EGFR/HER2 pathways) that, in addition to playing a prominent role as a driver of resistant cell growth, may also promote invasive response. It follows that targeting of these may be an effective means of suppressing the invasive phenotype in addition to cellular proliferation. However, pharmacological targeting of erbB signalling in these cells exerts only a modest inhibitory effect on the cells’ invasive capacity ([32]; L. Morgan, unpublished observations) suggesting that erbB signalling contributes to, but is not essential for, their invasive in vitro phenotype. Moreover, prolonged exposure to such inhibitors ultimately results in the development of a further drug-resistant state, with a further gain in cellular invasiveness [33]. We have now identified a number of key molecules that appear to play a central role as mediators of an intrinsic invasive phenotype in endocrine resistance in vitro. Intriguingly, data from in vitro co-culture systems is beginning to reveal that the overexpression of these molecules in endocrine resistant breast cancer cells appears to sensitize them to factors commonly found, and frequently overexpressed, within the tumour microenvironment. This raises the possibility that that the adverse phenotype of resistant cells may be further enhanced in an in vivo context. 2.1.1. c-Met Receptor One such case is exemplified by the c-Met receptor which we have identified as being overexpressed in fulvestrant-resistant MCF7 and T47D cells [31]. The c-Met receptor tyrosine kinase is the cell surface receptor for hepatocyte growth factor (HGF, also known as scatter factor (SF)) and its activation results in disruption of intercellular adhesion, cell migration and invasion and promotion of angiogenesis [37]. Subsequently, we have shown that co-culture of fulvestrant-resistant cells with stromal fibroblasts, known producers of HGF/SF [38], or in fibroblast-conditioned medium, results in the activation of Akt and the production of MMP2 and MMP9 (Y. Khirwadkar personal communication) and a further enhancement of these cells’ invasive behaviour [31]; although fibroblasts secrete a range of growth factors and cytokines that may modulate epithelial cell behaviour, our siRNA data demonstrated that these effects are specific to c-Met activation [31]. In vivo, the c-Met receptor is primarily expressed by epithelial cells and its overexpression in node-positive breast cancer identifies patients with poor clinical outcome [39]. This is not surprising given the ability of c-Met to be activated in a paracrine fashion by

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HGF/SF-secreting stromal fibroblasts. Indeed, this mechanism has been implicated as a major contributory factor for tumour progression with studies demonstrating the ability of HGF/SF to regulate EMT and metastasis [40]. Furthermore, the therapeutic value of c-Met in breast cancer has been demonstrated through studies that have used retroviral ribozyme transgenes to target HGF/SF expression in fibroblasts or the Met receptor in mammary cancer cells to inhibit paracrine stromal-tumour cell interactions [38]. Since tumour invasion and spread may thus be critically influenced by paracrine influences arising from the surrounding stroma, these observations suggest that, in vivo, overexpression of c-Met in anti-hormone-resistant epithelial breast cancer cells may significantly affect tumour progression. 2.1.2. CD44 In contrast to the overexpression of the c-Met receptor, which appears to be an effect specific to fulvestrant only, a common feature of acquired resistance to multiple endocrine agents (tamoxifen and fulvestrant) and to estrogen deprivation is the overexpression of cell surface receptors of the CD44 family [41], a group of transmembrane glycoproteins implicated in the progression and spread of breast cancer. Alternative splicing and variation in glycosylation results in structural and functional diversity amongst this group of proteins [42] with several CD44 variants being associated with invasive breast cancer. For example, expression of the CD44 variant 3 (CD44v3) correlates with lymphatic spread in breast cancers [43], soluble CD44v6 is associated with lymph node metastases [44] while CD44v7 is associated with a reduction in disease-free survival [45]. However, while a wealth of evidence implicates CD44 variants in tumour progression, the case for the standard form of CD44 (CD44s) is controversial. Whereas some studies report that increased expression of the CD44s correlates with patient survival [46], recent studies have demonstrated that expression of CD44s in non-metastatic MCF7 breast cancer cells promotes their migration and invasion in vivo [47]. In tamoxifen- and fulvestrant-resistant cell models, CD44s, together with the v3, v6 and v10 isoforms, are overexpressed at the gene and protein level. The relevance of overexpression of CD44 in these model systems has been demonstrated by siRNA knockdown experiments which reveal that loss of CD44 has an inhibitory effect on the cells’ intrinsic migratory capacity in vitro [48-50]. CD44 is also reported to associate, and form stable complexes with, a number of growth factor receptors including those of the erbB family providing a system through which cellular migration and invasion can be augmented [51, 52]. This is interesting in light of our knowledge that such receptors are also overexpressed in endocrine resistance [36]. Indeed, we have seen that CD44v3, and to a lesser extent CD44s, associate with the EGFR and HER2 in tamoxifen-resistant cells and the c-Met receptor in fulvestrant-resistant cells. The effect of this is to significantly augment the cellular invasive response to exogenous erbB ligands (in tamoxifen resistance) or HGF (in fulvestrant resistance) [48, 49]. A caveat to these data is that CD44 siRNA is not specific for any particular CD44 isoform but rather results in the knockdown of all forms of CD44 expressed. It is thus not possible to determine the relative contribution to the cell’s aggressive phenotype from individual CD44 family members. However, it is interesting to note that examination of CD44v3 protein expression in a small series (n=69) of clinical tissue revealed

