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Tumor Microenvironment: Signaling Pathways – Part B [1st ed.]
 9783030471880, 9783030471897

Table of contents :
Front Matter ....Pages i-xiii
Neuregulin Signaling in the Tumor Microenvironment (Ruxue Jia, Hu Zhao, Shuiliang Wang)....Pages 1-29
HGF/c-Met Signalling in the Tumor Microenvironment (Alberto Zambelli, Giuseppe Biamonti, Angela Amato)....Pages 31-44
Eph/Ephrin Signaling in the Tumor Microenvironment (Katsuaki Ieguchi, Yoshiro Maru)....Pages 45-56
SRC Signaling in Cancer and Tumor Microenvironment (Ayse Caner, Elif Asik, Bulent Ozpolat)....Pages 57-71
Purinergic Signaling Within the Tumor Microenvironment (Dobrin Draganov, Peter P. Lee)....Pages 73-87
TGFβ Signaling in the Tumor Microenvironment (Cassandra Ringuette Goulet, Frédéric Pouliot)....Pages 89-105
Wnt Signaling in the Tumor Microenvironment (Yongsheng Ruan, Heather Ogana, Eunji Gang, Hye Na Kim, Yong-Mi Kim)....Pages 107-121
Lysophospholipid Signalling and the Tumour Microenvironment (Wayne Ng, Andrew Morokoff)....Pages 123-144
Adenosine Signaling in the Tumor Microenvironment (Luca Antonioli, Matteo Fornai, Carolina Pellegrini, Vanessa D’Antongiovanni, Roberta Turiello, Silvana Morello et al.)....Pages 145-167
Androgen Signaling in the Tumor Microenvironment (Berna C. Özdemir)....Pages 169-183
Back Matter ....Pages 185-191

Citation preview

Advances in Experimental Medicine and Biology 1270

Alexander Birbrair  Editor

Tumor Microenvironment Signaling Pathways – Part B

Advances in Experimental Medicine and Biology Volume 1270 Series Editors Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux UMR 5287, Pessac Cedex, France Haidong Dong, Department of Urology and Immunology, Mayo Clinic, Rochester, IN, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Sciences, School of Life Science, Shanghai University, Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2018Impact Factor: 2.126. More information about this series at http://www.springer.com/series/5584

Alexander Birbrair Editor

Tumor Microenvironment Signaling Pathways – Part B

Editor Alexander Birbrair Department of Radiology Columbia University Medical Center New York, NY, USA Department of Pathology Federal University of Minas Gerais Belo Horizonte, MG, Brazil

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-47188-0    ISBN 978-3-030-47189-7 (eBook) https://doi.org/10.1007/978-3-030-47189-7 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

This book is dedicated to my mother, Marina Sobolevsky, of blessed memory, who passed away during the creation of this volume. Professor of Mathematics at the State University of Ceará (UECE), she was loved by her colleagues and students, whom she inspired by her unique manner of teaching. All success in my career and personal life I owe to her.

My beloved mom Marina Sobolevsky of blessed memory (July 28, 1959–June 3, 2020)

Preface

This book’s initial title was “Tumor Microenvironment.” However, due to the current great interest in this topic, we were able to assemble more chapters than would fit in one book, covering tumor microenvironment biology from different perspectives. Therefore, the book was subdivided into several volumes. This book “Tumor Microenvironment: Signaling Pathways – Part B” presents contributions by expert researchers and clinicians in the multidisciplinary areas of medical and biological research. The chapters provide timely detailed overviews of recent advances in the field. This book describes the major contributions of different signaling pathways in the tumor microenvironment during cancer development. Further insights into these mechanisms will have important implications for our understanding of cancer initiation, development, and progression. The authors focus on the modern methodologies and the leading-edge concepts in the field of cancer biology. In recent years, remarkable progress has been made in the identification and characterization of different components of the tumor microenvironment in several tissues using state-of-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of cancer progression within different organs. Thus, the present book is an attempt to describe the most recent developments in the area of tumor biology which is one of the emergent hot topics in the field of molecular and cellular biology today. Here, we present a selected collection of detailed chapters on what we know so far about the signaling pathways in the tumor microenvironment in various tissues. Ten chapters written by experts in the field summarize the present knowledge about distinct signaling pathways during tumor development. Shuiliang Wang and colleagues from Xiamen University School of Medicine discuss how neuregulin signaling shapes the tumor microenvironment. Angela Amato and colleagues from the Italian National Research Council describe the role of HGF/c-Met signaling in the tumor microenvironment. Katsuaki Ieguchi and Yoshiro Maru from Tokyo Women’s Medical University update us with what we know about Eph/ephrin signaling in the tumor microenvironment. Bulent Ozpolat and colleagues from The University of Texas MD Anderson Cancer Center summarize current knowledge on Src signaling in cancer and tumor microenvironment. Dobrin Draganov and Peter P. Lee from City of Hope Comprehensive Cancer Center address the importance of purinergic signaling within the tumor microenvironment. Cassandra Ringuette Goulet and Frédéric Pouliot from Laval University focus on how vii

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TGFβ signaling is contributing in the tumor microenvironment. Yong-Mi Kim and colleagues from the University of Southern California introduce our current knowledge about Wnt signaling in the tumor microenvironment. Wayne Ng and Andrew Morokoff from the University of Melbourne talk about the lysophospholipid signaling in the tumor microenvironment. Luca Antonioli and colleagues from the University of Pisa focus on the role of adenosine signaling in the tumor microenvironment. Finally, Berna C. Özdemir from Lausanne University Hospital gives an overview of the androgen signaling in the tumor microenvironment. It is hoped that the articles published in this book will become a source of reference and inspiration for future research ideas. I would like to express my deep gratitude to my wife Veranika Ushakova and Mr. Murugesan Tamilselvan from Springer, who helped at every step of the execution of this project. Belo Horizonte, MG, Brazil Alexander Birbrair

Preface

Contents

1 Neuregulin Signaling in the Tumor Microenvironment ��������������   1 Ruxue Jia, Hu Zhao, and Shuiliang Wang 2 HGF/c-Met Signalling in the Tumor Microenvironment��������������  31 Alberto Zambelli, Giuseppe Biamonti, and Angela Amato 3 Eph/Ephrin Signaling in the Tumor Microenvironment��������������  45 Katsuaki Ieguchi and Yoshiro Maru 4 SRC Signaling in Cancer and Tumor Microenvironment������������  57 Ayse Caner, Elif Asik, and Bulent Ozpolat 5 Purinergic Signaling Within the Tumor Microenvironment��������  73 Dobrin Draganov and Peter P. Lee 6 TGFβ Signaling in the Tumor Microenvironment������������������������  89 Cassandra Ringuette Goulet and Frédéric Pouliot 7 Wnt Signaling in the Tumor Microenvironment�������������������������� 107 Yongsheng Ruan, Heather Ogana, Eunji Gang, Hye Na Kim, and Yong-Mi Kim 8 Lysophospholipid Signalling and the Tumour Microenvironment���������������������������������������������������������������������������� 123 Wayne Ng and Andrew Morokoff 9 Adenosine Signaling in the Tumor Microenvironment���������������� 145 Luca Antonioli, Matteo Fornai, Carolina Pellegrini, Vanessa D’Antongiovanni, Roberta Turiello, Silvana Morello, György Haskó, and Corrado Blandizzi 10 Androgen Signaling in the Tumor Microenvironment ���������������� 169 Berna C. Özdemir Index���������������������������������������������������������������������������������������������������������� 185

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Contributors

Angela  Amato  Institute of Molecular Genetics (IGM); National Research Council (CNR), Pavia, Italy Luca Antonioli  Unit of Pharmacology and Pharmacovigilance, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy Elif Asik  Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA Giuseppe  Biamonti Institute of Molecular Genetics (IGM); National Research Council (CNR), Pavia, Italy Corrado  Blandizzi Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy Ayse Caner  Cancer Research Center, Ege University, Izmir, Turkey Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Vanessa  D’Antongiovanni Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy Dobrin Draganov  Calidi Biotherapeutics, San Diego, CA, USA Matteo  Fornai Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy Eunji Gang  Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA Cassandra Ringuette Goulet  Oncology Division, CHU de Québec Research Center, Quebec, QC, Canada Department of Surgery, Faculty of Medicine, Laval University, Quebec, QC, Canada György  Haskó  Department of Anesthesiology, Columbia University, New York, NY, USA Katsuaki Ieguchi  Department of Pharmacology, Tokyo Women’s Medical University, Tokyo, Japan

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Ruxue  Jia Fujian Provincial Key Laboratory of Transplant Biology, Affiliated Dongfang Hospital, Xiamen University School of Medicine, Fuzhou, Fujian Province, China Department of Urology, the 900th Hospital of the Joint Logistics Team, Fujian Medical University, Fuzhou, Fujian Province, China Hye Na Kim  Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA Yong-Mi Kim  Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA Peter P. Lee  Department of Immuno-Oncology, City of Hope Comprehensive Cancer Center, Duarte, CA, USA Yoshiro  Maru Department of Pharmacology, Tokyo Women’s Medical University, Tokyo, Japan Silvana Morello  Department of Pharmacy, University of Salerno, Fisciano, Italy Andrew  Morokoff  Centre for Medical Research, Department of Surgery, Royal Melbourne Hospital, Parkville, VIC, Australia Department of Surgery, Royal Melbourne Hospital Campus, University of Melbourne, Parkville, VIC, Australia Wayne  Ng  Department of Neurosurgery, Gold Coast University Hospital, Hospital Boulevard, Southport, QLD, Australia School of Medicine, Griffith University, Gold Coast Campus, Parkwood, QLD, Australia Heather  Ogana Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA Berna C. Özdemir  Department of Oncology, Lausanne University Hospital, Lausanne, Switzerland International Cancer Prevention Institute, Epalinges, Switzerland Bulent  Ozpolat  Department of Gynecologic Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Center for RNA Interference and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Carolina Pellegrini  Department of Pharmacy, University of Pisa, Pisa, Italy Frédéric  Pouliot Oncology Division, CHU de Québec Research Center, Quebec, QC, Canada

Contributors

Contributors

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Department of Surgery, Faculty of Medicine, Laval University, Quebec, QC, Canada Department of Surgery, CHU de Québec Research Center – Laval University, Quebec, QC, Canada Yongsheng  Ruan Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou, China Roberta Turiello  Department of Pharmacy, University of Salerno, Fisciano, Italy Shuiliang Wang  Fujian Provincial Key Laboratory of Transplant Biology, Affiliated Dongfang Hospital, Xiamen University School of Medicine, Fuzhou, Fujian Province, China Department of Urology, the 900th Hospital of the Joint Logistics Team, Fujian Medical University, Fuzhou, Fujian Province, China Alberto  Zambelli Unit of Oncology, Ospedale Papa Giovanni XXIII, Bergamo, Italy Hu Zhao  Fujian Provincial Key Laboratory of Transplant Biology, Affiliated Dongfang Hospital, Xiamen University School of Medicine, Fuzhou, Fujian Province, China Department of Urology, the 900th Hospital of the Joint Logistics Team, Fujian Medical University, Fuzhou, Fujian Province, China

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Neuregulin Signaling in the Tumor Microenvironment Ruxue Jia, Hu Zhao, and Shuiliang Wang

Abstract

Keyword

Neuregulins, members of the largest subclass of growth factors of the epidermal growth factor family, mediate a myriad of cellular functions including survival, proliferation, and differentiation in normal tissues through binding to receptor tyrosine kinases of the ErbB family. However, aberrant neuregulin signaling in the tumor microenvironment is increasingly recognized as a key player in initiation and malignant progression of human cancers. In this chapter, we focus on the role of neuregulin signaling in the hallmarks of cancer, including cancer initiation and development, metastasis, as well as therapeutic resistance. Moreover, role of neuregulin signaling in the regulation of tumor microenvironment and targeting of neuregulin signaling in cancer from the therapeutic perspective are also briefly discussed.

Neuregulins (NRGs)/heregulins (HRGs) · Signaling pathway · Paracrine · Tumor microenvironment (TME) · Receptor tyrosine kinases (RTKs) · ErbB family · Tumor biology · Tumorigenesis · Development of cancer · Epithelial–mesenchymal transition (EMT) · Metastasis · Genetics · Epigenetics · Therapeutic resistance · Target therapy

R. Jia · H. Zhao · S. Wang (*) Fujian Provincial Key Laboratory of Transplant Biology, Affiliated Dongfang Hospital, Xiamen University School of Medicine, Fuzhou, Fujian Province, China Department of Urology, the 900th Hospital of the Joint Logistics Team, Fujian Medical University, Fuzhou, Fujian Province, China e-mail: [email protected]

1.1

Introduction

Over the past two decades, tumor has increasingly been recognized as organ that results from the co-evolution of malignant cells and their direct environment [1, 2]. The tumor microenvironment (TME) encompasses extracellular matrix (ECM) and various non-transformed cells including fibroblasts, immune infiltrates, and vascular vessels recruited from nearby local or distant tissues [3]. Through providing matrices, cytokines, growth factors, as well as vascular networks for nutrient and waste exchange, the TME plays an essential role in tumor initiation, progression, invasion, metastasis, and resistance to therapy [4]. Cumulative studies have demonstrated that the cross talk between cancer cells and their TME

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_1

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R. Jia et al.

The structures and distribution of different isoforms of NRG had been well reviewed [8, 11]. All NRGs share a typical EGF-like domain, an important structure which it alone is sufficient for receptor binding and the activation of intracellular signaling pathways. This extracellular domain contains about 50 amino acids and is characterized by 3 pairs of cysteines that are important for its tertiary structure and biological function. The differences in EGF-like domain define the α and β isoforms of NRG. Among the four members of NRG gene, NRG-1 is the best characterized one whose biological functions had been extensively studied. Transcribed from the same NRG-1 gene located on human chromosome 8p12, the isoforms of NRG-1 mRNA differ in their coding segment composition due to initiation of transcription from different NRG-1 gene promoters and alternative splicing which give rise to six types of NRG-1 (types I, II, III, IV, V, and VI). These different types of NRG-1 are also divided based on the differences in their extracellular amino-terminal domains with types I, II, IV, and V sharing an immunoglobulin-like (IgG-like) 1.2 Overview of Neuregulin domain followed by a glycosylation-rich region Signaling (type I) or a GGF-specific (kringle) domain (type NRGs are a family of structurally related signal- II) while type III of NRG-1 containing a cysteine-­ ing proteins that mediate cell–cell interactions in rich domain (CRD) that loops back intracellua broad spectrum of tissues including breast, larly along with its N-terminal sequence. In heart, nervous system, and others [11, 12]. addition, whether the isoform is initially syntheIdentified independently over two decades ago by sized as a transmembrane or nonmembrane proseveral different research groups, these peptide tein adds more dimensionality to different growth factors were originally described as neu isoforms of NRG.  The human NRG-2 gene differentiation factor (NDF) [13], heregulins located on chromosome 5q31.2 encodes six iso(HRGs), acetylcholine receptor-inducing activity forms of NRG-2 (α, β, αν, βν, α*1, and α*2) [21, (ARIA) [14], glial growth factors (GGFs) [15], 28–30]. The human NRG-3 and NRG-4 genes and sensory and motor neuron-derived factor were mapped to chromosome 10q23.1 and (SMDF) [16]. NRG was first characterized in rat 15q24.2, respectively [31, 32]. While the NRG-1 and human as a putative ligand for the ErbB2 [13, transcripts were evidenced to be broadly 17, 18]; however, it was later showed that the expressed in multiple tissues, both NRG-2 and actual receptors of NRG were ErbB3 and ErbB4 NRG-3 were found to be mainly expressed in the [19, 20]. Currently, 4 NRGgenes, NRG-1, NRG-­ nervous system. Although the expression of 2, NRG-3, and NRG-4, encoding more than 30 NRG-4 mRNA has only been detected in the pandifferent isoforms through multiple promoter creas and to a lesser extent in muscle [24], NRG-4 usage and alternative splicing, have been protein expression was found in bladder as well described [7, 21–24]. Moreover, studies are as prostate cancer [32, 33]. Structurally, NRG-4 ­continually revealing that the family of NRGs is distinct from other NRGs, which contain no may be larger than currently known [25–27]. recognizable motifs other than the EGF-like involves reciprocal juxtacrine and paracrine signaling pathways. Among them, neuregulin (NRG) signaling has long been recognized as an important player in regulating tumor progression [5]. Neuregulins (NRGs) are members of the largest subclass of polypeptide factors that bind to receptor tyrosine kinases of the ErbB family and mediate a myriad of cellular functions including survival, proliferation, and differentiation in normal tissues [6]. However, there is mounting evidence that dysregulated neuregulin signaling plays important role in initiation and development of a variety of human cancers via regulating cancer cells and/or the TME [7, 8]. Thus, the neuregulin signaling is emerging as therapeutic target in exploring novel strategy against cancer [9, 10]. The aim of this chapter is to discuss how neuregulin signaling takes an important part in regulating the tumor biology. We also address the targeting of neuregulin signaling in cancer from the therapeutic perspective.