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an association with HER2 expression, poor survival and shortened response to endocrine therapy in ER+ patients [48, 50]. In addition to growth factors and cytokines, tumour cells are in contact with a number of extracellular matrix components in an in vivo situation. A number of these can act as ligands for cell surface receptors providing additional means through which the epithelial cell phenotype can be modulated. Our recent observations have revealed that activation of CD44 by hyaluronic acid (HA), an important structural component of extracellular matrices known to be concentrated in regions of high cell division and invasion [53], promotes erbB invasive signalling in tamoxifen-resistant cells (B. Baruha, unpublished observations) which may again promote an adverse cellular phenotype. Together these observations suggest that acquired resistant cells are sensitized to many factors commonly found within the tumour microenvironment such as erbB ligands, HGF/SF and the matrix components themselves. The fact that many of these factors are increased in breast cancer tissue and serum may have significant bearing on the progression of tumours following relapse on therapy.

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3. Antihormones Induce Pro-Invasive Responses during the Drug-Responsive Phase Which, in the Appropriate Cell Context, May Contribute to an Adverse Cell Phenotype An intriguing observation is that increased cellular invasiveness is observed in response to short term anti-hormone treatment in ER+, endocrine-sensitive breast cancer cells [54]. Although modest, these antihormone-induced, pro-invasive effects become significant under conditions of E-cadherin deficiency, where the gain in cellular invasion is greatly augmented [54]. Such data highlight a previously unreported effect of tamoxifen (and potentially further antiestrogens), in that these agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts); this may have major clinical implications for those patients with tumours where there is inherently poor intercellular adhesion. These observations suggest that pro-invasive gene/pathway changes can be induced by endocrine agents at an early stage, the effects of which may be further augmented by changes in cell context: for example, the presence of exogenous growth factors within the tumour microenvironment that may suppress cell-cell adhesive interactions. The interaction between tumour cells and the microenvironment can have a substantial effect on tumour cell behaviour by influencing cell-cell as well as cell-matrix contacts although the underlying molecular mechanisms are not well characterised at present. Several factors within the tumour microenvironment, including growth factors such as TGFβ and erbB ligands in addition to protein components of the surrounding extracellular matrix itself, have been identified as being able to cause the disruption of the E-cadherin adhesion complex and reduced E-cadherin expression [55]. These factors activate a number of pathways within the tumour cells (integrins, Src kinase, focal adhesionkinase and PI3K) that may regulate EMT like behaviour. Sensitization of breast cancer cells to these microenvironmental factors, brought about through acquired endocrine resistance, may further facilitate the complex

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interactions between tumour cells and the surrounding stroma may create conditions permissive for further pro-invasive actions of antihormones and ultimately promote disease progression and spread.

4. A Role for Src in Endocrine-Sensitive and Endocrine-Resistant Breast Cancer

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4.1. Endocrine-Sensitive Breast Cancer A number of studies have suggested a role for Src in breast cancer, revealing frequently elevated levels of Src kinase protein and activity in breast cancer cells and in tissue from breast cancer patients compared with corresponding normal tissue [56, 57]. Within these cells and tissues, Src has been shown to interact with a variety of molecules, such as growth factor receptors and the estrogen receptor ultimately impacting on cellular behaviour that is central to tumour progression [58]. Indeed, crosstalk between ER and growth factor signalling pathways has been shown to involve Src, particularly in the non-genomic activation of the ER. For example, in human breast cancer cells, ligand binding to the ER results in the rapid activation of the ERK and Akt pathways in a Src dependent manner [59] [60] [61]. Furthermore, in both ER-positive breast cancer cells and in cells which transiently express ER, oestradiol has been shown to induce rapid (within minutes) activation of Src-dependent signalling pathways [59, 60] which regulate cellular processes such as proliferation and survival [62, 63]. Additionally, EGF stimulation promotes formation of a membrane-bound ER-Src complex [64]. The physical interactions that occur between Src and the ER can enhance estrogen-mediated gene transcription and may be facilitated by intermediate adapter-molecules such as the recentlydescribed MNAR protein [65]. The EGF/E2-dependant assembly of an ER-Src complex requires interaction of the SH2 domain of Src with a phosphotyrosine residue of the ER allowing the ER-Src complex to stimulate further downstream signalling events culminating in DNA synthesis and cytoskeleton changes. Association between Src and the ER can be blocked by application of small peptides derived from ER sequences involved in the receptor interaction with Src with the resultant inhibition of ER-mediated DNA synthesis in vitro and tumour growth in vivo [66, 67], suggesting the potential usefulness of inhibition Interestingly, increases in Src activity can arise as a result of antihormone treatment [68, 69] and ; in such instances, elevated Src activity may subsequently contribute to the limitation of cellular response to endocrine agents given the involvement of Src in ER and growth factor signalling pathways; . Importantly, iin support of this, we have recently demonstrated that elevation of Src activity in endocrine-sensitive breast cancer cells promotes an endocrine insensitive state [70]. The use of Src inhibitors alongside endocrine therapy may therefore present a means through which such effects may be circumvented. Indeed, two recent studies provide compelling evidence to support such a hypothesis, demonstrating the short-term effectiveness of such combination treatments in preventing the growth of MCF7 cells [71, 72]. Importantly, studies from our own group demonstrate that targeting of Src

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kinase in ER-positive MCF7 and T47D cells using the novel Src/Abl inhibitor, AZD0530 [73] alongside the ER (using tamoxifen) significantly delays the emergence of tamoxifen resistance [74]. A further benefit of such combination therapy combining Src inhibition with tamoxifen was the ability to suppress the development of the highly migratory and invasive characteristics seen to accompany the development of resistance; this would have has significant implications in vivo, where the development of aggressive characteristics, even in the absence of cellular proliferation, may favour cell dissemination and tumour spread. These studies are now being extended to other ER-positive cell lines and with additional hormonal agents to further determine the effectiveness of co-targeting Src and the ER as a treatment strategy to circumvent the phenomena of acquired endocrine resistance.

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4.2. Endocrine-Resistant Breast Cancer Of particular importance is the emerging role of Src, and therefore the potential benefits of its inhibition, in endocrine-resistant breast cancer. A role for Src kinase in this context acquired endocrine resistance has been suggested by several groups, where it may contribute to cellular growth via regulation of Cas-mediated EGFR signalling [75] or through interplay with focal adhesion kinase (FAK) [76]. Additionally, our own studies have revealed elevated Src activity to be a unifying feature of acquired resistance to hormonal therapies [69, 77]. Furthermore, we have observed that the expression of constitutively-active Src is sufficient to confer resistance to tamoxifen in MCF7 breast cancer cells and, subsequently, inhibition of Src activity re-sensitises both these and tamoxifen-resistant MCF7 cells to tamoxifen (L. Morgan, unpublished data). These studies are confirmed by others who have demonstrated that co-targeting of Src and the ER results in sensitization of ER+ breast cancer cells to tamoxifen and a greater inhibition of cellular proliferation [71, 72] [78]. In these models of endocrine resistance, Src plays a key role in mediating cellular migration and invasion through its interplay with focal adhesion kinase (FAK) [76, 79]. Subsequently, pharmacological and siRNA-mediated inhibition of Src significantly suppresses the invasive phenotype of endocrine-resistant cells [69]. Interestingly, inhibition of Src activity in endocrine resistant cells appears to restore their morphology to that of their parental, endocrine-sensitive cells, a process which likely involves suppression of β-catenin tyrosine phosphorylation [80]. Further to these dataobservations, we have recently observed that endocrine resistant breast cancer cells also show increased expression of a number of angiogenic factors (e.g., VEGF, IL-8) in addition to a reduction in the expression of angiostatic factors. Our preliminary studies have shown that human umbilical vein endothelial cell (HUVEC) cultures stimulated by conditioned medium from resistant cells show enhanced proliferation compared with conditioned medium from endocrine-sensitive counterparts and this is accompanied by an elevation in HUVEC ERK1/2 activity. Significantly, conditioned medium from endocrinesensitive MCF7 cells engineered to express constitutively active Src also stimulate HUVEC proliferation whereas conditioned medium from antiestrogen-resistant cells treated with a Src kinase inhibitor fails to elicit angiogenic activity in HUVEC cultures. This is interesting in the light of recent reports that Src signalling via FAK has been identified as a mechanism for

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the production of VEGF and subsequent blood vessel growth in vivo [81]. Notably, pharmacological inhibition of Src can reduce FAK tyrosine phosphorylation in a number of tumour cell types, including our acquired resistant cells [69, 82], suppress VEGF and IL-8 expression [83, 84] and prevent VEGF-induced proliferation of endothelial cells [85].

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Conclusions There is increasing evidence to suggest revealing that prolonged exposure to endocrine agents results in a number of changes within breast cancer cells that favour an adverse, proinvasive phenotype in vitro. Such changes include the overexpression of a number of cell surface receptors that may sensitize these cells to factors found within the tumour microenvironment. Indeed, the concept that the development of endocrine resistance in breast cancer cells sensitizes these cells to stromal-produced factors is further supported by experimental data showing the ability of conditioned medium from primary fibroblast cells to promote the migration of endocrine-resistant breast cancer cells compared to their endocrinesensitive counterparts, although it is not currently clear which fibroblast-secreted factors and/or epithelial cell receptors are involved in this process. However, these observations have clear implications for the development and spread of tumours in an in vivo context. Several potential targets for intervention have been identified through which these adverse cellular features may be suppressed and, although there are few inhibitors available for c-Met, and CD44 is not yet developed as a target, the. The targeting potential of these individual molecules has been demonstrated through siRNA studies. A role is emerging for Src kinase in breast cancer, where its inihibtion may provide a broad therapeutic benefit given the role that Src plays in multiple signalling pathways that contribute to the growth and development of adverse tumour cell behaviour. Of particular interest are recent data supporting a role for Src in the context of endocrine response and resistance. Here there appears the potential to combine Src inhibitors with existing therapeutic agents chemotherapies in order to achieve greater response. Clinical studies are now needed in order to determine whether the in vitro success of Src inhibitors in breast cancer models translates out into clinical benefit. However, although Src inhibitors have shown encouraging effects in preclinical studies in breast cancer, further investigation of the clinical effectiveness of Src inhibitors is needed in selected patient groups in order to assess whether their use would provide benefit alongside existing endocrine therapies.