1  Neuregulin Signaling in the Tumor Microenvironment

domains. Most NRGs are synthesized as large transmembrane precursor proteins with the EGF-­ like domain connected to the transmembrane domain by a juxtamembrane linker. These NRGs can be activated and released from the membrane through proteolytic processing, which has also been extensively studied and reviewed [34–37]. In the current body of knowledge, NRGs mainly elicit their biological functions via binding of ErbB3 or ErbB4 in an autocrine, paracrine, or juxtacrine way (Fig. 1.1) [5, 38, 39]. The ErbB subfamily of transmembrane receptor tyrosine kinases (RTKs) consists of four closely related transmembrane receptors: ErbB1 (also known as EGFR or HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) [40]. Among all four ErbB subfamily members, NRGs can directly bind to the ErbB3 and ErbB4 receptors. All products of four NRG genes can bind ErbB4, whereas only NRG-1 and NRG-2 proteins can bind ErbB3 [41, 42]. Binding of ligands to the extracellular domain of ErbB3 or ErbB4 induces the formation of kinase active homo- or hetero-oligomers, which further induces transphosphorylation of the ErbB dimer partner and stimulates down-

Fig. 1.1  Schematic diagram of acting models of NRGs

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stream intracellular pathways including RAS/ RAF/MEK/ERK, PI3K/Akt/mTOR, Src kinases, and JAK/STAT [43, 44]. To date, there is no known ligand for ErbB2; however, ErbB2 is constantly in a conformation that resembles a ligandactivated state and favors dimerization [45, 46]. Unlike other ErbB receptors, ErbB3 can bind ATP and catalyze autophosphorylation, whereas it has a weak kinase activity [47]. Interestingly, ErbB3 possesses most tyrosine residues in its intracellular domain ready to be phosphorylated. It must interact with other RTKs to exhibit its biological functions [48]. Among many interactive partners of ErbB3, ErbB2 is the most important one [49]. Upon ligand binding, ErbB3 triggers the formation of ErbB2/ErbB3 heterodimer, in which ErbB3 benefits from ErbB2’s strong kinase activity for phosphorylation on its intracellular tyrosine residues. Thus, the “dumb” ErbB3 (weak kinase activity, but has ligands) and the “deaf” ErbB2 (no known ligand, but has kinase activity) make a perfect sense to form a potent ErbB2/ErbB3 heterodimer leading to activation of the downstream signaling pathways (Fig.  1.2) [42]. Accordingly, co-expression of

R. Jia et al.

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Fig. 1.2  Schematic diagram of NRG signalings and their biological consequences

ErbB3 and ErbB2 in normal or cancer tissues is frequently observed [50, 51], and ErbB3 has been increasingly evidenced to play a critical role in ErbB2-mediated cancer development as well as therapeutic resistance [52–56].

1.3

 ole of Neuregulin Signaling R in Hallmarks of Cancer

NRG is initially identified as an important regulator in the development of nervous system [57]; however, NRG-induced signaling has also been shown to play a pivotal role in a wide variety of tissues, including breast [58, 59], heart [60], lung [61], muscle [62], and stomach [63]. Given the importance of ErbB receptors in hallmarks of cancer [64, 65], it has been evidenced that aberrant activation of the NRG signaling acts as a key

player in the development and progression of various human cancers, which will be discussed in detail in the following section of this chapter (Table 1.1) [5, 7, 8].

1.3.1 R  ole of Neuregulin Signaling in Cancer Initiation and Development Cancer is a genetic disease. Several lines of evidence indicate that tumorigenesis is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives [66]. Among the eight distinctive and complementary capabilities—hallmarks of cancer— enabling tumor growth and metastatic dissemination proposed by Hanahan et al. [2, 66],

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Table 1.1  Neuregulin genes, products, and expression in human tumors NRG gene NRG-1

Location on chromosome 8p12

Protein product NRG-1/HRG-1/NDF/ ARIA/GGF/SMDF

Type of tumor Breast cancer Colon cancer

NRG-2

5q31.2

NRG-2/HRG-2

NRG-3 NRG-4

10q23.1 15q24.2

NRG-3/HRG-3 NRG-4/HRG-4

Endometrial cancer Gastric cancer Glioma Hepatocellular carcinoma Lung cancer Medulloblastoma Melanoma Ovarian cancer Pancreatic cancer Papillary thyroid cancer Prostate cancer Squamous cell carcinomas of the head and neck Vestibular schwannoma Breast cancer Schwannoma Breast cancer Bladder cancer Prostate cancer

Representative references Aguilar et al. [83] Venkateswarlu et al. [124] Srinivasan et al. [148] Noguchi et al. [63] Westphal et al. [139] Hsieh et al. [149] Gollamudi et al. [106] Gilbertson et al. [150] Buac et al. [151] Gilmour et al. [152] Kolb et al. [154] Fluge et al. [155] Leung et al. [157] Shames et al. [158] Hansen et al. [141] Carraway et al. [21] Stonecypher et al. [142] Hijazi et al. [159] Memon et al. [32] Hayes et al. [33]

NRG neuregulin, HRG heregulin; NDF, neu differentiation factor,;ARIA, acetylcholine receptor-inducing activity, GGF, glial growth factor, SMDF, sensory and motor neuron-derived factor

sustaining proliferative signaling is fundamental in cancer initiation and development. In normal tissues, the production and release of growth-promoting signals governing cell growth and proliferation is strictly controlled, thereby ensuring a homeostasis of cell number and thus maintenance of normal tissue architecture and function. In contrast, malignant cells acquire the ability to sustain proliferative signaling via accumulating genetic as well as epigenetic alterations. The mitogenic signals are largely conveyed by growth factors that bind cell surface receptors such as RTKs, typically containing intracellular tyrosine kinase domains. The RTKs proceed to elicit signals via branched intracellular signaling pathways that regulate progression through the cell cycle as well as cell growth [67, 68]. Over the past several decades, it has become evident the ErbB subfamily members of RTKs

have a prominent role in the initiation and development of several types of cancer. The evidence for a role of ErbB receptorsin cancer was first inferred from a study on ErbB2, a human ortholog of rat Neu, as forced overexpression of human ErbB2 was shown to transform diploid cells [69]. Almost at the same time, the EGFR was initially identified as an oncogene owing to its homology to v-erb-B [70]. Overexpression of wild-type Neu or ErbB2 under the control of a mammary-­ specific promoter was latterly shown to lead to metastatic mammary tumors in transgenic mice [71, 72]. Consistently, the amplification and/or overexpression of ErbB2 was found to be significantly and independently associated with a worse prognosis for breast cancer patients [73, 74]. Less frequent activating mutations of ErbB2 in several cancer types without gene amplification were further unveiled [75]. Comparably, the causal role of EGFR in tumorigenesis of a rela-

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tively broad spectrum of cancers was well recognized thereafter its first identification as oncogene [76–78]. Although mutations in both ErbB3 and ErbB4 were identified in human cancers [79–81], the involvement of ErbB3 and ErbB4  in cancer was mainly revealed as partners in promoting signaling from EGFR and ErbB2 currently [44]. As aforementioned, most of the time, homoor hetero-dimerization of ErbB receptors triggered by binding of ligand is prerequisite for fulfilling their biological function. Ever since NRGs were identified as the ligands of ErbB3 and/or ErbB4, the role of NRGs in cancer initiation and development has attracted much research interest. While forced overexpression of NRGs in the mammary gland of transgenic mice was shown to provoke the development of breast adenocarcinomas [82], NRGs were also evidenced to function as potent stimulators of proliferation of both malignant human breast and ovarian epithelial cells in vitro [83]. Moreover, development of an autocrine NRG signaling loop results in transformation of breast epithelial cells into malignant derivates [84]. Consistently, NRG is overexpressed in about 30% of breast cancer biopsies that do not overexpress ErbB2, and this overexpression is sufficient to promote tumorigenesis and metastasis of breast cancer cells [85, 86]. In addition to growth factors, the development of breast cancer is also regulated by a plethora of signals mediated by steroid receptors such as estrogen receptor (ER) and progestogen receptor (PR) [87, 88]. Importantly, although NRG was showed to induce an estrogen-independent phenotype of breast cancer cells [85, 89], the interactions between progestins and NRG signaling pathways were unveiled in the development of mouse mammary adenocarcinomas [90]. NRG not only induces transcriptional activation of the progesterone receptor via an ErbB2-dependent manner in breast cancer cells [91] but also drives breast cancer growth through the co-option of progesterone receptor signaling [92]. Mechanistically, while the extracellular region of NRG was shown to promote mammary gland proliferation and tumorigenesis [93], a recent study demonstrated that the Ig-like region of NRGs may exert an important role in their capa-

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bility to activate ErbB receptors and mitogenic responses [94]. Consequently, circulating NRG1 has been demonstrated to be additional biomarkers indicative of prognosis and outcomes for breast cancer patients [95]. Lung cancer remains the most commonly diagnosed cancer and the leading cause of cancer-­ related deaths worldwide [96]. Of all pathological types, non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers [97]. Extensive genomic characterization of NSCLC has led to the identification of molecular subtypes of NSCLC that are oncogene addicted, including overexpression and/or activating mutations in epidermal growth factor receptor (EGFR) [98]. However, there is mounting evidence that substantial molecular and clinical heterogeneity exists within oncogenic driver-defined subgroups of NSCLC [99, 100]. In comparison with a higher prevalence of EGFR mutations in NSCLC [78], the aberrations of other ErbB subfamily members have also been observed in NSCLC but with a relative low frequency [101]. The mutations of ErbB2, ErbB3, and ErbB4 were reported to occur in about 4% [102], 1% [79], and 5.4% [103] of human primary lung tumors, respectively. Nevertheless, in addition to EGFR, the overexpression of other ErbB subfamily members was frequently detected in NSCLC [102, 104]. While ErbB2, ErbB3, ErbB4, and NRG were found to be differentially expressed in normal bronchial epithelial and NSCLC cell lines [105], an autocrine activation of ErbB2/ErbB3 receptor complex by NRG-1 was further unraveled in NSCLC cells [106]. These studies raised the potential role of NRG signaling in initiation and development of NSCLC. More importantly, through transcriptome sequencing of 25 lung adenocarcinomas of never smokers, a novel somatic gene fusion, CD74–NRG1, was identified [107]. The CD74– NRG1 was demonstrated to give rise to the extracellular expression of the EGF-like domain of NRG1 III-β3 which acts as the ligand for ErbB2/ EebB3 receptor complexes. Thereafter, NRG1 fusions were reported in different populations with different frequencies in NSCLC [108–111]. In addition to canonical NRG signaling, a recent study demonstrated that EGF and NRG may

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induce phosphorylation of ErbB3 by EGFR using was shown to induce expression of vascular distinct oligomeric mechanisms which even endothelial growth factor (VEGF) in colon canbroadens our understanding of NRG signaling in cer cells, which might further affect the growth of cancer development [112]. The increasing recog- colon cancer in vivo [126]. ErbB4 is the least recnition of the role of NRG signaling in lung cancer ognized ErbB subfamily member. However, has raised the possibility that specific inactivation phosphorylated ErbB4 and NRG have also been of NRG signaling may have therapeutic potential demonstrated to contribute to poorer patient in this malignant disease [113]. More detailed prognosis in CRC [127, 128]. information regarding the NRG signaling-­ The worldwide mortality of pancreatic cancer targeted therapy for cancer treatment will be dis- ranks the seventh among all cancers in both sexes cussed in the rear part of this chapter. accounting for about 4.5% of all cancer deaths Dysregulated ErbB signaling has also been [96]. Extensive genetic studies have revealed that reported to be involved in development of neo- during the progression of three broad stages of plasms of gastrointestinal tract, which mainly pancreatic cancer, acquired somatic mutations in include esophageal carcinoma, gastric cancer, oncogenes and tumor suppressor genes accumuliver cancer, colorectal cancer (CRC), and pan- late and account for initiation and aggressive creatic cancer accounting for more than 26% and development of this malignant disease. These 35% of all new cancer cases and deaths world- mutations occur most frequently in KRAS, wide [44, 96]. Overexpression of ErbB2 in gas- CDKN2A, TP53, and SMAD4 [129–131]. tric cancer was firstly reported in 1986 [114]. Activating mutations of the KRAS oncogene, Thereafter, accumulated data indicates the asso- which encodes a member of the RAS family of ciation of ErbB2 and/or ErbB3 overexpression GTP-binding proteins, are the most common with poor prognosis of patients with this disease genetic abnormality presenting in approximately [115–117]. While HRG-α was showed to affect 95% of pancreatic tumors analyzed [132, 133]. In epithelial cell proliferation through mesenchy- addition, wild-type KRAS is also normally actimal–epithelial interaction in the gastric mucosa vated in response to the binding of extracellular [63], an interesting study demonstrated that over- signals such as growth factors to RTKs [134]. expression of NRG1 could promote progression Accumulating evidence shows that the ErbB of gastric cancer by regulating the self-renewal of receptors are overexpressed in approximately cancer stem cells [118]. In contrast to EGFR and 60% of pancreatic cancers [135]. Recently, sevErbB2, which have been widely studied in CRC eral studies have uncovered that recurrent gene [119], the role of ErbB3 and ErbB4 in CRC is rearrangements such as NRG1 fusions are prevalargely underestimated previously. Although the lent in patients with KRAS wild-type pancreatic expression of ErbB3  in CRC was reported to cancer [136, 137]. Thus, in a subset of KRAS range from 36 to 89% [120, 121], the information wild-type pancreatic cancer cases, NRG signalregarding its prognostic role in CRC was ever ing may act as targetable oncogenic driver, prolimited. This situation changed when two groups viding a potential treatment strategy in this reported that ErbB3 overexpression was signifi- disease [138]. cantly associated with decreased time to CRC Given the role of NRG in the development of progression [122, 123]. Actually, autocrine nervous system, it is thus not surprising that aberheregulin has been demonstrated to generate rant NRG signaling could result in development growth factor independence and block apoptosis of glioma and schwannoma [139–142]. Gliomas in CRC cells as early as in 2002 [124]. are the leading cause of death among adults with Interestingly, bone marrow-derived mesenchy- primary brain malignancies due to lack of effecmal stem cells (MSCs) in TME were evidenced tive remedy [143, 144]. In an attempt to search to promote CRC progression through paracrine for novel target therapy against glioma, an RNAi-­ NRG1/ErbB3 signaling [125]. In addition to act- based inhibition of presenilin 2 was demonstrated ing on cancer cells themselves, NRG signaling to inhibit glioma cell growth and invasion via

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regulation of NRG1/ErbB signaling [145]. MicroRNA is emerging as important player in tumor biology [146]. It was revealed that the expression of miR-125a-3p was significantly decreased in malignant glioma and lower expression of miR-125a-3p was associated with a poor prognosis of patients with glioblastoma. Further mechanistic study suggested that miR-125a-3p might perform an important role in development of glioma via direct targeting of NRG-1 [147]. In addition to aforementioned cancer types, the aberrant NRG signaling was also involved in development of many other cancers derived from different tissues as listed in Table 1.1 which will not be discussed in detail [148–159].

1.3.2 R  ole of Neuregulin Signaling in Cancer Metastasis Currently, metastatic disease is largely incurable and remains the main cause of cancer-related deaths worldwide [160]. Metastasis is the end result of a multistage process that includes acquisition of invasive phenotype of primary tumor cells, local invasion, intravasation into the blood or lymphatic system, survival in circulation, arrest at a distant organ, extravasation, survival in a new environment, and adaptation and proliferation to form metastases [161]. Each of these steps in the complex metastatic cascade depends on the genetic and epigenetic alterations acquired by primary tumor cells, as well as interactions with the host microenvironment and the immune system [162, 163]. Change in cell phenotype between epithelial and mesenchymal states, defined as epithelial– mesenchymal transition (EMT), has been increasingly recognized as an initial step of tumor metastasis [164]. The EMT program is generally induced in epithelial cells by heterotypical signals, among which the transforming growth factor-β (TGFβ) family of cytokines are the best characterized inducers of EMTs [165]. In addition, RTK signaling has also been shown to play crucial roles in the induction of EMT [166]. NRG-1 was able to enhance motility and migration of human glioma cells via not only activation

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of focal adhesion kinase (FAK) [167] but also induction of expression of cell adhesion molecule L1 [168]. While the constitutive activation of ERBB3-dependent signaling driven by an NRG-1/ERBB3 autocrine mechanism was strongly associated with microscopic vascular invasion and poor prognosis of hepatocellular carcinoma (HCC) [149], a recent study further demonstrated that miR-296-5p suppressed EMT of HCC via targeting NRG1/ErbB2/ErbB3 signaling [169]. Metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant [170]. Accumulating data suggest that metastatic dissemination often occurs early during tumor formation [171, 172]. Progesterone-induced signaling was shown to trigger migration of cancer cells from early lesions shortly after HER2 activation in a HER2-driven mouse mammary tumor model which exhibited capability for early metastatic dissemination [173]. Using the same mammary tumor model, a subpopulation of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells with invasive ability to spread to target organs was identified in the early lesions of the transgenic mice [174]. These studies strongly suggested that aberrant ErbB2 signaling played a pivotal role in early dissemination of breast cancer cells. Furthermore, while the NRG expression in breast tumors was evidenced to be associated with lymph node invasion and poor patient outcome, it was demonstrated that NRG expression favored in situ tumor growth, local spreading, and metastatic dissemination via an ERK1/2 kinasedependent upregulation of collagenase-­3 [175]. Tumor-associated macrophages (TAMs), derived from recruited circulating monocytes by a wide variety of growth factors such as colony-­ stimulating factor 1 (CSF1), are critical for regulating processes of tumor including various steps in the metastatic cascade [176–179]. The first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in  vivo was provided in 2004 [180]. Macrophages are conventionally subdivided into antitumor pro-inflammatory M1 or pro-tumor immune-suppressive M2 phenotypes; however, the diversity of macrophage types in different tis-

1  Neuregulin Signaling in the Tumor Microenvironment

sues and cancers indicates that this classification is an oversimplification [181]. Accumulating data have revealed different TAM behaviors linked to their locations, including migration-associated streaming and perivascular populations [182– 184]. Perivascular macrophages are an essential component of structures termed TMEM (tumor microenvironment of metastasis) that consist of a TAM in direct contact with a Mena-overexpressing tumor cell and endothelial cell [185]. While the EGF–CSF1 paracrine loop between the tumor cells and macrophages was shown to mediate pairing and stream formation [186], it was further demonstrated that the hepatocyte growth factor (HGF) supplied by endothelial cells is required for sustained directional migration of both tumor cells and macrophages toward blood vessels [184]. Moreover, the temporal aspects of macrophage subtype specification within primary tumors and the possibility of interconversion among subtypes had been verified by Arwert and colleagues whose study indicate that a unidirectional transition from migratory to perivascular macrophage is required for tumor cell intravasation [187]. Notably, the EGF/CSF-1 paracrine invasion loop has been reported to be triggered by HRG-β1 and CXCL12 [188]. In addition, heregulin/ErbB3 signaling has also been shown to enhance breast cancer cell motility via hypoxia-inducible factor 1α (HIF-1α)-dependent upregulation of CXCR4, the receptor of SDF-1/ CXCL12 [189]. The Notch signaling pathway regulates many aspects of cancer biology, including metastasis [190]. Upon ligand binding, the transmembrane Notch receptor is cleaved sequentially, leading to the release of the Notch intracellular domain (NICD). The NICD translocates to the nucleus where it orchestrates the transcription of specific genes. Ligands of Notch receptor expressed by the signal-sending cell are transmembrane proteins of the Delta/Serrate/LAG-2 (DSL) family which comprises three delta-like ligands (Dll1, Dll3, and Dll4) and two jagged ligands (Jagged 1 and Jagged 2) in mammals. It has been demonstrated that tumor-derived Jagged 1 (JAG1) could promote osteolytic bone metastasis of breast cancer by engaging Notch signaling in bone cells

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[191]. Interestingly, stimulation of macrophages by tumor cell-derived NRG-1 results in upregulation of JAG1, which in turn enhances transendothelial migration and intravasation of breast cancer cells [192]. The cross talk between different signaling pathways may play a synergistic role in tumor biology. Through a transcriptome meta-analysis, higher number of gene fusions affecting core members of the Hippo pathway, Neurofibromatosis 1 (NF1), and NRG1 genes was shown to be an independent prognostic factor for poor survival in lung cancer [193]. A direct evidence of cross talk between Hippo and NRG signaling in regulating aggressive behavior of tumor cells was provided by Haskins and colleagues, whose study demonstrated that NRG 1-activated ErbB4 interacted with YAP to induce Hippo pathway target genes and promoted cell migration [194]. The dysregulated NRG signaling in development of lung cancer has been described in the front part of this chapter. In respect to metastasis, some significant differences in ErbB family receptor-related abnormalities have been shown in NSCLC brain metastases in comparison with primary lung tumors [195]. As aforementioned, oncogene fusions have increasingly been identified as driver mutations in lung adenocarcinoma. In an attempt to identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, Nakaoku and colleagues found that two oncogenic fusions involving NRG1 (CD74–NRG1 and SLC3A2–NRG1) occurred mutually exclusive with KRAS mutations [196]. The SLC3A2–NRG1 fusion was further demonstrated to increase cell migration via promoting ErbB2–ErbB3 phosphorylation and heteroduplex formation and activating the downstream PI3K/Akt/mTOR pathway through paracrine signaling [197].

1.3.3 R  ole of NRG Signaling in Therapeutic Resistance in Cancer Rapid progress in our understanding of cancer biology has dramatically improved our therapeu-

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tic strategies against cancer. In addition to the conventional operations, such as chemotherapy and radiotherapy, molecularly targeted therapies and immunotherapy recently emerge as the principal modes of cancer treatment. However, resistance to these therapies frequently occurs and currently represents a major clinical problem. Therapeutic resistance can be divided into two broad categories: primary (de novo) or acquired. Primary resistance is usually caused by resistance-­mediating factors pre-existing in the bulk of tumor cells, which make the therapy ineffective. Acquired resistance can develop during the treatments of cancers that are initially sensitive. Both primary and acquired resistances can occur at many levels, including increased drug efflux and decreased drug influx, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis [198]. Acquired resistance can arise through therapy-­ induced selection of a resistant subpopulation of cells that is present in the original tumors with a high degree of heterogeneity [199]. It can also be caused by mutations arising during the treatments, by increased expression of the therapeutic target, as well as through various other adaptive responses such as activation of the compensatory signaling pathways [200].

1.3.3.1 Resistance to Chemotherapy Chemotherapy, as an important conventional treatment for human cancers, usually induces cancer cell death by cytotoxicity, thereby reducing the tumor bulk [201]. The mechanism of antitumor activity of chemotherapeutic drugs is complex and involves various biological pathways, including apoptosis, autophagy, necrosis, and mitotic catastrophe [202]. Consequently, the mechanisms of chemoresistance are intricate and not fully understood. Taking into consideration that many clinically used chemotherapeutic drugs mainly exert antitumor activity via induction of apoptosis, it is understandable that cancer cells with enhanced survival signaling and/or defects in apoptotic pathways may escape from those therapies [203]. Since the PI3K/Akt pathway is an important survival signaling and readily acti-

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vated by RTKs, including ErbB3, it is rational that aberrant NRG signaling may result in chemoresistance in cancer treatment [204]. In an early study, the co-expression of ErbB2 and ErbB3  in human breast cancer cell lines was shown to be associated with an increased resistance to multiple chemotherapeutic agents, such as paclitaxel, doxorubicin, 5-fluorouracil, etoposide, and camptothecin, via activation of PI3K/ Akt signaling [205]. In our own attempt to identify the key downstream mediator of ErbB3 signaling that contributed to chemoresistance, we discovered that elevated expression of ErbB3 conferred paclitaxel resistance in ErbB2-positive breast cancer cells via PI3K/Akt-dependent upregulation of Survivin [56]. MSCs are connective tissue progenitor cells that contribute to fibrotic reactions during tissue remodeling and repair in places of wounding and inflammation. In response to chemokines from tumor cells, MSCs are continuously recruited to and become integral components of the tumor microenvironment [206, 207]. MSCs in tumor microenvironment have been shown to influence multiple hallmarks of cancer, including resistance to chemotherapy [208, 209]. More recently, we demonstrated that the aforementioned ErbB2/ ErbB3  →  PI3K/Akt  →  Survivin signaling axis underlying paclitaxel resistance in breast cancer cells could also be activated via the paracrine stimulation by MSCs-derived NRG-1 [210]. Doxorubicin is another widely used drug in chemotherapy against multiple types of cancer. It was reported that treatment with doxorubicin resulted in activation of the ErbB3–PI3K–Akt signaling cascade in ovarian cancer cells through transcription-dependent upregulation of NRG1 and specific blockade of ErbB3 enhanced the doxorubicin-induced apoptosis [211]. These data suggest that an activated NRG1/ErbB3 autocrine loop may account for doxorubicin resistance in ovarian cancer cells.

1.3.3.2 Resistance to Targeted Therapy Multiple processes of cancer initiation and development involve the progressive acquisition of genetic mutations and epigenetic abnormalities in the expression of various genes with highly

1  Neuregulin Signaling in the Tumor Microenvironment

diverse functions. Nonetheless, the observation that some cancer cells can seemingly exhibit dependence on a single oncogenic pathway or protein for their sustained proliferation and/or survival has led to a new concept referred to as oncogene addiction [212]. The notion of oncogene addiction reveals a possible “Achilles’ heel” within the cancer cells that can be therapeutically targeted [213, 214]. Owing to the improved understanding of the oncogenic driver mutations in NSCLC, targeted therapy has been extensively exploited while combating this malignant disease. Systematic analysis has revealed that almost two-thirds of patients with NSCLC harbor an oncogenic driver mutation, approximately half of whom have a therapeutically targetable lesion [215]. Although treatment with a targeted therapy improves outcomes in patients with NSCLC, responses to the therapeutic agents are generally incomplete and temporary followed by resistance [216]. Cancer cells develop several mechanisms of resistance to targeted therapy, which can be classified as “on-­ target” or “off-target” [217]. Alterations of the primary target of the drug typically result in on-­ target resistance [218]. In the circumstance of off-target resistance, activation of collateral signaling events that are parallel to, or downstream of, signaling by the driver oncoprotein bypass the requirement of the driver oncoprotein for cell survival and growth. Among the expanding spectrum of oncogenic driver mutations identified in NSCLC, somatic activating mutations in EGFR are the most common ones occurring in ~16% of patients with advanced lung adenocarcinoma [219]. Currently, two strategies including blocking antibody such as cetuximab and tyrosine kinase inhibitors (TKIs, such as gefitinib, lapatinib, and erlotinib) are mainly used in EGFR-­ targeted therapy in clinic [220]. Cumulative data have indicated that activation of NRG/ErbB3 signaling is one of the major mechanisms contributing to the resistance to EGFR-targeted therapy [221–224]. It has been reported that MET amplification leads to gefitinib resistance in lung cancer treatment by activating ErbB3 signaling [225]. Besides, NRG/ErbB3 signaling was also shown to induce resistance to lapatinib-mediated

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growth inhibition of ErbB2-amplified gastric cancer cells and cetuximab-based therapy in colorectal cancer, respectively [226, 227]. The EML4–ALK fusion gene was detected in 3% to 7% of patients with NSCLC [228, 229]. It has been demonstrated that crizotinib, an ALK inhibitor, shows remarkable antitumor effect in EML4–ALK-­positive lung cancer [230]. However, patients receiving treatment with crizotinib eventually acquire resistance to this drug. While paracrine receptor activation by ligands from the microenvironment was demonstrated to trigger resistance to ALK inhibitors in EML4–ALK lung cancer cells [231], an analysis of patient-derived cancer cell further revealed that activation of neuregulin/ErbB3 signaling accounted for crizotinib resistance in EML4–ALK lung cancer [232]. In addition, paracrine effect of NRG1 was also evidenced to drive resistance to MEK inhibitors in metastatic uveal melanoma [233]. The finding that amplification and/or overexpression of ErbB2 occurs in approximately 25% of invasive breast cancer and is significantly associated with a worse prognosis for breast cancer patients has made ErbB2 an attractive therapeutic target [73, 74, 234]. ErB2-targeted therapy in breast cancer is thus another paradigm in cancer research. As the first ErbB2-targeted agent approved by the US Food and Drug Administration (FDA), trastuzumab (also known as Herceptin, a humanized monoclonal antibody against ErbB2) has demonstrated significant activity in the treatment of both early-stage and metastatic ErbB2-­ overexpressing (ErbB2+) breast cancer [235–238]. Subsequently, lapatinib, an orally bioavailable small molecule TKI dual targeting ErbB2 and EGFR, was also approved by FDA for the treatment of patients with advanced ErbB2+ breast cancer in combination with capecitabine [239]. Unfortunately, resistance to both trastuzumab and lapatinib has greatly limited the efficacy of ErbB2-targeted therapy [240]. Numerous mechanisms including loss of phosphatase and tensin homolog (PTEN) and activating mutations in genes coding for components of the PI3K/Akt/ mTOR pathway have been proposed that may mediate de novo and acquired resistance to trastuzumab and lapatinib [241, 242]. Moreover,

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our studies and others have demonstrated that increased activation of ErbB3 signaling plays a critical role in the resistance to ErbB2-targeted therapy in breast cancer [243–245]. These findings are further confirmed by a study indicating that ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating ErbB3 [246]. Recently, to explore the influence of microenvironment signals on HER2-targeted TKI response, Watson et al. assessed the effects of >2500 different combinations of 56 soluble and 46 matrix microenvironmental proteins on outgrowth of lapatinib-treated HER2+ breast cancer cells using an emerging technology termed microenvironment microarrays (MEMA) [247]. It was demonstrated that NRG1β and hepatocyte growth factor (HGF) are the most significant factors to enhance outgrowth of HER2+ breast cancer cells in the presence of lapatinib. Specifically, NRG1β attenuated the response of the luminal-like HER2+ (L-HER2+) cells to lapatinib, whereas HGF attenuated the lapatinib sensitivity in the basal-like (HER2E) cells. This elegant work suggests that different mechanisms underlying resistance to HER2-TKI lapatinib may be due to the complexity of microenvironment as well as differences in signaling network wiring and architecture in different subtype cells. These findings reinforce the notion that tissue microenvironments are remarkably complex and intertwined in cancer biology [248].

1.3.3.3 Resistance to Other Therapy Endocrine therapy such as tamoxifen, an antiestrogen agent, is commonly used in the treatment of patients with estrogen receptor-positive (ER+) breast cancer, a subtype accounting for about 80% of all breast cancers [249]. Although patients benefit a lot from tamoxifen treatment, resistance to this agent is a serious problem in clinic [250]. In the current body of knowledge, multiple mechanisms responsible for endocrine resistance have been proposed and include deregulation of various components of the ER pathway itself [251], alterations in cell cycle regulators such as MYC or cyclin D1, and the activation of alternative signaling pathways [252]. Among these, increased expression or

aberrant activation of ErbB signaling has been extensively investigated and associated with both experimental and clinical endocrine therapy resistance [204, 253]. ER+ breast cancer patients with co-expression of ErbB2 and ErbB3 were significantly more likely to relapse on tamoxifen [254]. Direct evidence of the involvement of neuregulin/ErbB3  in tamoxifen resistance was provided by Liu et al. whose studies showed that downregulation of ErbB3 by a siRNA abrogated ErbB2-mediated tamoxifen resistance in breast cancer cells [55]. Furthermore, overexpression of HRG-β2 was reported to induce hormoneindependent phenotype of ER+ breast cancer cells and resistant to tamoxifen via constitutive activation of ErbB signaling [89].

1.4

 ole of Neuregulin Signaling R in Regulation of Tumor Microenvironment

As an important part of immune infiltrates in TME, tumor-educated macrophages at the primary site promote tumor initiation and malignant progression via supporting tumor-associated angiogenesis; enhancing tumor cell invasion, migration, and intravasation; as well as suppression of antitumor immune responses [255, 256]. As earlier mentioned, a paracrine interaction involving reciprocal signaling between carcinoma cells and macrophages involving EGF and CSF-1 has been demonstrated to be required for tumor cell migration in mammary tumors [180, 257]. The finding that this EGF/CSF-1 paracrine invasion loop can be triggered by HRG-β1 [188], along with the fact that tumor cell-derived NRG-1 induces upregulation of JAG1  in macrophages [192], indicates that macrophages are subjected to a fine tune regulated by NRG signaling. Nerves in the TME also play roles in tumor progression [258, 259]. It has been shown that, in addition to vascular and lymphatic systems, nerve system may serve as an alternative route for dissemination of cancer cells [260]. Cancer cells can grow around existing nerves and eventually invade them, in a process defined as perineural invasion (PNI) [261]. To date, PNI has

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been observed in a variety of cancers, including pancreatic, prostate, colorectal, and others. PNI is generally associated with a poor prognosis and can cause severe pain in patients [262]. In a landmark publication, Magnon and colleagues provided experimental evidence showing that autonomic nerve sprouting in prostate tumors was essential for the progression of prostate cancers [263]. Later on, vagal innervation was demonstrated to contribute to gastric tumorigenesis via M3 receptor-mediated Wnt signaling in the stem cells [264]. Further investigation revealed that Dclk1+ tuft cells and nerves were the main sources of acetylcholine (ACh) within the gastric mucosa [265]. While cholinergic stimulation was evidenced to induce nerve growth factor (NGF) expression in the gastric epithelium, NGF/Trk signaling in turn was demonstrated to regulate mucosal innervation and promote carcinogenesis [265]. Considering the importance of NRG signaling in the development of nervous system [12, 266], these findings have shed a new light on the possible nerve-dependent mechanism of NRG signaling in regulating tumorigenesis [267]. Inducing angiogenesis is one of the key hallmarks of both primary and metastatic cancer [2]. When the primary tumor or metastases grow to a certain extent, oxygen and nutrient supply and the discharge of metabolic wastes and carbon dioxide are insufficient. Thus, the diameter of cancer can rarely exceed 2–3 mm without neovascularization. Hypoxia in TME is a key driver of transition from pre-vascular hyperplasia to highly vascularized and progressively outgrowing tumors termed “angiogenic switch,” a process being fine-tuned by factors that either induce or inhibit angiogenesis [268, 269]. Among many reported pro-angiogenic proteins, vascular endothelial growth factor (VEGF) mainly secreted by cancer cells is now known to be central to this process [270]. Moreover, a large body of evidence indicates that angiogenic programming of tumor tissue is regulated by TME in concert with cancer cells [271]. Once the blood vessels grow into the tumor, the way of supplying nutrients and oxygen to the tumor tissue changes from peripheral diffusion to blood perfusion, and its metabolic wastes can be eliminated in time. In addition, tumor blood vessels can also determine

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the pathophysiology, growth, invasion, and metastasis of cancer, as well as its response to various treatments [272]. In normal condition, the neuregulin signaling has been demonstrated to play an important role in development of cardiovascular system [273, 274]. In a recent study, Nrg4 was also showed to be an angiogenic factor involved in maintaining adipose tissue vasculature [275]. Given the findings that the expression of pro-angiogenic proteins can be induced by several activated oncogenes including ErbB2 [276, 277], the role of neuregulin signaling in the process of tumor angiogenesis has attracted much research interest as early as in the very beginning of this century. It was revealed by Yen et al. for the first time that HRG β1 can selectively upregulate the expression of VEGF in ErbB2-­overexpressing breast cancer cells [278]. The induced expression of VEGF by HRG β1 was further confirmed by two independent research groups thereafter [279, 280]. Furthermore, HRG signaling-induced VEGF expression was also found in colon cancer [281]. Upon activation, normally quiescent vasculature sprouts new vessels to sustain the expanding growth of tumor. During this process, the recruitment of pericytes by platelet-derived growth factor-­B (PDGFB) in TME was demonstrated to play an important role in maintaining appropriate vascular morphogenesis [282–284]. Pericytes vary not only in their morphology but also in protein markers they expressed as well as their origin [285]. Importantly, only type-2 pericytes were demonstrated to participate in normal and tumoral angiogenesis [286]. Taking into consideration the role of HIF signaling in regulating the secretion of PDGFB by endothelial cells [287], as well as the reported cross talk between HER2 and HIF signalings [288, 289], it is of great interest to explore the more exact role of NRG signaling in tumor angiogenesis.