References [1] [2]

Nicholson RI, Johnston SR: Endocrine therapy--current benefits and limitations. Breast Cancer Res. Treat. 2005, 93 Suppl 1:S3-10. Conte P, Guarneri V, Bengala C: Evolving nonendocrine therapeutic options for metastatic breast cancer: how adjuvant chemotherapy influences treatment. Clin. Breast Cancer 2007, 7(11):841-849.

158 [3] [4]

[5]

[6]

[7]

[8]

[9]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[10]

[11]

[12]

[13]

[14]

[15]

Stephen Hiscox, Liam Morgan, Nicola Jordan et al. Howell A, Bundred NJ, Cuzick J, Allred DC, Clarke R: Response and resistance to the endocrine prevention of breast cancer. Adv. Exp. Med. Biol. 2008, 617:201-211. Nicholson RI, Hutcheson IR, Jones HE, Hiscox SE, Giles M, Taylor KM, Gee JM: Growth factor signalling in endocrine and anti-growth factor resistant breast cancer. Rev. Endocr. Metab. Disord 2007, 8(3):241-253. Arpino G, Wiechmann L, Osborne CK, Schiff R: Crosstalk between the estrogen receptor and the HER tyrosine kinase receptor family: molecular mechanism and clinical implications for endocrine therapy resistance. Endocr. Rev. 2008, 29(2):217233. Nicholson RI, Staka C, Boyns F, Hutcheson IR, Gee JM: Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy. Endocr. Relat. Cancer 2004, 11(4):623-641. Pietras RJ, Arboleda J, Reese DM, Wongvipat N, Pegram MD, Ramos L, Gorman CM, Parker MG, Sliwkowski MX, Slamon DJ: HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene 1995, 10(12):2435-2446. Tang CK, Perez C, Grunt T, Waibel C, Cho C, Lupu R: Involvement of heregulin-beta2 in the acquisition of the hormone-independent phenotype of breast cancer cells. Cancer Res. 1996, 56(14):3350-3358. McClelland RA, Barrow D, Madden TA, Dutkowski CM, Pamment J, Knowlden JM, Gee JM, Nicholson RI: Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182,780 (Faslodex). Endocrinology 2001, 142(7):2776-2788. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, Barrow D, Wakeling AE, Nicholson RI: Elevated levels of epidermal growth factor receptor/cerbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifenresistant MCF-7 cells. Endocrinology 2003, 144(3):1032-1044. Guvakova MA, Surmacz E: Tamoxifen interferes with the insulin-like growth factor I receptor (IGF-IR) signaling pathway in breast cancer cells. Cancer Res. 1997, 57(13):2606-2610. Stephen R, Darbre PD: Loss of growth inhibitory effects of retinoic acid in human breast cancer cells following long-term exposure to retinoic acid. Br. J. Cancer 2000, 83(9):1183-1191. Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK: Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res. Treat. 1992, 24(2):85-95. Nicholson RI, Hutcheson IR, Knowlden JM, Jones HE, Harper ME, Jordan N, Hiscox SE, Barrow D, Gee JM: Nonendocrine pathways and endocrine resistance: observations with antiestrogens and signal transduction inhibitors in combination. Clin. Cancer Res. 2004, 10(1 Pt 2):346S-354S. Hutcheson IR, Knowlden JM, Madden TA, Barrow D, Gee JM, Wakeling AE, Nicholson RI: Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells. Breast Cancer Res. Treat. 2003, 81(1):81-93.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Targeting Adverse Features of Hormone-Resistant Breast Cancer