1.5

Targeting of Neuregulin Signaling in Cancer Therapy

Given the pivotal oncogenic role of aberrant NRG signaling in a wide variety of human cancers, therapeutic targeting of NRG signaling in

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cancer treatment has long been proposed and extensively studied. Strategies developed for NRG signaling-targeted therapy are different owing to the broad spectrum of casual mutations/ aberrations unraveled. Some of them have been successfully approved for clinic application in cancer therapy and achieved favorable outcome. Since both EGFR- and ErbB2-targeted therapies have been extensively reviewed [290–292], our discussion mainly focuses on, but is not restricted to, NRG/ErbB3-targeted therapy in the following section. Unlike its close relatives EGFR and ErbB2, less attention has been paid to the oncogenic functions of ErbB3 due to its weak kinase activity. However, during the last two decades, ErbB3 has been shown to be a direct therapeutic target in cancer treatment because of its role in tumor biology [293]. Currently, strategies developed for ErbB3-targeted therapy are mainly focused on blocking its activation through antibodies. The various ErbB3-targeted antibodies, including seribantumab (also known as MM-121/ SAR256212) [294], LJM716 [295], and patritumab (or U3–1287) [296], have been demonstrated to inhibit ErbB3 pathway activation via different ways. MM-121, a fully humanized monoclonal antibody that interferes in binding of HRG to ErbB3, has been shown to effectively block ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET [297]. In our own studies, we found that targeting of ErbB3 with MM-121 not only potentiated antitumor activity of paclitaxel against ErbB2+ breast cancer [298] but also had a potential of reversing trastuzumab resistance [299]. Recently, patritumab was evidenced to overcome ligand-­ mediated resistance to trastuzumab in ErbB2+ breast cancer via synergistically targeting of ErbB2/ ErbB3 signaling axis [300]. With the significant advances in antibody engineering technologies, strategies that simultaneously target multiple receptors with bispecific or multi-­ specific antibodies have been developed and demonstrated to circumvent the limitations of conventional mono-specific therapies and achieved enhanced therapeutic efficacy [301]. MEHD7945A, a monoclonal antibody that

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dually targets EGFR and ErbB3 [302], has been shown to be more effective than a combination of cetuximab and anti-HER3 antibody at inhibiting both EGFR/HER3 signaling and tumor growth [303]. Although multiple phase I and II trials have been opened for various ErbB3targeted antibodies [304, 305], clinical benefits of the antibodies as single agents have not been reported. In a randomized phase II trial of paclitaxel with or without seribantumab in patients with advanced platinum-resistant or platinumrefractory ovarian cancer, although no difference in progression-­free survival (PFS) between the two arms was reported, the authors’ exploratory analyses suggested that high HRG and low ErbB2 might be predictive of seribantumab benefit [306]. More recently, it was reported that the combination of SAR256212 and PI3K inhibitor SAR245408 for patients with metastatic or locally advanced solid tumors resulted in stable disease as the best response without effect on the pharmacokinetics of either drug, and the side effects seen in combination were similar to the profiles of each individual drug [307]. These results of clinical trials indicate that deliberate selection of patient and combinatory application with other treatments will be critical for the clinical practice of ErbB3-­targeted therapy in the future [308]. In addition to blocking antibody, novel approaches targeting ErbB3 have also been proposed [309]. EZN-3920, a locked nucleic acid (LNA)-based ErbB3 antisense oligonucleotide, has been demonstrated to inhibit growth of xenograft models of breast and lung cancer cell lines via specific downregulation of ErbB3 [310]. As alterations in chromatin structure by histone modification and/or DNA methylation have been extensively correlated with cancer development, progression, and resistance to therapy [311, 312], epigenetic targeting is emerging as a promising therapeutic strategy for cancer treatment [313]. Histone deacetylases (HDACs), whose deregulation is evidenced to play an important role in aberrant gene expression in tumorigenesis, have long been recognized as druggable targets [314]. We previously reported that the class I HDAC inhibitor entinostat (also known as MS-275 or

1  Neuregulin Signaling in the Tumor Microenvironment

15

SNDX-275) specifically enhanced expression of xenografts [322]. Large body of functional studmiR-125a, miR-125b, and miR-205, which acted ies has confirmed that miRNA dysregulation in concert to downregulate ErbB2 and ErbB3 and plays a causal role in many cases of cancer. selectively induced apoptosis in ErbB2-­Insights into the roles of miRNAs in cancer have overexpressing breast cancer cells [315–317]. made them attractive tools and targets for novel Recently, we found that valproic acid (VPA), a therapeutic approaches [323]. MiRNAs may act safely used anticonvulsant drug with reported as tumor suppressors or oncogenes (oncomiRs), HDACi capability, held an antitumor activity and miRNA mimics and molecules targeted at selectively against EGFR/ErbB2/ErbB3-­ miRNAs (antimiRs) have shown promise in precoexpressing pancreatic cancer via induction of clinical development. In a recent study, miRErbB family members-targeting microRNAs 296-5p was demonstrated to be significantly [318]. Thus, miRNA-mediated epigenetic regula- downregulated in HCC tissues, and introduced tion may represent a new mechanism inactivating miR-296-5p was able to suppress EMT of HCC ErbB2/ErbB3 [319]. via direct targeting of NRG1 [169]. Thus, these Since ligand binding is indispensable for findings have raised the possibility of miRNA successful eliciting of NRG/ErbB signaling, it is therapeutics in NRG1-targeted therapy, which rational that specific downregulation of NRG deserves further investigation. In addition, with and/or blocking its binding to ErbB3 should be the landmark finding of CD74–NRG1 gene an attractive strategy for interrupting this signal- fusions in lung adenocarcinoma in 2014 [107], ing. Actually, in the very beginning of this cen- much effort has been made in exploring NRG1 tury, it was discovered that blockade of HRG fusion-targeted therapy in cancer management expression using an antisense nucleotide tech- [324]. Although strategies proposed for NRG1 nology resulted in inhibition of cell prolifera- fusion-targeted therapy are still focused on tion and anchorage-independent growth, as well interruption of downstream ErbB signaling as suppression of invasive potential of breast [325–327], whether NRG1 fusion itself may act cancer cells in  vitro [320]. Targeting of as a direct druggable target is of great interest. ADAM17, a major ErbB ligand sheddase, was also reported to inhibit ErbB3 and EGFR pathConclusions and Future ways through preventing the processing and 1.6 activation of multiple ErbB ligands [38]. Perspectives Neutralization antibodies have been extensively explored in targeting excess ligands in cancer Although extensive effort has concentrated on therapeutics. By using two self-developed elucidating the role of NRG signaling in the tumor blocking antibodies of NRG1, Hegde et  al. microenvironment, several critical questions have reported a successful inhibition of NRG1 sig- been raised owing to the progress in some rapidly naling in NSCLC, leading to an enhanced dura- evolving fields of cancer research. Firstly, with tion of the response to chemotherapy [321]. the increasing recognition of link between the Moreover, a NRG1-specific antibody 7E3 has microbiota and cancer [328], the identification of been demonstrated as a promising antitumor intratumor microbiota has broadened the dimenagent against pancreatic cancer recently. 7E3 sion of complexity of TME [329]. Microbiota has not only promotes antibody-dependent cellular been demonstrated to play a key role in carcinocytotoxicity (ADCC) in NRG1-positive pancre- genesis and regulation of the response to therapy atic cancer cells and cancer-associated fibro- through a variety of mechanisms such as bacterial blasts (CAFs) and inhibits NRG1-associated dysbiosis, production of genotoxins, pathobionts, signaling pathway induction in vitro; it also sup- and disruption of the host metabolism [330]. In a presses migration and growth of pancreatic can- cross-sectional study, Tsay et  al. reported that cer cells co-cultured with CAFs, both in  vitro enrichment of the lower airway microbiota with and in  vivo using orthotopic pancreatic cancer oral commensals was associated with upregula-

16

tion of the PI3K signaling pathway in lung cancer [331]. Since the underlying mechanism of microbiota-induced upregulation of PI3K pathway remains undiscovered, whether NRG signaling is involved in this biological process deserves further exploration. Secondly, although anticancer immunotherapies involving the use of immune checkpoint inhibitors or adoptive cellular transfer have achieved promising clinical outcome, resistance to immunotherapy is still a major obstacle to be overcome [332, 333]. As has been revealed by two independent groups, loss of PTEN results in resistance to T-cell-mediated immunotherapy [334, 335]. These findings may hint a causal role of aberrant NRG signaling in resistance to immunotherapy. Thus, it will be of special interest to figure out whether simultaneous targeting of NRG signaling may enhance the efficacy of immunotherapy. Finally, exosome-mediated communication within TME has been recognized as an important player in regulating tumor progression [336]. An improved understanding of whether NRGs may be present in exosome and act locally and/or distantly on eliciting downstream signaling will be of great help in the guidance of developing novel NRG-targeted therapy, such as eliminating oncogenic exosomes with aptamerfunctionalized nanoparticles [337]. In conclusion, we believe that the answer of these questions will offer a clearer understanding of the role of NRG signaling in the TME and point out the future direction of mechanism-based NRG signalingtargeted therapy. Acknowledgments  We thank Dr. Bolin Liu at Department of Genetics, Stanley S.  Scott Cancer Center, Louisiana State University (LSU) Health Sciences Center, for his critical comments on the manuscript. Work of Shuiliang Wang was supported in part by the National Natural Science Foundation of China No. 81772848 and Joint Funds for the Innovation of Science and Technology from Fujian Province No.2017Y9127.

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2

HGF/c-Met Signalling in the Tumor Microenvironment Alberto Zambelli, Giuseppe Biamonti, and Angela Amato

Abstract

Recently, it has become clearer that tumor plasticity increases the chance that cancer cells could acquire new mechanisms to escape immune surveillance, become resistant to conventional drugs, and spread to distant sites. Effectively, tumor plasticity drives adaptive response of cancer cells to hypoxia and nutrient deprivation leading to stimulation of neoangionesis or tumor escape. Therefore, tumor plasticity is believed to be a great contributor in recurrence and metastatic dissemination of cancer cells. Importantly, it could be an Achilles’ heel of cancer if we could identify molecular mechanisms dictating this phenotype. The reactivation of stem-like signalling pathways is considered a great determinant of tumor plasticity; in addition, a key role has been also attributed to tumor microenvironment (TME). Indeed, it has been proved that cancer cells interact with different cells in the

surrounding extracellular matrix (ECM). Interestingly, well-established communication represents a potential allied in maintenance of a plastic phenotype in cancer cells supporting tumor growth and spread. An important signalling pathway mediating cancer cell-TME crosstalk is represented by the HGF/c-Met signalling. Here, we review the role of the HGF/c-Met signalling in tumor-stroma crosstalk focusing on novel findings underlying its role in tumor plasticity, immune escape, and development of adaptive mechanisms. Keywords

HGF/c-Met signalling · Tumor microenvironment (TME) · Cellular crosstalk · Tumor heterogeneity · Cancer cell plasticity · Cancer stem cells (CSCs) · Mesenchymal stem cells (MSCs) · Adipokines · Hepatokines · Immune escape · Metabolic stress · Drug resistance · Metastasis · Inflammation · Neo-angiogenesis

A. Zambelli Unit of Oncology, Ospedale Papa Giovanni XXIII, Bergamo, Italy G. Biamonti · A. Amato (*) Institute of Molecular Genetics (IGM); National Research Council (CNR), Pavia, Italy e-mail: [email protected] © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_2

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2.1

Introduction

2.1.1 T  umor Plasticity, Cancer Stem Cells, and Tumor Microenvironment (TME) Traditionally, tumors from the same anatomical site are considered as one tumor entity due to the identification of common markers and shared clinical features. However, genome-wide analysis enabled molecular profiling of cancer cells showing that tumors of the same origin site could be divided into different subgroups based on distinct patterns of gene expression as in breast cancer [1, 2] and colon cancer [3–5]. Stochastic genetic changes have long been considered a trigger for tumor heterogeneity [6]; however, they well explain intra- and intra-tumor heterogeneity but they hardly mirror the remarkable plasticity that cancer cells frequently exhibit. Importantly, tumor plasticity is considered a key determinant of adaptive response to a changing microenvironment [7]. Therefore, it should depend on a dynamic mechanism conferring on cancer cells the ability to “sense” microenvironment conditions and to “change” themselves accordingly. In this view, the theory of cancer cells acquiring stem-like features (cancer stem cells (CSCs)) better explains the high adaptive capability which is common to cancer cells of different tissues. In addition, the reactivation of stem-like signalling pathways has been considered pivotal in driving self-renewal and tumor plasticity. Recently, it has become clearer that tumor plasticity increases the chance that cancer cells could develop new mechanisms to escape immune surveillance, become resistant to conventional drugs, and spread to distant sites [8–11]. In this scenario, a significant contribution comes from tumor microenvironment (TME), the extracellular space surrounding cancer cells. TME comprises several cell types able to establish a communication with tumor cells. It has been argued that they represent a potential allied in tumor growth and spread. An important signalling pathway mediating this crosstalk is represented by the HGF/c-Met

signalling. It is a molecular signalling activated after establishment of cell-cell communication, and it is essential for proper embryogenesis and organogenesis. In adult tissue, its activation is restricted to adult cells with regenerative properties as hepatocytes. Interestingly, it is reactivated in human tumors demonstrating that cancer cells frequently hijack embryonic mechanisms to sustain recurrence and metastatic invasion. Indeed, we have a deep comprehension of the role of c-Met in tissue invasion and metastasis (for which readers are referred to [12]); therefore, this review focuses on novel insights showing the pivotal role of HGF/c-Met signalling in development of adaptive responses to a challenging TME. Importantly, we report recent evidence highlighting its role in tumor heterogeneity, immune escape, neo-angiogenesis, response to metabolic stress, and drug resistance.

2.2

Main Text

2.2.1 T  he HGF/c-Met Signalling in Development HGF is a cytokine produced by stromal fibroblasts and able to stimulate migration of epithelial cells. It is produced as an inactive precursor (pro-HGF). A proteolytic cleavage is required by stromal trypsin-like proteases as hepsin, matriptase, or HGF activator. Interestingly, pro-HGF and active HGF compete for c-Met binding; therefore, the balance between the two peptides represents a fine-tuned mechanism for regulation of c-Met signalling. HGF binding to c-Met receptor induces its homo-dimerization, autophosphorylation of tyrosine residues, and activation of the catalytic domain. Phosphorylated tyrosines create a docking site for several signalling effectors including GRB2, SHC, CRK, PI3K, PLCγ, SRC, SHIP2, and STAT3 [13–15]. The complete plethora of downstream signalling of c-Met has not been fully elucidated: among others, activation of K-RAS and, consequently,

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the MAPK cascade occurs through the binding of SHC and GRB2 [16]; conversely, survival signalling is triggered through interaction with PI3K and activation of AKT [17]. Importantly, activated c-Met can directly bind to STAT3, the main player in c-Met-driven invasive program [18]. Different mechanisms limit c-Met activation contributing to regulation of its physiological role. For example, several phosphatases inhibit c-Met activation, as in the case of LAR or PTP1B [19]. Conversely, phosphorylation of tyrosine residues in the juxtamembrane domain determines ubiquitination of the receptor through CBL [20]. In addition, ADAM metalloproteases provide a mechanism of c-Met degradation which affects paracrine c-Met signalling: the metalloprotease cleaves the extracellular domain of c-Met which is able to sequester HGF in TME or bind the full-length c-Met in neighboring cells preventing its activation [21]. Met activation induces various biological responses including proliferation, motility, cell survival, morphogenesis, and angiogenesis. Knockout mice have demonstrated that c-Met plays a physiological role in embryogenesis. Effectively, since its discovery, many studies have linked HGF/c-Met signalling to migration programs in both embryonic and adult stem cells [22]. During early embryogenesis, the activation of HGF/c-Met supports migration of mesenchymal stem cells. At later stages, it is involved in the neural induction of ectoderm and in myoblast migration during differentiation of the skeletal muscles [23]. Basically, mesenchymal stem cells secret high amount of HGF and stimulate c-Met-­ expressing progenitor cells (ectodermal cells, myoblasts, or epithelial cells) [24]. The importance of the HGF/c-Met signalling during embryogenesis and organogenesis implies that the expression of both hgf and c-met genes is temporally and spatially controlled [25, 26]. Mouse models confirmed the paramount role of this pathway during development showing that homozygous null mice for either hgf or c-met die in utero [27]. In adult tissues, HGF/c-Met signalling is restricted to adult stem cells, hepatocytes, pancreatic β-cells, skeletal and cardiac muscle, renal epithelial cells, neurons, and immune cells.