159

[16] El-Shewy HM, Kelly FL, Barki-Harrington L, Luttrell LM: Ectodomain sheddingdependent transactivation of epidermal growth factor receptors in response to insulinlike growth factor type I. Mol. Endocrinol. 2004, 18(11):2727-2739. [17] Roudabush FL, Pierce KL, Maudsley S, Khan KD, Luttrell LM: Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells. J. Biol. Chem. 2000, 275(29):22583-22589. [18] Song RX, Zhang Z, Chen Y, Bao Y, Santen RJ: Estrogen signaling via a linear pathway involving insulin-like growth factor I receptor, matrix metalloproteinases, and epidermal growth factor receptor to activate mitogen-activated protein kinase in MCF-7 breast cancer cells. Endocrinology 2007, 148(8):4091-4101. [19] Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS: Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF1/estrogen receptor cross-talk. J. Biol. Chem. 2002, 277(10):8531-8537. [20] Nicholson RI, Hutcheson IR, Harper ME, Knowlden JM, Barrow D, McClelland RA, Jones HE, Wakeling AE, Gee JM: Modulation of epidermal growth factor receptor in endocrine-resistant, oestrogen receptor-positive breast cancer. Endocr. Relat. Cancer 2001, 8(3):175-182. [21] Freiss G, Vignon F: Antiestrogens increase protein tyrosine phosphatase activity in human breast cancer cells. Mol. Endocrinol. 1994, 8(10):1389-1396. [22] Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS: Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc. Natl. Acad. Sci. U S A 1992, 89(10):4658-4662. [23] Hutcheson IR, Knowlden JM, Jones HE, Burmi RS, McClelland RA, Barrow D, Gee JM, Nicholson RI: Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy. Endocr. Relat. Cancer 2006, 13 Suppl 1:S89-97. [24] Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, McClelland RA, Jordan N, Wakeling AE, Nicholson RI: The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro. Endocrinology 2003, 144(11):5105-5117. [25] Gee JM, Robertson JF, Ellis IO, Nicholson RI: Phosphorylation of ERK1/2 mitogenactivated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer. Int. J. Cancer 2001, 95(4):247-254. [26] Britton DJ, Hutcheson IR, Knowlden JM, Barrow D, Giles M, McClelland RA, Gee JM, Nicholson RI: Bidirectional cross talk between ERalpha and EGFR signalling pathways regulates tamoxifen-resistant growth. Breast Cancer Res. Treat. 2006, 96(2):131-146. [27] Wilson CA, Slamon DJ: Evolving understanding of growth regulation in human breast cancer: interactions of the steroid and peptide growth regulatory pathways. J. Natl. Cancer Inst. 2005, 97(17):1238-1239. [28] Wells A, Kassis J, Solava J, Turner T, Lauffenburger DA: Growth factor-induced cell motility in tumor invasion. Acta. Oncol. 2002, 41(2):124-130.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

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Stephen Hiscox, Liam Morgan, Nicola Jordan et al.

[29] Ueno Y, Sakurai H, Tsunoda S, Choo MK, Matsuo M, Koizumi K, Saiki I, Arora P, Cuevas BD, Russo A et al.: Heregulin-induced activation of ErbB3 by EGFR tyrosine kinase activity promotes tumor growth and metastasis in melanoma cells. Int. J. Cancer 2008, 123(2):340-347. [30] Arora P, Cuevas BD, Russo A, Johnson GL, Trejo J: Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion. Oncogene 2008, 27(32):4434-4445. [31] Hiscox S, Jordan NJ, Jiang W, Harper M, McClelland R, Smith C, Nicholson RI: Chronic exposure to fulvestrant promotes overexpression of the c-Met receptor in breast cancer cells: implications for tumour-stroma interactions. Endocr. Relat. Cancer 2006, 13(4):1085-1099. [32] Hiscox S, Morgan L, Barrow D, Dutkowskil C, Wakeling A, Nicholson RI: Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib ('Iressa', ZD1839). Clin. Exp. Metastasis 2004, 21(3):201-212. [33] Jones HE, Goddard L, Gee JM, Hiscox S, Rubini M, Barrow D, Knowlden JM, Williams S, Wakeling AE, Nicholson RI: Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells. Endocr. Relat. Cancer 2004, 11(4):793-814. [34] Nicholson RI, Hutcheson IR, Hiscox SE, Knowlden JM, Giles M, Barrow D, Gee JM: Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer. Endocr. Relat. Cancer 2005, 12 Suppl 1:S29-36. [35] Jones HE, Gee JM, Taylor KM, Barrow D, Williams HD, Rubini M, Nicholson RI: Development of strategies for the use of anti-growth factor treatments. Endocr. Relat. Cancer 2005, 12 Suppl 1:S173-182. [36] Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Burmi R, Barrow D, Nicholson RI: Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation. Int. J. Cancer 2006, 118(2):290301. [37] Comoglio PM, Giordano S, Trusolino L: Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat. Rev. Drug. Discov. 2008, 7(6):504516. [38] Jiang WG, Grimshaw D, Martin TA, Davies G, Parr C, Watkins G, Lane J, Abounader R, Laterra J, Mansel RE: Reduction of stromal fibroblast-induced mammary tumor growth, by retroviral ribozyme transgenes to hepatocyte growth factor/scatter factor and its receptor, c-MET. Clin. Cancer Res. 2003, 9(11):4274-4281. [39] Lengyel E, Prechtel D, Resau JH, Gauger K, Welk A, Lindemann K, Salanti G, Richter T, Knudsen B, Vande Woude GF et al.: C-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. Int. J. Cancer 2005, 113(4):678-682. [40] Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2002, 2(6):442-454.