Formerly, this signalling has been related to the regenerative properties of hepatocytes, as revealed also by the name of c-Met receptor ligand: hepatocyte growth factor (HGF). Indeed, loss of c-met in hepatocytes severely affects the adaptive response of the liver to injury. Although conditional c-met knockout generated mice with normal liver in terms of size and histological architecture, however, the liver accumulated lipid vesicles as soon as 6 months after c-met loss; in addition, partial hepatectomy highlighted dramatically decreased regenerative properties [26, 27]. Similarly, the HGF/c-Met signalling sustains renewal processes in the kidney. Indeed, in a mouse model of renal injury, c-Met knockout is associated with increased interstitial fibrosis, inflammatory cell infiltration, and acute tubular necrosis [28]. Furthermore, these mice exhibited a reduced tubular cell proliferation and kidney regenerative capacity. Interestingly, several studies have demonstrated that HGF has regulatory effects on glucose transport and metabolism in different insulin-sensitive cells. Effectively, it has been shown that it stimulates glucose uptake and metabolism in skeletal muscle cells [29], pancreatic β-cell [30], and hepatocytes [31]. Therefore, it could participate in the physiological regulation of glycemia and insulinemia during the starved-fed circadian rhythm and could have a role in insulin resistance and obesity [as reviewed in 32]. Effectively, plasmatic HGF levels are high in obese patients [32]. In addition, specific c-met knockout in pancreatic β-cell is associated with reduced glucose tolerance and low plasma levels of insulin [33]. Lastly, the HGF/c-Met signalling is required for development of erythroid, myeloid, and lymphoid lineage cells in concert with other hematopoietic growth factors. Moreover, it influences cytokine secretion by monocytes and macrophages during tissue repair [34] and contributes to endothelial cell differentiation during wound healing [35]. Surprisingly, it is conceivable that an involvement of HGF/c-Met signalling exists in brain disorders since it seems to regulate neuronal development and establishment of synapsis.

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Effectively, reduced c-Met expression has been detected in postmortem temporal lobe of individuals with autism and Rett syndrome [36].

2.2.2 HGF/c-Met Signalling in Cancer Pathological regulation of HGF/c-Met signalling confers invasive properties on cancer cells [12]. In addition, it is a trigger for activation of multiple downstream signalling pathways which contribute to cancer cell proliferation, migration, and survival. Moreover, HGF/c-Met signalling has been involved also in generation of CSCs through epithelial-mesenchymal transition (EMT) and in neo-angiogenesis [37]. Effectively, constitutive c-Met activation (despite the binding of HGF ligands) has been found as a consequence of c-met gene amplification in several human cancers as glioblastomas, medulloblastomas, NSCLC, and colon cancer [38–42]. Some c-met mutations have been found in hereditary papillary renal carcinomas [43] and sporadic ovarian [44], gastric [45], lung [46], thyroid [47], and hepatocellular carcinoma [48]. Also, mutations affecting HGF or HGF-activating proteases are frequently found in human cancers [49–51]. Interestingly, high levels of plasmatic HGF have been observed in patients with stage II–III colon cancer and have been associated with lymph node positivity and metastatic spread in both colon and breast cancer patients [52–55]. Effectively, genetic mutations affecting HGF or c-Met expression are less frequent in primary tumors in comparison with metastatic diseases [56]. For example, nearly 4–10% of upper gastrointestinal carcinomas show c-met amplification; surprisingly, 50% of patients with advanced gastric cancer show c-met overexpression [57]. This observation correlates with the invasive behavior and aggressiveness of cancer cells after activation of HGF/c-Met signalling, as reviewed in [12]. Moreover, we should also consider that wild-­ type c-Met can interact with several cell surface proteins. This reveals that c-Met could contribute to oncogenic signals sustained by mutated receptors which are able to heterodimerize with c-Met itself.

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For example, the interaction between c-Met and CD44 links HGF/c-Met signalling to EMT program which accounts for migration of embryonic cells during embryogenesis, but it is also required for maintenance of CSCs [58, 59]. Importantly, CSCs are believed to better explain tumor plasticity due to the activation of embryonic pathways which confer stem-like properties to cancer cells.

2.2.3 T  he HGF/c-Met Crosstalk: A Trigger of Tumor Inflammation and Neo-angiogenesis It has been proved that cancer cells of the same tumor bulk interact with different cells in the surrounding extracellular matrix (ECM): fibroblasts, endothelial cells and their progenitors, pericytes, immune cells, mesenchymal stem cells (MSCs), and adipocytes (Fig. 2.1). It is now well established that a crosstalk between cancer cells and TME could affect several aspects of tumorigenesis such as neoplastic transformation, growth and dissemination of cancer cells, neo-angiogenesis, and immune surveillance escape. This effect relies on the activation of specific pathways which drive phenotypic changes in cancer cells. The establishment of a HGF/c-Met crosstalk supports this process of tumor transformation toward a more aggressive phenotype; therefore, tumor cells set up different mechanisms to ­activate c-Met receptors. For example, they produce and secrete HGF in TME allowing both autocrine and paracrine activations of c-Met. Conversely, cancer cells might benefit from HGF produced by surrounding stromal cells (Fig. 2.2). Effectively, it has been proved that cancer cells are able to recruit mesenchymal stem cells (MSCs) at tumor site exploiting their secretome. This is an important cue sustaining inflammation and neoangiogenesis. Interestingly, inhibition of this HGF/c-Met crosstalk severely affected the contribution of MSCs to tumor growth. Surprisingly, failure in establishment of an HGF/c-Met-mediated crosstalk with MSCs resulted in loss of tumorigenic potential in NOD/SCID mice [60].

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Fig. 2.1  Cancer cells grow and develop and are surrounded by several cell types: mesenchymal stem cells (MSC), fibroblasts, endothelial progenitors, and immune cells such as T-cells, B-cells and tumor-associated macrophages

(TAM). An established communication of these cells with cancer cells is detrimental in tumor progression and migration, which is also supported by extracellular matrix (ECM) represented by proteoglycans and collagen fibers

Fig. 2.2  Cancer cells could produce high amount of HGF which is secreted into TME. Secreted HGF acts in an autocrine fashion creating a positive loop for maintenance of

activated HGF/c-Met signalling in cancer cells; simultaneously, it could act paracrinally to stimulate production of HGF activating c-Met receptors in surrounding stromal cells

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Similarly, there is a synergism between HGF/ Simultaneously, it stimulates differentiation of c-Met signalling and some cytokines: for exam- macrophages toward M2 phenotype with inflample, G-CSF enhances the recruitment of cells matory and immunosuppressive properties [68]. from peripheral blood [61], and SDF-1α and Interestingly, in a mouse model of melanoIGF-1 contribute to guide dissemination of can- mas, c-Met signalling suppresses T-cell-mediated cer cells, as in myeloma tumor model [62]. immune response through the recruitment of neuIn addition, the paracrine role of HGF could be trophils into lymph node and tumor tissues. also sustained by adipocytes which, in some tissues, Surprisingly, different immunotherapy live in close proximity to cancer cells, as in breast approaches administered with concomitant c-Met and ovarian tissue. Indeed, it has been proved that inhibitors increased T-cell infiltration in tumors adipocytes produce high amount of HGF both in improving immune system response. Accordingly, murine models and in human tissues [63]. Moreover, high HGF serum levels correlated with increasadipose tissue seems to account for high HGF ing neutrophil counts and poor responses to plasma levels suggesting a potential endocrine checkpoint blockade therapies in a subgroup of patients with metastatic melanomas [69]. mechanism for the hepatokine [reviewed in 64]. Cytotoxic T-cells are critical mediators of an Basically, HGF/c-Met signalling is key in recruitment of fibroblasts, MSCs, tumor-­ effective antitumor response evoked by the immune associated macrophages (TAMs), and tumor-­ system. An interesting study has shown that HGF associated natural killers (TANKs) at the tumor could affect T-cell activation by inhibiting dendritic site contributing to the maintenance of an cells and their function as antigen-­presenting cells inflammatory microenvironment. As previously [70]. Moreover, in a later issue, the same authors positive T-cells shown, the activation of inflammatory pathways identified a subgroup of c-Met-­ is a key step which sustains activation of self- which were highly cytotoxic; however, their activrenewal mechanisms and neo-angiogenesis; ity could be restrained by HGF [71]. Altogether, these findings suggest that secreted indeed, a tumor has been defined “a wound HGF, accumulating in TME, could evoke an never healing” [65]. immunosuppressive role in different c-Met-­ expressing immune cells. 2.2.4 The Immunosuppressive Role Importantly, we should consider that these of the HGF/c-Met Crosstalk inflammatory and immunosuppressive mechanisms mediated by HGF/c-Met signalling could The mutual cooperation between cancer cells and also be activated in an endocrine fashion. Indeed, stromal cells seems to protect cancer cells from it has been recently highlighted that both the liver immune system and anticancer drugs. and adipose tissue secrete and release HGF into It seems to be dependent on the ability of HGF blood circulation. to act as a paracrine factor recruiting immune cells and, simultaneously, influencing their behavior. Indeed, in  vitro studies showed that 2.2.5 The Pivotal Role of HGF/c-Met Signalling in Cancer Cell secreted HGF in TME recruits and stimulates Plasticity monocytes for production of chemokines (MIP-1β and MIP-2α) and interleukins (IL-6, IL-8, IL-10) in order to promote a nonspecific Cancer cell plasticity has been recently associated cellular inflammatory response [66, 67]. with the ability of cancer cells to adapt in a chalSimilarly, HGF/c-Met signalling has been lenging TME. Indeed, as tumor growth proceeds, proven to contribute to progression of chronic cancer cells should be able to survive in a hypoxic lymphocytic leukemia (CLL); indeed, HGF microenvironment and acquire new mechanisms secreted by stromal cells is a trigger for AKT to tolerate nutrient deprivation. In addition, they activation and survival of leukemia cells. should develop adaptive responses to microenvi-

2  HGF/c-Met Signalling in the Tumor Microenvironment

ronment modifications induced by prolonged treatment with anticancer drugs or radiotherapy [9]. Importantly, a high adaptive potential is also required during different steps of metastatic dissemination: detachment from ECM (extracellular matrix), migration through blood vessels, extravasation, and homing at metastatic site [72, 73]. Therefore, it is conceivable that tumor plasticity could account for the intrinsic adaptive capabilities frequently acquired by cancer cells. Hence, it represents a great determinant in tumor relapse, chemoresistance, and metastatic spread, severely contributing to a poor outcome in cancer patients.

2.2.5.1 HGF/c-Met Signalling Drives Adaptive Response of Cancer Cells to Metabolic Stress Several studies recently focused on the ability of HGF to stimulate glucose uptake in hepatic cells, pancreatic β-cells, and myotubes through a mechanism inducing overexpression and/or translocation of GLUT-2 and GLUT-4 to plasma membrane [29–31, 74]. Therefore, HGF/c-Met signalling contributes to the physiological role of the liver in regulation of glucose metabolism and in pancreatic control of insulin release. Importantly, this metabolic function could be exploited by cancers to survive metabolic stress. Effectively, it has been argued that tumor plasticity gives cancer cells the ability to establish new mechanisms for competitive glucose uptake or, more interestingly, to stimulate oxidative phosphorylation (OXPHOS) of glucose in surrounding stromal cells so that they, in change, could provide onco-metabolites to cancer cells [75]. For example, a study in colon cancer mouse models showed that cancer cells survive starvation due to HGF/c-Met signalling. Namely, paracrine HGF increases glucose uptake through GLUT-1, sustains glycolysis, and ensures survival of starved cancer cells by activation of autophagy. The glycolytic pathway is the preferential metabolic path for cancer cells. Glycolysis-­ related genes can be activated by hypoxia-­ inducible factor-1α (HIF-1α). Importantly, HIF-1α is a well-known sensor of hypoxia; how-

37

ever, it has been also acknowledged as a sensor of metabolic stress in normoxic conditions [76]. Indeed, it could be also activated after decrease of nutrient availability [77]. Interestingly, HIF-1α is also a direct activator of c-met expression and represents a nodal switch for survival of cancer cells because, on one hand, it could trigger neo-­ angiogenesis and, on the other hand, it could sustain the activation of signalling pathways driving cancer cell to escape from tumor bulk. In both cases, HIF-1α exploits c-Met for recruitment of progenitor endothelial cells for new vessel formation or activation of invasive program required for migration to distant sites. This mechanism could drive tumor resistance to inhibitors of anti-­ angiogenesis [78]. Similarly, a metabolic symbiosis established in TME has been acknowledged as the cause of cancer cell resistance to EGFR inhibitors. Chemoresistance is the result of an adaptive response generated in cancer cells after prolonged exposure to cancer drugs, for example, tyrosine kinase inhibitors (TKI). A recent study showed that cancer cells benefit from a metabolic shift toward increased glycolysis and lactate secretion in response to TKI inhibitors in NSCLCs. Moreover, secreted lactate in TME induced cancer-associated fibroblasts (CAFs) to produce HGF in a NFkB-dependent signalling. Interestingly, increased stromal HGF and cancer cell lactate correlated with onset of TKI resistance in NSCLC patients [79]. The above finding suggests that a crosstalk between CAFs and c-Met-expressing cancer cells could be mediated by metabolites (in this case, “lactate”) eliciting mechanisms of adaptive resistance to anticancer drugs (Fig. 2.3).

2.2.5.2 HGF/c-Met Signalling Contributes to CSCs Maintenance in Tumor Bulk Recently, the HGF/c-Met signalling has been acknowledged as a key pathway for CSC maintenance in several cancers including colorectal [80], breast [81], prostate [82], pancreatic [83], and glioblastoma [84]. A HGF/c-Met-mediated crosstalk between pancreatic stellate and cancer cells promotes

38

A. Zambelli et al.

Fig. 2.3  The HGF/c-Met signalling has a pivotal role in tumor-stroma crosstalk. Novel insights reveal that it could control glucose metabolism enhancing glycolysis and reducing oxidative phosphorylation (OXPHOS). In addition, secreted lactate acts as a signalling molecule evoking the support of stromal cells in drug resistance and maintenance of activated HGF/c-Met signalling. Moreover, increased glucose uptake stimulates survival signalling

from surrounding stromal cells also activating autophagy in order to ensure survival of cancer cells during metabolic stress. Similarly, HIF-1α responding to metabolic stress (as nutrient deprivation or hypoxia) enhances c-Met signalling through inducing c-met gene expression which triggers an adaptive response of cancer cells in TME as neo-angiogenesis and metastatic escape

the expression of CSC markers as NANOG, OCT-4, and SOX-2  in pancreatic cancer cells through HIF-1α stabilization. In addition, it induces the expression of hexokinase 2 (HK2) sustaining the preferential glycolytic metabolism [85]. Similarly, in colon cancer, an HGF/c-Met crosstalk drives the activation of stemness-related genes and pathways as wnt/β-catenin [86]. In addition, it seems that simultaneous activation of c-Met and wnt/β-catenin pathway contributes to breast cancer bone metastases [87]. In hepatocellular carcinoma (HCC), CAFs are important regulators of CSC self-renewal through activation of a c-Met/FRA1/HEY1 cascade. Indeed, the amount of α-SMA-expressing

CAFs correlates with poor prognosis in HCC patients [88]. Noteworthy, HIF-1α activation, which is usually activated upon inflammation in TME, also promotes self-renewal of CSCs and, simultaneously, sustains c-met expression [89–91]. Furthermore, c-Met activation contributes to epithelial-mesenchymal transition (EMT) which, on one hand, maintains CSC amount in the tumor bulk and, on the other hand, favors metastatic dissemination of cancer cells through activation of migration. High levels of c-Met and Snail, a key regulator of EMT, correlate with highly invasive tumor phenotypes and poor prognosis in basal breast cancer [92].

2  HGF/c-Met Signalling in the Tumor Microenvironment

39

sites [99]. In this scenario, pericytes play a key role. Indeed, they are elongated and branched cells surrounding the vessel wall. In a physiological context, they provide support to endothelial cells contributing to blood vessel maturation and branching [100]; however, during tumorigenesis, pericytes contribute to early stage of metastasis because they facilitate the invasion of blood vessel lumen. In addition, they seem to contribute to later stages of the metastatic process supporting the formation of a pre-metastatic niche at distant sites [101]. Effectively, clinical studies have correlated the extension of pericyte coverage on tumor microvessels with cancer prognosis [102– 104]. For example, low pericyte coverage of blood vessel has been associated with invasive 2.2.5.3 The HGF/c-Met Signalling breast cancer and correlated with decreased Regulates Neo-Angiogenesis patient survival. Noteworthy, brain cancer has a high neo-­ One hypothesis sustains that pericytes may angiogenic potential; indeed, they are frequently suppress metastatization of cancer cells because highly vascularized. Accordingly, HGF/c-Met they may act as a barrier that prevents cancer signalling controls angiogenesis at multiple cells entering vessel lumen. Effectively, inhibitsteps: (a) c-Met regulates expression of vegfa ing interaction between pericytes and endothelial (vascular endothelial growth factor A) and inhib- cells, through knockout of the adhesion-related its tsp1 (thrombospondin 1), the angiogenesis protein NCAM, severely affected the stability of suppressor [95], and (b) HGF/c-Met crosstalk in tumor vasculature leading to increased metastatiTME enhances proliferation, migration, and zation in a mouse model of pancreatic tumor differentiation of progenitor endothelial cells. [105]. In addition, a recent study proved that dif­ Accordingly, inhibition of c-Met severely ferent subpopulation of pericytes exist and only affected tumor growth by inhibition of angiogen- some of them (expressing Nestin and NG2) in the esis [96, 97]. tumor bulk are recruited to support neo-­ An important c-Met allied in tumor angiogen- angiogenesis [106, 107]. This may suggest that esis is STAT3, which sustains endothelial cell different subpopulation of pericytes may play proliferation and tubule morphogenesis [18]. different physiological roles and this should be Noteworthy, it seems that differentiated endothe- kept in consideration for development of effeclial cells establish a chemotactic gradient of HGF tive anti-angiogenic treatment. which sustains migration of cancer cells toward So far, the underlying molecular mechablood vessels. This is an important mechanism nisms whereby pericytes may limit tumor supporting the first step of the metastatic process metastasis have not been entirely elucidated. A in a breast cancer model. Effectively, inhibition recent study has shown that pericyte depletion of the HGF/C-Met pathway severely affected the reduced tumor growth likely due to a defective established crosstalk between endothelial cells tumor vasculature in a mouse model of breast and tumor cells suggesting c-Met as a target to cancer; however, it increased the metastatic treat metastatic breast cancers [98]. spread of cancer cells owing to hypoxia-driven Moreover, it has been shown that metastatic EMT and activation of c-Met signalling. Indeed, spread of cancer cells requires also an increased pharmacological inhibition of c-Met or silencvessel permeability enabling cancer cells to move ing of Twist suppressed both hypoxia and to the blood vessel lumen and colonize distant metastasis [108].

In the central nervous system, physical interaction between astrocytes and tumor cells enhances the CSC fraction in brain malignancies. Interestingly, activation of c-Met enhances proliferation of glioma cells [93] through activation of downstream cascades such as c-Myc and STAT3. In addition, c-Met not only supports brain cancer growth but also homing of cancer cells (i.e., originating from breast cancer) into the brain. Indeed, in breast cancer brain metastases, tumor-associated astrocytes (TAAs) stimulate the c-Met pathway in cancer cells activating angiogenesis in the metastatic site. Accordingly, c-Met inhibition significantly reduced the metastatic growth in the brain [94].

A. Zambelli et al.

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This work suggests that pericyte targeting and c-Met inhibition could be a valuable strategy to control primary tumor growth and prevent metastatic spread and progression to a worse outcome.

2.3

Future Trends or Directions

It is a well-established concept that a crosstalk of tumor cells with the surrounding cells in TME could have a detrimental effects on tumor progression and dissemination. Several evidences have shown that the oncogenic role of TME could be realized in different ways, depending on activated signalling. Interestingly, HGF/c-Met has been identified as a main player in tumor-stroma crosstalk. This signalling pathway governs cellular migration during embryogenesis and is hijacked by tumor cells to evoke an oncogenic role in surrounding microenvironment. It has been proved that HGF/c-Met signalling is key in maintenance of self-­ renewal mechanisms, development of chemoresistance, and metastatic dissemination. Recent novel insights have also suggested that HGF/c-Met signalling could support mechanisms for immune escape of cancer cells. In addition, it could also regulate glucose metabolism in cancer cells interfering with glucose uptake and availability of cell metabolites in TME. This evidence holds the promise to provide new markers for cancer diagnosis and prognosis; moreover, they could suggest new targets for development of specific molecular inhibitors addressing the oncogenic role of TME in tumor progression.

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3

Eph/Ephrin Signaling in the Tumor Microenvironment Katsuaki Ieguchi and Yoshiro Maru

Abstract

Keywords

The Eph/ephrin system plays a vital role in diverse physiological events such as neurogenesis, vasculogenesis, and cell adhesion. Expression analysis of mRNA and protein in clinical samples revealed the involvement of the Eph/ephrin system in tumorigenesis, Alzheimer’s disease, and atherosclerosis. Therefore, the Eph/ephrin system is considered a promising therapeutic target. However, no molecularly targeted drug against Ephs and ephrins is being used in the clinic thus far. Tumors are composed of various types of cells, including fibroblasts, immune cells, and endothelial cells. Recent studies showed the contribution of these cells to tumor growth, tumor progression, drug resistance, and metastasis. In this chapter, we discuss the role of Eph/ephrin system in the tumor microenvironment and describe its functions in tumor initiation, angiogenesis, cancer stem cell, tumor immunity, and also the metastatic environment.

Eph · Ephrin · Angiogenesis · Metastasis · Prognosis · Breast cancer · Colon cancer · Tumor Progression · Immune therapy · PD-L1 · Cancer stem cell · ADAM · Ras · MAPK · RhoA

K. Ieguchi (*) · Y. Maru (*) Department of Pharmacology, Tokyo Women’s Medical University, Tokyo, Japan e-mail: [email protected]; maru.yoshiro@twmu. ac.jp

3.1

General Overview

EphA1 a putative oncogene was isolated from an erythropoietin-producing human hepatocellular carcinoma cell line ELT1 by Hirai et al. in 1987 [1]. The Eph receptors are the largest family of receptor tyrosine kinases and divided into two subgroups, EphAs (A1–A8, A10) and EphBs (B1–B4, B6), based on receptor-ligand preferences. The EphA and EphB structures share a similarity in cysteine-rich, two fibronectin type III repeat, transmembrane, tyrosine kinase, and SAM domains (Fig. 3.1) [2, 3]. A ligand for the EphA receptors termed as ephrin-A1 was identified initially as a soluble factor upregulated by TNFα stimulation [4]. Ephrins also comprise of two families, ephrin-As (A1–A6) and ephrin-Bs (B1–B3), based on their structure. Ephrin-A is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein, and ephrin-B is a transmembrane protein (Fig. 3.1). An interaction

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_3

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Fig. 3.1  Eph/ephrin system activation mechanism in a juxtacrine fashion. The representative domain structure is shown. Eph binds to ephrin present on an adjacent cell. Ephs transmit downstream signal in an Eph-

expressing cell termed as “forward signal,” and ephrins transmit intracellular signals in an ephrin-expressing cell termed as “reverse signal” mediated by Src family kinases

between types A and B families was shown earlier. For instance, ephrin-B2 promiscuously binds to the EphA receptors over the family such as EphA4 and EphA5 [3]. Ephrins form dimers or oligomers and bind to the receptors. Ligand-­ bound Eph receptors usually form homodimers and induce tyrosine phosphorylation via their

tyrosine kinase activity. However, some reports showed that kinase-inactive Eph receptors form heterodimers and cooperate with kinase-active Eph complexes such as EphA7/EphA10 [5] and EphB1/EphB6 [6]. The specific functions of these dimers are undefined and under investigation. Recent studies showed the involvement of

3  Eph/Ephrin Signaling in the Tumor Microenvironment

Ephs and ephrins in various physiological functions such as artery/vein development and neurogenesis [7, 8]. Moreover, dysregulation of Eph/ ephrin expression leads to diseases such as cancer and atherosclerosis. We will not describe all the published Eph/ephrin results here. This chapter discusses the well-characterized functions of the Eph/ephrin system in the tumor microenvironment.

3.2

Eph/Ephrin Expression in Tumors

Expression profiling of mRNA or Eph/ephrin protein in biopsy samples of cancer patients suggests the correlation of Eph/ephrin expression levels with tumor malignancy, tumor progression, or metastasis in various types of cancer [9]. Therefore, Eph/ephrin molecules are considered as promising therapeutic targets. Unfortunately, no Eph−/ephrin-targeted drug is currently in clinical use. Genetic alterations often dysregulate mRNA or protein expression and lead to tumorigenesis. Similarly, in the Eph/ephrin system, chromosomal abnormalities, gene methylation, and changes in transcription regulators induce dysregulation of the Eph/ephrin expression and tumorigenesis [9]. In vulvar squamous carcinoma, expression of EphA2 and ephrin-A1 positively correlated with poor survival [10, 11], whereas EphA2, but not ephrin-A1, expression positively correlated with poor survival and ephFig. 3.2 Differential expression of EphA2 and ephrin-A1 in tumors. The Ras/MAPK signaling regulates EphA2 and ephrin-A1 expression

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rin-­ A1 expression inversely correlated with EphA2 expression in metastatic breast cancer [12]. The complementary expression of EphA2 and ephrin-A1 observed in breast cancer is dependent on the Ras/MAPK activity (Fig. 3.2) [13]. As seen in these cases, the results from expression profiling of Eph/ephrin from various types of cancer are controversial and not consistent. Hence, further analysis is needed to unravel the function of the Eph/ephrin system in each cancer type.

3.3

Roles of the Eph/Ephrin System in Tumor Initiation

The function of the Eph/ephrin system in the initiation of colorectal and breast cancer has been analyzed in detail. EphB2/EphB3 and ephrin-B1/ ephrin-B2 are expressed as a gradient along the crypt-villus axis. EphB2 expression is mainly in the progenitor cells present in the crypt bottom and gradually decreases toward the top of the villus. EphB2 expression controls the positions of ephrin-B-positive cells at the crypt side and the villus [14]. EphB3 expression is limited to the crypt bottom and regulates the Paneth cell position. Ephrin-B1/ephrin-B2 is mainly expressed in the villus, and its expression gradually decreases toward the crypt bottom. The polarized expression of EphB2/EphB3 and ephrin-B1/ephrin-B2 tightly controls the crypt-villus axis (Fig.  3.3) [14] and the proliferation of the intestinal stem cell niche in the crypt bottom of the mouse intes-

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Fig. 3.3 Compartmentalized expression of EphBs and ephrin-Bs in mouse small intestine. A schematic representation of expression patterns of EphBs and ephrin-Bs is shown

tine [15]. Loss of polarized expression pattern accelerated tumorigenesis in the intestine [16]. In human pathological settings, higher EphB2 expression correlated with prolonged survival [17] and a decreased expression associated with higher tumor stage and grade [18]. The β-catenin signal is a critical regulator of EphB2 expression in the intestine. A converse expression was observed between EphB2 and EphB4 differentially regulated by β-catenin/p300 and β-catenin/ CBP, respectively. Higher EphB4 expression conferred resistance to TNFα-induced apoptosis in tumor cells. Double-knockout EphB2 and EphB3 mice accelerated intestinal tumorigenesis, and siRNA-mediated knockdown of EphB4 expression showed decreased tumor growth and metastasis [19]. Taken together, EphB2/EphB3 and ephrin-B1/ephrin-B2 compartmentalize progenitor cells, and ephrin-B-positive differentiated cells to the intestinal crypt and the villus. Loss of polarized expression of EphB/ephrin-B by genetic alterations of a transcriptional regulator such as β-catenin leads to tumor initiation and progression. Earlier reports showed the contribution of EphB4 and its preferred ligand, ephrin-B2, to mammary gland development. EphB4 was expressed predominantly in myoepithelial cells,

and ephrin-B2 expression was mutually exclusive with EphB4 and limited to luminal epithelial cells [20, 21]. The EphB4 expression is regulated strictly during mammary gland development at puberty, pregnancy, and lactation. Therefore, unscheduled and unexpected expression of EphB4 by a genetic modification such as the transgene leads to morphological defects in mammary alveoli and lobules. Abrogation of ephrin-B2 expression in mouse mammary epithelial cells during late pregnancy and lactation showed loss of integrity in the lactating mammary gland [22]. In EphB4 transgenic mice, apoptosis of mammary epithelium, which was essential for lactational involution, was negligible after weaning. The MMTV-NeuT mice crossed with EphB4 transgenic mice (MMTV-­ EphB4) showed an early tumor appearance than those in MMTV-NeuT mice and metastasis to lungs that were not seen before in MMTV-NeuT mice [21]. Tumor histology was similar in MMTV-EphB4 and MMTV-NeuT mice. Therefore, EphB4 expression is a possible trigger for tumor promotion in the mouse mammary gland but does not affect tumor histology or malignancy. EphB4 controls the progenitor cell differentiation of luminal lineage in the mammary gland [23]. Altogether, the differential

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expression of EphB4 during mammary gland MCF10A mammary cell line, but not in development and lactation is essential for mam- MDA-MB-231, a malignant breast cancer cell mary gland homeostasis. Another study indicated line with metastatic potential [30]. Ephrin-A1-­ that EphB4 forward signaling regulated tumor induced EphA2 activation leads to autophosphorcell growth and motility via the Abl/Crk pathway ylation of a tyrosine residue in EphA2 followed in a xenograft model of breast cancer [24]. by c-Cbl-dependent degradation [31]. Therefore, Involvement of EphA2 in tumorigenesis of the loss of ephrin-A1 expression leads to upregulamammary gland was reported earlier [25], and tion of EphA2 expression. Knockdown of ephrinaccumulating evidence shows a positive correla- ­A1 by siRNA in MCF10A resulted in increased tion of overexpression of EphA2 with worse EphA2 expression [29]. tumor grade and prognosis in breast cancer [26]. In a two-stage chemical carcinogenesis model, EphA2 and ephrin-A1 are expressed in the mam- EphA2 played a tumor suppressor role in the mary gland and regulate the development of skin, although EphA2 contributed to tumorigenmammary epithelium. EphA2 contributed to esis in the breast cancer mouse model [32]. HGF-induced branching morphogenesis via the EphA2 was expressed in the hair follicles, interRhoA/ROCK signaling pathway in the mammary follicular epidermis, and sebaceous glands of gland [27]. EphA2 ablation resulted in delayed healthy skin; however, the skin of EphA2 knockonset of tumor progression and reduced metasta- out mice showed no abnormal development. sis frequency in the lungs of MMTV-Neu but not However, EphA2 expression-lacking mice in MMTV-PyMT mice [28]. In human and mouse showed rapid tumor appearance in a two-stage breast carcinoma cells, EphA2 forms a complex carcinogenesis model than those of wild-type or with ErbB2 and enhanced the Ras/Erk and RhoA the heterogeneous mice. In the tumors, cell prosignaling pathway resulting in the acquisition of liferation increased as evaluated by Ki67, and migratory activity in vitro [28]. Expression anal- Erk signal was not downregulated in EphA2-­ ysis of the Eph/ephrin using cultured human lacking tumors, although ephrin-A1-dependent breast cancer cell lines showed a positive EphA2 EphA2 activation led to downregulation of Erk and negative ephrin-A1 correlation with tumor activity and lower cell proliferation in primary malignancy [29]. Recent studies showed EphA2 keratinocytes. Moreover, histological analysis regulating the development of mammary gland, showed papilloma phenotypes for most tumors in as mentioned above. However, the role of ephrin- the wild-type mouse skin, whereas aggressive ­A1  in the mammary gland development is still phenotypes progressing to squamous cell carciunclear. In pathological settings, ephrin-A1 defi- noma were found frequently in EphA2-lacking ciency accelerated tumor development in MMTV-­ tumors. However, the function of EphA2 in tumor PyMT mice suggesting that ephrin-A1 functioned progression in the mouse model remains to be as a tumor suppressor. Indeed, overexpression of elucidated [32]. ephrin-A1 reduced the tumor volume in vivo and spheroid formation in vitro. Moreover, upregulation of glutamine metabolism resulted in 3.4 Roles of the Eph/Ephrin enhanced cell proliferation and increased lipid System in Angiogenesis droplet formation in ephrin-A1-null Her2-­ positive tumor cells. The above suggests ligand-­ Tumor angiogenesis is required for nutrients and independent EphA2 function regulating the O2 supply for tumor growth. When tumor size tumor glutamine metabolism, which is modu- reaches 1–2  mm in diameter, O2supply is shut lated by ephrin-A1-induced EphA2/RhoA/ down, and hypoxic conditions are established ROCK signaling pathway [29]. Differential inside tumors [33, 34]. Thereby, the angiogenic expression of EphA2 and ephrin-A1 was switch is turned on, and upregulation of various observed in some breast cancer cell lines. genes controlled by hypoxia-inducible factor 1α Ephrin-A1 was expressed significantly in (HIF1α) occurs in the tumors. Then endothelial