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Targeting Adverse Features of Hormone-Resistant Breast Cancer

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[41] Harper ME, Smith C, Nicholson RI: Upregulation of CD44s and variants in antihormone resistant breast cancer cells. Eur. J. Cancer 2005, 3:A71. [42] Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI: Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. U S A 1992, 89(24):12160-12164. [43] Rys J, Kruczak A, Lackowska B, Jaszcz-Gruchala A, Brandys A, Stelmach A, Reinfuss M: The role of CD44v3 expression in female breast carcinomas. Pol. J. Pathol. 2003, 54(4):243-247. [44] Mayer S, Zur Hausen A, Watermann DO, Stamm S, Jager M, Gitsch G, Stickeler E: Increased soluble CD44 concentrations are associated with larger tumor size and lymph node metastasis in breast cancer patients. J. Cancer Res. Clin. Oncol. 2008. [45] Watanabe O, Kinoshita J, Shimizu T, Imamura H, Hirano A, Okabe T, Aiba M, Ogawa K: Expression of a CD44 variant and VEGF-C and the implications for lymphatic metastasis and long-term prognosis of human breast cancer. J. Exp. Clin. Cancer Res. 2005, 24(1):75-82. [46] Gong Y, Sun X, Huo L, Wiley EL, Rao MS: Expression of cell adhesion molecules, CD44s and E-cadherin, and microvessel density in invasive micropapillary carcinoma of the breast. Histopathology 2005, 46(1):24-30. [47] Ouhtit A, Abd Elmageed ZY, Abdraboh ME, Lioe TF, Raj MH: In vivo evidence for the role of CD44s in promoting breast cancer metastasis to the liver. Am. J. Pathol. 2007, 171(6):2033-2039. [48] Goddard L, Jordan N, Smith C, Nicholson RI, Hiscox S: Overexpression of CD44 accompanies acquired tamoxifen resistance and potentiates Her2 invasive signaling. Submitted 2008. [49] Jordan NJ, Smith C, Gee J, Nicholson RI, Hiscox S: CD44 is overexpressed in fulvestrant-resistant breast cancer cells: potentiation of response to HGF. Submitted 2008. [50] Hiscox S, Jordan NJ, Goddard L, Smith C, Harper M, Gee J, Nicholson R: Overexpression of CD44 augments tamoxifen-resistant breast cancer cell response to heregulin. Breast Caner Research 2008, 10(2):S19. [51] Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC: Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation. J. Biol. Chem. 1997, 272(44):27913-27918. [52] Tsatas D, Kanagasundaram V, Kaye A, Novak U: EGF receptor modifies cellular responses to hyaluronan in glioblastoma cell lines. J. Clin. Neurosci. 2002, 9(3):282288. [53] Toole BP, Slomiany MG: Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells. Semin. Cancer Biol. 2008, 18(4):244-250. [54] Borley AC, Barrett-Lee PJ, Gee JMW, Shaw V, Nicholson RI, Hiscox SE: Antiestrogens promote an invasive phenotype in intercellular adhesion deficient breast cancer cells. Breast Cancer Res. Treat. 2007, 106(s1):24. [55] Giehl K, Menke A: Microenvironmental regulation of E-cadherin-mediated adherens junctions. Front Biosci. 2008, 13:3975-3985.

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Stephen Hiscox, Liam Morgan, Nicola Jordan et al.

[56] Ottenhoff-Kalff AE, Rijksen G, van Beurden EA, Hennipman A, Michels AA, Staal GE: Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product. Cancer Res. 1992, 52(17):4773-4778. [57] Wilson GR, Cramer A, Welman A, Knox F, Swindell R, Kawakatsu H, Clarke RB, Dive C, Bundred NJ: Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity. Br. J. Cancer 2006, 95(10):1410-1414. [58] Hiscox S, Nicholson RI: Src inhibitors in breast cancer therapy. Expert Opin. Ther. Targets 2008, 12(6):757-767. [59] Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F: Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiolreceptor complex in MCF-7 cells. EMBO J. 1996, 15(6):1292-1300. [60] Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, Fiorentino R, Varricchio L, Barone MV, Auricchio F: PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO J. 2001, 20(21):6050-6059. [61] Wessler S, Otto C, Wilck N, Stangl V, Fritzemeier KH: Identification of estrogen receptor ligands leading to activation of non-genomic signaling pathways while exhibiting only weak transcriptional activity. J. Steroid Biochem. Mol. Biol. 2006, 98(1):25-35. [62] Castoria G, Barone MV, Di Domenico M, Bilancio A, Ametrano D, Migliaccio A, Auricchio F: Non-transcriptional action of oestradiol and progestin triggers DNA synthesis. EMBO J. 1999, 18(9):2500-2510. [63] Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C et al.: Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation. EMBO J. 2000, 19(20):5406-5417. [64] Hitosugi T, Sasaki K, Sato M, Suzuki Y, Umezawa Y: Epidermal growth factor directs sex-specific steroid signaling through Src activation. J. Biol. Chem. 2007, 282(14):10697-10706. [65] Wong CW, McNally C, Nickbarg E, Komm BS, Cheskis BJ: Estrogen receptorinteracting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proc. Natl. Acad. Sci. U S A 2002, 99(23):14783-14788. [66] Madak-Erdogan Z, Kieser KJ, Kim SH, Komm B, Katzenellenbogen JA, Katzenellenbogen BS: Nuclear and extranuclear pathway inputs in the regulation of global gene expression by estrogen receptors. Mol. Endocrinol. 2008, 22(9):2116-2127. [67] Varricchio L, Migliaccio A, Castoria G, Yamaguchi H, de Falco A, Di Domenico M, Giovannelli P, Farrar W, Appella E, Auricchio F: Inhibition of estradiol receptor/Src association and cell growth by an estradiol receptor alpha tyrosine-phosphorylated peptide. Mol. Cancer Res. 2007, 5(11):1213-1221. [68] Acconcia F, Barnes CJ, Kumar R: Estrogen and tamoxifen induce cytoskeletal remodeling and migration in endometrial cancer cells. Endocrinology 2006, 147(3):1203-1212.

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[69] Hiscox S, Morgan L, Green TP, Barrow D, Gee J, Nicholson RI: Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells. Breast Cancer Res. Treat. 2005:1-12. [70] Morgan L, Gee J, Pumford S, Farrow L, Smith C, Robertson J, Ellis I, Kawakatsu H, Nicholson RI, Hiscox S: Elevated Src kinase activity attenuates endocrine response in MCF7 breast cancer cells and is associated with poor endocrine response clinically. submitted 2008. [71] Herynk MH, Beyer AR, Cui Y, Weiss H, Anderson E, Green TP, Fuqua SA: Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor-positive human breast cancer cells. Mol. Cancer Ther. 2006, 5(12):3023-3031. [72] Planas-Silva MD, Hamilton KN: Targeting c-Src kinase enhances tamoxifen's inhibitory effect on cell growth by modulating expression of cell cycle and survival proteins. Cancer Chemother Pharmacol 2006. [73] Hennequin LF, Allen J, Breed J, Curwen J, Fennell M, Green TP, Lambert-van der Brempt C, Morgentin R, Norman RA, Olivier A et al.: N-(5-chloro-1,3-benzodioxol-4yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. J. Med. Chem. 2006, 49(22):6465-6488. [74] Hiscox S, Jordan NJ, Smith C, James M, Morgan L, Taylor KM, Green TP, Nicholson RI: Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells. Breast Cancer Res. Treat. 2008. [75] Riggins RB, Thomas KS, Ta HQ, Wen J, Davis RJ, Schuh NR, Donelan SS, Owen KA, Gibson MA, Shupnik MA et al.: Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b. Cancer Res. 2006, 66(14):7007-7015. [76] Planas-Silva MD, Bruggeman RD, Grenko RT, Stanley Smith J: Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer. Biochem. Biophys. Res. Commun. 2006, 341(1):73-81. [77] Hiscox S, Morgan L, Green T, Nicholson RI: Reduction of in vitro metastatic potential of tamoxifen-resistant breast cancer cells following inhibition of src kinase activity by AZD0530. Eur. J. Cancer 2004, 2(8):121-122. [78] Fan P, Wang J, Santen RJ, Yue W: Long-term Treatment with Tamoxifen Facilitates Translocation of Estrogen Receptor {alpha} out of the Nucleus and Enhances its Interaction with EGFR in MCF-7 Breast Cancer Cells. Cancer Res.. 2007, 67(3):13521360. [79] Hiscox S, Jordan NJ, Morgan L, Green TP, Nicholson RI: Src kinase promotes adhesion-independent activation of FAK and enhances cellular migration in tamoxifenresistant breast cancer cells. Clin. Exp. Metastasis 2007, 24(3):157-167. [80] Wadhawan A, Smith C, Jordan NJ, Barrett-Lee P, Hiscox S: Inhibition of Src kinase restores cell-cell adhesion and suppresses the aggressive phenotype of endocrineresistant breast cancer cells. Breast Cancer Res. 2008, Sumbitted.

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[81] Mitra SK, Schlaepfer DD: Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr. Opin. Cell Biol. 2006, 18(5):516-523. [82] Summy JM, Gallick GE: Treatment for advanced tumors: SRC reclaims center stage. Clin. Cancer Res. 2006, 12(5):1398-1401. [83] Trevino JG, Summy JM, Lesslie DP, Parikh NU, Hong DS, Lee FY, Donato NJ, Abbruzzese JL, Baker CH, Gallick GE: Inhibition of SRC expression and activity inhibits tumor progression and metastasis of human pancreatic adenocarcinoma cells in an orthotopic nude mouse model. Am. J. Pathol. 2006, 168(3):962-972. [84] Han LY, Landen CN, Trevino JG, Halder J, Lin YG, Kamat AA, Kim TJ, Merritt WM, Coleman RL, Gershenson DM et al.: Antiangiogenic and antitumor effects of SRC inhibition in ovarian carcinoma. Cancer Res. 2006, 66(17):8633-8639. [85] Ali N, Yoshizumi M, Yano S, Sone S, Ohnishi H, Ishizawa K, Kanematsu Y, Tsuchiya K, Tamaki T: The novel Src kinase inhibitor M475271 inhibits VEGF-induced vascular endothelial-cadherin and beta-catenin phosphorylation but increases their association. J. Pharmacol. Sci. 2006, 102(1):112-120.