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cells are recruited into tumors to extend vessels from preexisting vessels. The critical role of the VEGF-VEGFR axis as a regulator of angiogenesis is well established [35]. The functions of the Eph/ephrin system are analyzed well and documented, especially its precise control of vasculogenesis and angiogenesis and the roles of EphB4 and ephrin-B2  in artery/vein development. EphB4 and ephrin-B2 specify the vascular fate of artery and vein by regulating cell adhesion and migration of endothelial cells [7, 8]. Therefore, ephrin-B2 and EphB4 are used often as a maker for the artery and vein, respectively [36, 37]. Ephrin-B2 mutant mice lacking the intracellular domain showed impairments in retinal and tumor angiogenesis in intracranially injected astrocytoma. Ephrin-B2 localized at filopodia formed on tip cells, and the reverse signal controlled the extension of filopodia fibers resulting in vessel sprouting during vasculogenesis. In ephrin-­B2-­ deficient endothelial cells, VEGFR2 failed to internalize in response to VEGF stimulation and induced the downstream signals [38]. Syntenin, also known as syndecan binding protein, was characterized as a scaffold protein of the ephrinB2/VEGFR2 complex. Syntenin was initially ­ isolated from human melanoma cells and termed as melanoma differentiation-associated gene-9 (MDA-9) [39]. Syntenin interacted with syndecan by the C-terminus and regulated membrane cytoskeleton organization [40]. VEGFR2 interacts with ephrin-B1 by syntenin since ablation of syntenin in HUVECs blocked co-­ immunoprecipitation of ephrin-B2 and VEGFR2 complex. Moreover, syntenin lacking endothelial cells were defective in ephrin-B2-induced VEGFR2 signal, VEGFR2 internalization, VEGF-induced endothelial cell migration, permeability, proliferation, and tube formation [41]. Knockdown of syntenin expression resulted in a significant reduction of proangiogenic genes such as Vegf and Mmp2. Subcutaneously injected B16 melanoma cells into syntenin knockout mice grew slower than the wild-type mice. However, tumor angiogenesis was not tested in the study, and the mechanism of reduced tumor growth was unclear. However, we speculated that it was caused by impairment of VEGF-induced angio-

K. Ieguchi and Y. Maru

genesis since syntenin controlled VEGF-induced angiogenesis and the intracellular signals mediated by ephrin-B2 as mentioned above. Syntenin-­ deficient mice also showed a decrease in lung metastasis in an experimental lung metastasis model. The inhibitory effect was due to lower infiltration of myeloid-derived suppressor cells (MDSCs) and not inhibition of tumor angiogenesis. MDSC-derived inflammatory cytokines decreased in the tumors of syntenin knockout mice, suggesting impairment in the establishment of a pre-metastatic niche by inflammatory cytokines such as TNFα and IL-6  in the mice. Differences in MDSCs infiltration was absent 15 days after tumor injection in mice suggesting the involvement of syntenin in early-phase establishment of a metastatic environment in the lungs [42]. In human pathological settings, the ephrin-A1 expression is downregulated in malignant tissues of diverse tumors. Indeed, ephrin-A1 expression was downregulated in MDA-MB-231, a highly metastatic breast tumor cell line compared with MCF10A, a benign breast epithelial cell line [30]. Knockdown of ephrin-A1 expression in 4  T1, a mouse mammary adenocarcinoma cell line, diminished tumor vascular density and lung metastasis. Ephrin-A1 positively regulated VEGF expression in 4 T1 and enhanced endothelial cell migration by an unknown mechanism. Ephrin-­ A1-­ induced angiogenesis and endothelial cell migration are mediated by EphA2 [26]. Administration of Fc-fused EphA2 extracellular domain to decoy membrane-anchored ephrin-A1 reduced vascular density in 4  T1 tumors. Expression patterns of ephrin-A1 in experimental breast tumors were different from human-derived breast tumors. Therefore, we propose that stromal ephrin-A1 expression in inflammatory cells and fibroblasts may regulate VEGF expression and endothelial cell migration in human pathological settings. Further investigation to elucidate the function of ephrin-A1 expression in angiogenesis is required. Increased ephrin-A1 decreases EphA2 expression mediated by c-Cbl-­ dependent degradation. Slit2 expression was elevated in EphA2-deficient mice, whereas overexpression of EphA2 reduced Slit2 expres-

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sion. Moreover, elevated Slit2 expression attenuated tumor volume and VEGF-induced Src and Rac1 activity. Inhibition of Slit2  in EphA2-­ deficient mice rescued VEGF-induced angiogenesis [30, 43]. EphA2 was overexpressed in many types of cancer, such as breast and lung cancer, and showed a positive correlation with malignancy [9]. Collectively, we propose Slit2 could be a promising target for cancer therapy in EphA2 overexpressing tumors.

use of EphA3  in cancer therapy, and no report exists on the use of EphA3-mediated immunotherapy so far. The most well-analyzed immunotherapy of the Eph/ephrin system targets EphA2. The potential EphA2 epitope recognized by CD4+CD8+ T cells was identified by computational analysis using an algorithm for putative HLA-A2 binding site [46]. DC-based vaccine using the peptide corresponding to mouse EphA2 sequence attenuated tumor volume in EphA2-positive glioma cells and unexpectedly reduced lung metastasis and angiogenesis in EphA2-negative B16-BL6 cells [47]. The authors propose that the unexpected inhibition of EphA2-negative tumors was due to nonspecific induction of CD4+CD8+ T cells by DC-based vaccination using EphA2 peptide [48]. The DC-based vaccination using EphA2 peptide markedly inhibited liver metastasis in a mouse model [49]. Coadministration of immunological adjuvants such as ovalbumin (OVA) enhances the antitumor effects of the peptide vaccine. However, the peptide vaccine-based immune therapy often limits the antitumor effect because of adjuvant toxicity or too weak a potency to enhance immune reactivity. A new adjuvant using nanoparticles (NPs) was developed to overcome the difficulties mentioned above [50]. The NPs consist of poly(γ-glutamic acid) and L-phenylalanine. The adjuvant is biodegradable and is distributed widely in the body. The NPs enable the presentation of EphA2 peptides to DCs and macrophages with excellent capacity. EphA2 peptide-conjugated NPs effectively enhanced immunity and induced CD8+ T cells and significantly decreased tumor volume in the liver [51]. Programmed cell death-1 (PD-1) is an important checkpoint protein and a promising therapeutic target for advanced human cancers [52]. PD-1 is widely expressed in various types of tumors and is involved in immune surveillance escape in the tumor microenvironment. Hence, checkpoint inhibitors such as anti-PD-1/anti-PD­L1 and CTLA-4 antibodies bring positive outcomes in cancer patients. It was reported that the Eph/ephrin system regulated PD-L1 expression in breast tumors [53]. In human cultured breast

3.5

 he Eph/Ephrin System T in Tumor Immunity

The immune system in tumor microenvironment controls various actions such as cytokine production and endothelial recruitment. Tumors often evade attacks from natural killer (NK) cells and cytotoxic T lymphocytes (CTL) in the microenvironment to promote tumor growth and progression. Therefore, tumor immunity is considered one of the notable targets for cancer therapy. Dendritic cells (DCs) are antigen-presenting cells that initiate the immune system. DC-based vaccination renders naïve T cells to differentiate to CD8+ antigen-specific CTL or CD4+ T cell and attacks tumor cells without damage to healthy tissues or cells. Today DC-based treatment is considered an effective antitumor therapy. Tumor-associated antigen (TAA) is present abundantly in blood and is an autologous cell antigen that is recognized by autoantibodies in cancer patients [44]. HEK293 cells were transfected with a cDNA library of a metastatic melanoma cell derived from a cancer patient with MHC class II trans-activator to characterize cell surface antigens recognized by CD4+ T cell with melanoma cell-lysing ability. Then, some parts of the EphA3 gene encoding the region between cysteine-­ rich and fibronectin type III repeat domains were found. EphA3 was highly expressed in the brain but lower in other organs and tissues with no expression in hematopoietic cells. However, high EphA3 expression was detected in most malignant tumors, including melanoma, SCLC, sarcoma, kidney, and brain tumors [45]. Further research is needed for the

52

cancer cell lines such as BT-549 and MDA-MB-231, PD-L1 expression was upregulated in dense culture, whereas the expression level was low when cells were seeded sparsely. Furthermore, ephrin-A3, a preferred ligand for EphA10 induced PD-L1 expression, and knockout of EphA10 abolished increased PD-L1 expression. Altogether, these results suggest that upregulation of PD-L1 expression caused by juxtacrine signaling is mediated by the association of EphA10 and ephrin-A3 at the cell boundaries. Surprisingly, PD-L1 expression was upregulated when either EphA2 or EphA4 was knocked down [53]. Therefore, EphA2 and EphA4 can be considered as a negative regulator of PD-L1 expression in breast cancer. EphA10 is a kinase-dead receptor for ephrin-As and cannot induce forward signal. Ephrin-A3 is a shared ligand among EphA2, EphA4, and EphA10. Collectively, we propose that EphA10 may sequester ephrin-A3 from EphA2, EphA4, or both to indirectly inhibit their reverse signals.

K. Ieguchi and Y. Maru

ment of tumor-propagating ability in an orthotopic mouse xenograft model and lower survival rate compared to mice injected with EphA2low cell population. Fc-fused ephrin-A1 stimulation-­ induced EphA2 degradation  resulted  in loss of hGBM stem cell-like properties such as sphere formation and self-renewal ability followed by a loss of stem cell markers and astroglial differentiation. Loss of stem cell-like characteristic features correlated with the decreased expression of EphA2. EphA2 inhibition by either ephrin-­A1-­ stimulation or siRNA-mediated knockdown resulted in a significant reduction of cell proliferation in  vitro and tumor growth in  vivo, suggesting that the EphA2-ephrin-A1 axis controls the stemness in GBM [55]. EphA3 also seemed to be a hallmark of cancer stem cell in hGBM. Ablation of EphA3 reduced cell proliferation and tumor growth compared to EphA2-regulated hGBM. Knockdown of EphA3 attenuated stem cell marker expression with an increase of MAPK activity that positively regulates differentiation of neural precursor cells leading to differentiation [56]. The abovementioned studies described the 3.6 Roles of the Eph/Ephrin function of EphA2 and EphA3 in maintenance of cancer stem cell properties. However, expression System in Cancer Stem Cells profiles and functional relationships between Cancer stem cell (CSC), also known as tumor-­ EphA2 and EphA3  in hGBM are unknown. initiating cell or tumor-propagating cell, has self-­ Expression of EphA3 did not affect EphA2 renewal property and maintains itself in an expression, since there was no difference in undifferentiated state. The characteristics of CSC EphA2 expression between EphA3low and to form nonadherent cell cluster called a sphere EphA3high populations. Further investigations are shows high tumorigenic and metastatic activity. needed to assess whether functional blocking of Moreover, CSC often exhibits resistance to che- either EphA2 or EphA3, but not both, is suffimotherapy and radiotherapy [54]. Functions of cient to abolish stemness of hGBM. the Eph/ephrin system in CSC are mostly unknown. In human glioblastoma multiforme (hGBM), EphA2 and EphA3 functions were well 3.7 Roles of EphA2/Ephrin-A1 investigated. Both EphA2 and EphA3 were in the Metastatic expressed highly at the plasma membrane in Environment acutely isolated hGBM cells from patients. Furthermore, EphA2 and its preferred ligand Expression analysis of protein and mRNA from ephrin-A1 are present in the non-necrotic core of the clinical samples showed a positive correlation hGBM, but not in the periphery and healthy tis- of ephrin-A1 overexpression with poor prognosis sue, and their functions examined. hGBM is in liver and colorectal cancers [57, 58]. The progdivided into two subpopulations, EphA2low and nosis of cancer patients is dependent on the presEphA2high, based on EphA2 expression levels. ence of distant organ metastasis. However, the The EphA2high population showed an enhance- mechanism of ephrin-A1 modulation of metasta-

3  Eph/Ephrin Signaling in the Tumor Microenvironment

sis at the primary site is not fully understood. Recently, we discovered the relationship between overexpression of ephrin-A1 and metastasis. We showed cleavage of membrane-anchored ephrinA1 by a disintegrin and metalloproteinase 12 ­ (ADAM12) in subcutaneously injected primary tumors and enhanced lung vascular permeability by the tumor-derived cleaved ephrin-A1. The soluble form of ephrin-A1 rearranged tight and adherence junction molecules such as claudin and VE-cadherin. Ephrin-A1-induced EphA2 activation and degradation led to the loss of membrane localization of VE-cadherin and claudin-­ 5 in  vitro [59, 60]. Degradation of VE-cadherin generated gaps among cell-cell junctions, resulting in hyper-permeable vasculatures. Tumor cells thereby easily intravasate into the lungs. Treatment with the neutralizing antibody against cleaved ephrin-A1 markedly inhibited lung metastasis as well as tumor growth. Taken together, cleaved ephrin-A1 competes for the preexisting EphA2/ephrin-A1 complex at cell boundaries and internalizes EphA2 leading to c-Cbl-dependent degradation [31, 61], whereas membrane-anchored ephrin-A1 cooperates with EphA2 to maintain cell-cell boundaries and vascular integrity. Tumor-derived cleaved ephrin-A1 would be a molecular target for cancer therapy.

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no significant phenotype in mice [63, 64]. Nevertheless, the Eph/ephrin system contributes to tissue development [65, 66]. In contrast, the redundancy makes it challenging to develop molecularly targeted drugs that specifically recognize single Eph or ephrin. Moreover, expression analysis of Eph/ephrin from biopsy samples of cancer patients is controversial. Development of specific Eph or ephrin targeting drugs is relatively hard because of the reasons mentioned above. Consequently, experimental evidence is required to show the function of each Eph or ephrin in the stroma, tumorigenesis, tumor progression, and acquisition of metastatic capacity and molecular targeting drugs against the Eph/ephrin system that recognize specifically single Eph or ephrin for successful anti-­ Eph/ephrin therapy. If these are overcome, expression analysis of Eph/ephrin in each tumor sample might enable tailor-made treatment. Expanding research with increased efforts into the Eph/ephrin system will successfully lead to development of Eph/ephrin targeting drugs with specific inhibitory effect on selected Eph or ephrin. Eventually, agonist and antagonist peptides against single Eph receptor were identified, and their efficacy is under investigation [67]. Although the mechanism of EphA10-dependent elevation of PD-L1 expression is unknown, inhibition of Eph-mediated CTL functions is a prom3.8 Perspectives ising target for the development of anti-Eph/ ephrin drugs, since anti-PD-1/anti-PD-L1 have Emerging evidence showed a strong association shown dramatic antitumor efficacy in various between dysregulation of Eph/ephrin expression types of tumors [68, 69]. with tumor malignancy, tumor progression, and Soluble forms of ephrin-A1 are present in poor prognosis. Therefore, molecularly targeted human serum of hepatocellular carcinoma drugs for Eph/ephrin system have been ­developed. patients [70]. Furthermore, we recently showed However, no molecularly targeted drug against an elevation of serum ephrin-A1 levels in tumor-­ the Eph/ephrin system is used currently in the bearing mice compared to control mice [60], sugclinic. Indeed, an EphA2 molecularly targeted gesting the use of ephrin-A1 as a biomarker. As drug underwent a phase I clinical trial but halted mentioned before, increased expression of ephdue to side effects [62]. The reason is the Eph/ rin-­A1, a potent metastatic factor that induces ephrin system composes the largest family in lung hyper-permeability, positively correlated receptor tyrosine kinases and redundant Eph-­ with poor prognosis in cancer patients. Therefore, ephrin interactions. The redundancy protects monitoring serum ephrin-A1 levels in cancer against impairments in essential physiological patients might be useful for the prediction of metfunctions as other Ephs or ephrins substitute. astatic risk. Currently, some ongoing clinical triSingle-gene knockout of Eph or ephrin showed als using Eph or ephrin drugs (https://clinicaltrials.

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gov/) are in progress. Although Ephs and ephrins were considered as undruggable targets decades ago, we propose that anti-Ephs or ephrins therapy will be established in due course.