In: Drug Resistant Neoplasms Editors: Ethan G. Verrite

ISBN: 978-1-60741-255-7 ©2009 Nova Science Publishers, Inc.

Chapter VIII

Systematic Analysis of Patterns of Cross Resistance between Anticancer Agents

1

Britta Stordal∗1 and Ross Davey2 National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland 2 Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital and the University of Sydney, St. Leonards NSW 2065, Australia

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Abstract The cross resistance relationship between two chemotherapy agents is often decided by testing a new chemotherapy agent in cells resistant to an older agent. This can be very useful in determining the role for the newer agent and indeed be part of the process to approve the newer agent for use in the clinic. However, if only a few cell models are analysed, this can lead to incorrect conclusions about the activity of a given agent. The most rigorous method to evaluate the pattern of cross resistance between two chemotherapy agents is to perform a systematic review of all drug-resistant cell models reporting toxicity data for the two agents in question. This systematic review process must be updated regularly and fulfill certain criteria to be a valid analysis of the crossresistance relationship between two agents. We have performed systematic reviews examining the cross-resistance relationships between the platinums (cisplatin, carboplatin and oxaliplatin) and the taxanes (paclitaxel and docetaxel). In this process we have observed three broad patterns of cross-resistance relationships: complete cross resistance, incomplete cross resistance and inverse resistance. These different patterns can give insight into mechanisms of resistance and suggest the sequence in which drugs should be administered in the clinical treatment of cancer. ∗

Corresponding Author: Dr. Britta Stordal, National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Email: [email protected]

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Introduction The development of drug resistance is the primary reason that chemotherapy fails to cure cancer. We can improve our understanding of drug resistance as it occurs in the clinical treatment of cancer through the analysis of drug-resistant cell models. Acquired drugresistant cell models are developed in the laboratory by repeatedly exposing cancer cells in culture to chemotherapy. The surviving resistant cells are then compared to the parental sensitive cells using a cell viability assay such as the MTT or clonogenic assay. The sensitivity of these paired cell lines to any given chemotherapy agent is usually determined by exposing them to a range of drug concentrations and then assessing cell viability. The IC50 (drug concentration causing 50% growth inhibition) for these paired cell lines can be used to determine the increase in resistance known as fold resistance by the following equation:

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Fold Resistance = IC50 of Resistant Cell Line / IC50 of Parental Cell Line Some drug-resistant cell lines are developed in a clinically relevant way, using lower doses of a drug and delivering the drug in a pulsed manner to mimic the cycles of chemotherapy given in the clinic. This allows the cells to recover in a drug-free environment. Continuous drug treatment and escalating the dose of drug will produce more highly drugresistant models. These highly-resistant models are often more stably resistant in culture than their low-level counterparts. However, although these are valuable models for studying potential resistance mechanisms, they are less appropriate for studying the activity of drugs at clinically relevant levels of resistance. The cross-resistance relationship between two chemotherapy agents is often determined by testing a new chemotherapy agent in cells resistant to an older agent. This can be very useful in determining the role for the newer agent and indeed be part of the process to approve the newer agent for use in the clinic. However, if only a few cell models are analysed this can lead to incorrect conclusions about the activity of a given agent. An example of this is the cross-resistance relationship between cisplatin and oxaliplatin, where highly drugresistant models were first examined [1]; this will be discussed in further detail later in the chapter. The most rigorous method to evaluate the pattern of cross resistance between two chemotherapy agents is to perform a systematic review of all drug-resistant cell models reporting toxicity data for the two agents in question. This systematic review process must be updated regularly and fulfill certain criteria to be a valid analysis of the cross resistance relationship between two agents. The methodology of the systematic review process is described below using a comparison between two hypothetical drugs, Drug A and Drug B (Figure 1). Medline and/or Pubmed was systematically searched for all papers reporting resistance data for Drug A and Drug B in cellular models of acquired drug resistance. The following keywords were used: “Drug A”, “Drug B”, any other commonly-used names for Drug A and Drug B such as trade names, resistant, resistance, cross-resistance, toxicity, and cell line. The search results were then limited to the English language and review articles and clinical trials removed.

Systematic Analysis of Patterns of Cross Resistance between Anticancer Agents 167

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Figure 1. Cross Resistance Analysis of Drug A vs. Drug B. The dotted line at 1 indicates the level of resistance of the parental cell lines. The solid line at 2 indicates the level of clinical drug resistance. The dotted and dashed line at 10 indicates the boundary between high and low-level resistance. Cell models are divided up into four categories. Cross resistant, >2 fold resistant to both agents ({). Non-cross resistant > 2 fold resistant to one agent, no change to the other (z). Hypersensitive, > 2 fold resistant to one agent, sensitivity (