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56 53. Yang WH, Cha JH, Xia W, Lee HH, Chan LC, Wang YN et  al (2018) Juxtacrine signaling inhibits antitumor immunity by upregulating PD-L1 expression. Cancer Res 78(14):3761–3768 54. Cooper J, Giancotti FG (2019) Integrin signaling in cancer: mechanotransduction, stemness, epithelial plasticity, and therapeutic resistance. Cancer Cell 35(3):347–367 55. Binda E, Visioli A, Giani F, Lamorte G, Copetti M, Pitter KL et  al (2012) The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-­ propagating cells from human glioblastomas. Cancer Cell 22(6):765–780 56. Day BW, Stringer BW, Al-Ejeh F, Ting MJ, Wilson J, Ensbey KS et al (2013) EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme. Cancer Cell 23(2):238–248 57. Wada H, Yamamoto H, Kim C, Uemura M, Akita H, Tomimaru Y et al (2014) Association between ephrin­A1 mRNA expression and poor prognosis after hepatectomy to treat hepatocellular carcinoma. Int J Oncol 45(3):1051–1058 58. Yamamoto H, Tei M, Uemura M, Takemasa I, Uemura Y, Murata K et al (2013) Ephrin-A1 mRNA is associated with poor prognosis of colorectal cancer. Int J Oncol 42(2):549–555 59. Larson J, Schomberg S, Schroeder W, Carpenter TC (2008) Endothelial EphA receptor stimulation increases lung vascular permeability. Am J Physiol Lung Cell Mol Physiol 295(3):L431–L439 60. Ieguchi K, Tomita T, Omori T, Komatsu A, Deguchi A, Masuda J et  al (2014) ADAM12-cleaved ephrin-­ A1 contributes to lung metastasis. Oncogene 33(17):2179–2190 61. Sabet O, Stockert R, Xouri G, Bruggemann Y, Stanoev A, Bastiaens PIH (2015) Ubiquitination switches

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4

SRC Signaling in Cancer and Tumor Microenvironment Ayse Caner, Elif Asik, and Bulent Ozpolat

Abstract

Pioneering experiments performed by Harold Varmus and Mike Bishop in 1976 led to one of the most influential discoveries in cancer research and identified the first cancer-causing oncogene called Src. Later experimental and clinical evidence suggested that Src kinase plays a significant role in promoting tumor growth and progression and its activity is associated with poor patient survival. Thus, several Src inhibitors were developed and approved by FDA for treatment of cancer A. Caner Cancer Research Center, Ege University, Izmir, Turkey Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA E. Asik Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA B. Ozpolat (*) Department of Gynecologic Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Center for RNA Interference and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA e-mail: [email protected]

patients. Tumor microenvironment (TME) is a highly complex and dynamic milieu where significant cross-talk occurs between cancer cells and TME components, which consist of tumor-associated macrophages, fibroblasts, and other immune and vascular cells. Growth factors and chemokines activate multiple signaling cascades in TME and induce multiple kinases and pathways, including Src, leading to tumor growth, invasion/metastasis, angiogenesis, drug resistance, and progression. Here, we will systemically evaluate recent findings regarding regulation of Src and significance of targeting Src in cancer therapy. Keywords

Cancer · Oncogene · Src · Kinase · Targeted therapy · Proliferation · Invasion · Migration · Motility · Metastasis · Apoptosis · Microenvironment · Macrophages · Pericytes · Fibroblasts

4.1

Introduction

The Src family of protein tyrosine kinases encompasses 11 members, including Src, Fyn, Yes, Lyn, Hck, Fgr, Blk, Lck, Brk, Srm, and Frk [1]. Src family plays key roles in regulating signal transduction by a diverse set of cell surface

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_4

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receptors in a wide range of cellular events [2, 3]. The Src family kinases are widely expressed in most tissue types and regulate many cellular events including proliferation, survival, cytoskeletal alterations, differentiation, adhesion, invasion, and migration [4, 5]. Src was the first cancer-causing gene that has been the subject of intense investigation for four decades due to its association with malignant transformation and oncogenesis [6, 7]. Rous sarcoma virus (RSV), Src gene isolated from chicken tumor, was discovered in 1911 by Peyton Rous [8]. In 1976, pioneering experiments performed by Harold Varmus and Mike Bishop led to one of the most influential discoveries in cancer research and identified the first cancer-causing oncogene, src gene of RSV (v-src) [9, 10]. This discovery established the concept that activating mutations of cellular proto-oncogenes can lead to cancer. For the landmark discovery of the cellular origin of retroviral oncogenes, Bishop and Varmus were awarded the Nobel Prize in Physiology or Medicine in 1989 [7]. A great deal of knowledge about the function and mechanism of action of v-src showed that various mutations can alter the transformation capacity of this gene [11]. In contrast, c-Src, the cellular homologue in human genome, encodes a non-receptor tyrosine kinase that was also the first gene product discovered to have intrinsic protein tyrosine kinase activity [12]. The Src and Src-related proteins with the broad spectrum of activities have been strongly implicated in the development, cellular growth, progression, and metastasis of a large number of human malignancies [13]. The elevated protein levels and cata-

lytic activity of Src have been detected in a number of human cancers, including lung, skin, colon, breast, ovarian, endometrial, colon, and head and neck malignancies [13, 14]. Consequently, the activity of Src is known to play an important role in malignant transformation and oncogenesis; thus several agents targeting Src are developed for clinical use in cancer patients [15–17].

Fig. 4.1  Organization of human Src. The Src molecule is composed of a myristoylation sequence attached to the SH4 domain, a unique region followed by SH3 domain and

SH2 domain that contains Arg 175, a SH2-kinase linker, a SH1 domain (kinase domain) that contains Tyr419, and a regulatory domain that contains Tyr530 and 531 residues

4.2

The Structure of Src

The Src is approximately 60-kDa protein that is composed of seven functional regions including a 14-carbon myristoyl group (Src homology domain 4: SH4), a unique region, SH3 and SH2 domains, an SH2-kinase linker region, a SH1 domain (protein kinase catalytic domain), and a C-terminal regulatory segment [11] (Fig.  4.1). The N-terminal region containing myristoylation facilitates the attachment of Src to the inner surface of the cell membrane and is required for Src signaling in cells [18]. However, myristoylation alone is not sufficient to anchor Src to the cell membrane. The membrane binding of myristoylated proteins requires a second signal. This signal is polybasic amino acids, which interact with acidic phospholipids on the inner of the membrane bilayer [16, 19]. Myristoylation, together with palmitoylation, requires 16-carbone fatty acid palmitate attachment to cysteine residues at the N terminal region, forming a dual signal motif that targets Src kinase to the inner membrane [20, 21]. Mutation studies have shown that there is a clear relationship between the

4  SRC Signaling in Cancer and Tumor Microenvironment

myristoylation and Src-induced malignant transformation [22, 23]. The SH4 domain is linked to SH3 domain by a segment of 50–90-residue long region, called the unique region. This region shows a significant sequence divergence and provides a functional specificity to each member in the Src family [24]. Following the unique region is an SH3 domain (≈60 amino acid residues), which interacts with polyproline residues (the PxxP motif) located in the SH2-kinase linker region. The SH3 functions as an adaptor function and plays a critical role in the intracellular localization of Src and binding to the Src substrates [17, 25]. The SH2 domain (≈100 amino acid residues), which stabilizes the inactive state of Src kinase, binds to the phosphorylated tyrosine residues on Src and other proteins [17, 26]. SH2 domain preferentially binds to the PYEEI motif in other sequences and thus forms two recognition pockets: (i) the p-Tyr-binding pocket in N-terminal that interacts with the phosphorylated tyrosine, which is quite highly conserved among SH2 domains, and contains a universally conserved arginine residue (Arg 175) and (ii) the hydrophobic pocket in C-terminal that provides specificity toward a hydrophobic residue in a peptide ligand [16]. Molecular dynamics simulations and mutational analyses showed that the SH2 and SH3 domains cooperate in regulating Src kinase catalytic activity in C-terminal [17]. SH1 domain (also known as a protein kinase domain) represents catalytic activity of Src in the C-terminal. This domain has an overall double-lobe structure, with a small ATP-binding N-terminal and a large peptide-binding C-terminal lobes [27]. Autophosphorylation at Tyr-419 that localized in the activation loop between the lobes in the SH1 domain is required for the full activation of Src kinase, which alters the conformation and increases the intrinsic kinase activity [28]. Besides, the C-terminal tail contains the negative-regulatory Tyr-530, which is one of the most important regulatory phosphorylation sites in Src and leads to a less active conformation [11, 28]. When the C-terminal tyrosine is dephosphorylated at Tyr-530, Src is considered in activated form.

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4.3

Regulation of Src Activity

Src activity is regulated by conformational changes following phosphorylation and dephosphorylation of tyrosine residues [29]. The enzymatic activity of Src is mainly controlled by two phosphorylation sites including in Tyr-530 and Tyr-419. Src kinase is normally present in its inactive form by phosphorylation of Tyr-530 [4]. This is consistent with the fact that v-Src is structurally active due to the absence of the C-terminal regulatory region containing Tyr-527 (homologue of Tyr-530 human) [11]. By contrast, Tyr416 (homologue of Tyr-419 human) in the catalytic domain is highly phosphorylated in v-Src, suggesting that these two tyrosine phosphorylation sites are involved in a reciprocal regulatory mechanism [30, 31]. In resting cells, Src kinase is mostly phosphorylated at Tyr-530 and converted to the inactive conformation, which is stabilized by two intramolecular interactions, including binding of phosphorylated Tyr-530 to the SH2 domain and binding of the SH2-kinase linker to the SH3 domain [16]. These intramolecular interactions affect the configuration of the catalytic pocket and cause activation of the enzyme, resulting in intermolecular autophosphorylation of Tyr419 in the activation loop. This autophosphorylation interchanges the catalytic domain into the active conformation and provides recruitment of substrates to the active site [31, 32]. There are two important protein tyrosine kinases in this process which are c-Src kinase (Csk) and its homologue Csk-homologous kinase (Chk). The phosphorylation of Tyr-530 is mediated by these kinases, which act as major negative regulators of Src [33]. The studies have shown that the reduced expression of Csk plays a role in the activation of Src, and overexpression of Csk decreases tumor metastasis in some cancer models [34, 35]. Another mechanism of Csk regulation occurs through the transmembrane adaptor protein, Cskbinding protein (Cbp). After Cbp undergoes into tyrosine phosphorylation by Src, the resulting phosphotyrosine motif binds Csk and allows its recruitment to the plasma membrane where active Src creates a negative-regulatory loop [36],

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leading to suppression of Src-mediated cell transformation and tumorigenesis [37]. In addition to the phosphorylation, several protein tyrosine phosphatases (PTP) have been implicated in regulation of Src. The tyrosine phosphatases, including PTP1B, PTPα, PTPα, PTPα, SH2-containing phosphatase 1 (SHP1), and SHP2, are involved in the Src activity through the dephosphorylation of the C-terminal tyrosine in Src [28, 38, 39]. Overexpression of PTPα has been shown to dephosphorylate Src in human epidermoid carcinoma cells and increase cell adhesion [40]. Similarly, PTP1B is upregulated in human colon cancer cells and reduce phosphorylation Src at Y530, while there was no significant change in the level of phosphorylation at Y419 [41]. Also, PTP1B is a downstream target of ErbB2 in human breast epithelial cells and can activate Src and stimulate Src-dependent phenotype [42]. Src kinase can be also regulated by intramolecular interactions that alter the stabilized inactive conformations and promote the phosphorylation [31]. The SH2/3 domains of Src bind to various tyrosine-phosphorylated proteins by recognizing specific phosphopeptide sequences [43]. Thus, the phosphorylated proteins form new binding sites for other adaptors and effectors, providing amplification of signals [31]. When molecular mechanisms inducing Src kinase activity is terminated, activated Src kinase is quickly degraded by the ubiquitin-proteasome pathway or inactivated by phosphorylation at Tyr-530 [31, 44]. In many cancers, the ubiquitinproteasome degradation system is deregulated, resulting in elevating levels of the activated Src [45]. Although mutations and genomic amplifications leading to the constitutive activation of Src in human cancers have been shown, they are rare [17, 46]. However, the deletion of region containing Tyr-530 is the primary reason of conversion to a transforming protein, leading to constitutive activation of Src kinase [47]. Also, rare mutations at codon 531 in Src have been reported in some cases of colon cancer patients [48]. The Src mutations on 531 result in the production of a stop at codon, causing the deficiency in

C-terminal regulatory region and constitutive active activation [47].

4.4 TME

Regulation of Src Activity by

The dynamic cross-talk between tumor cells and the surrounding cells in TME is known to promote tumor growth and progression. Crosscommunication network between cancer cells and the surrounding microenvironment and stroma is mediated by growth factors, cytokines, and extracellular matrix [49]. Therefore, TME is considered one of the major targets in cancer treatment as targeting only cancer cells has failed to induce long-term complete remissions and patient survival in aggressive cancers, such as lung, ovarian, breast and pancreatic cancer and melanoma. Because Src signaling network is a key pathway that regulates TME and vice versa, Src represents a common and an important molecular target. Src has been shown to play multiple roles in macrophage activation and phagocytosis including the production of inflammatory cytokines, migration, and activation of macrophages [50]. Macrophages are involved in innate immunity and inflammatory diseases. Src has been proposed as a potential molecular target to treat macrophage-mediated inflammatory effects. Thus, Src inhibitors with immunosuppressive and antiinflammatory properties have been shown to protect macrophage-mediated inflammatory diseases. Pericytes are perivascular cells that envelop and make intimate connections with adjacent capillary endothelial cells. Pericytes have a profound impact in vessel formation and angiogenesis [51]. The kinase domains of PDGFRα phosphorylate tyrosine residues of the receptor’s cytoplasmic domain, which act as docking sites for phosphatidylinositol 3-kinase and Src family kinases. Birbrair et al. have characterized two distinct pericyte populations, including type-1 pericytes that are able to generate adipocyte and fibroblasts [52]. Tumorassociated adipocytes [53] and fibroblast [54] are known to contribute to tumor growth. Interactions

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between adipocytes and breast cancer cells stimulate cytokine production through Src-mediated malignant progression [54]. Src can phosphorylate substrates and mediators of numerous molecular pathways that promote tumor cell survival, proliferation, migration, invasion, and angiogenesis [55] (Fig.  4.2). Src kinase is activated by multiple growth factor signaling pathways, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and integrins and Eph receptor (i.e., EphA2) [49, 57]. Src cooperates with multiple receptors modulating downstream signaling and acts as a signal transducer induced by the cell surface receptors as part of

outside-in signaling through sequential phosphorylation of tyrosine residues [58]. Src kinase is an important mediator of integrin signaling in cancer cells and regulates the activity of several proteins, including focal adhesion kinase (FAK), epidermal growth factor receptor (EGFR), Akt/PI 3-kinase, and Rho/ROCK signaling, that are frequently deregulated in cancer cells. Src also interacts with numerous cellular factors, adhesion molecules, cytoskeleton components, steroid hormone receptors, components of some pathways, and the adaptor proteins such as BCAR1, Cas family scaffolding protein (p130Cas), and Src homology and collagen (Shc). The most of them play a crucial role in tumorigenesis and some of them are highlighted below [4, 47, 59].

Fig. 4.2  Activation of Src signaling network. Src is activated by numerous receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and integrins. Activation of Src leads to activation of MAPK-MEK1/2 and ERK1/2 through RAS and RAF, leading to the transcription of genes promoting cell growth, proliferation, survival, and invasion. Signal transducer and activator of transcription 3 (STAT3) activation regulates gene expres-

sion of VEGF, IL10, and FoxP3, inducing angiogenesis and immunosuppression. PI3K/Akt signaling promotes protein synthesis, cell growth, survival, and drug resistance. Activation of Rho GTPases/Rho-associated protein kinase (ROCK) and myosin-light chain (MLC), leading to actin cytoskeleton remodeling and cell motility and stromal feedback and extracellular matrix deposition, respectively. Activation of Arp2/3 and WAVE results in the formation of new actin polymers and the inhibition of depolarization of actin and cell motility and migration. (Adapted from Parkin et al. [56])

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Platelet-derived growth factor (PDGF), acting as a vascular endothelial growth factor, is closely associated with tumor development. The latest findings indicate that PDGF signaling pathway can regulate many cellular processes, including cell proliferation, migration, invasion, angiogenesis, and metastasis in cancer cells [60– 62]. Src is an important component of the PDGF receptor signaling pathway that causes cell cycle progression in fibroblasts, contributing to cancerassociated fibroblast (CAF) activity [63, 64]. Ligand activation of the PDGF receptor β-subunit leads to activation of Src through SH2 domain binding to specific phosphotyrosine residues in PDGF receptor, which is upregulated in various cancer types [28, 63, 65]. Moreover, the integrin signaling pathway is activated by Src and extracellular signal-regulated kinase (ERK)-mediated collagen engagement, leading to enhanced PDGF production, which mediates fibroblast recruitment in tumor [66]. Besides, PDGF stimulation induces expression of Myc in addition to activation of Src kinase, and both of them are required for PDGF-induced mitogenesis [67, 68]. Signal transducer and activator of transcription-3 (STAT-3) is induced rapidly by PDGF in Src -dependent manner in tumor cells [46, 69]. Also, STAT activation by tyrosine phosphorylation of Src contributes to Myc mitogenic pathway, inducing VEGF expression and angiogenesis, and plays a role in invasion and metastasis [70, 71]. Moreover, Src is capable of enhancing tumor growth through the induction of Bcl-xL anti-apoptotic protein expression by STAT3, contributing to oncogenic signaling by preventing apoptosis and stimulating cell cycle progression [13]. Overall, studies define the signaling pathway in the following order, PDGF » Src » STAT » Myc, which is important in PDGFinduced mitogenesis [69]. Epidermal growth factor receptor (EGFR) family regulates differentiation, survival, proliferation, motility, and angiogenesis in the initiation and progression of cancer [72]. Mostly, Src is co-overexpressed with members of the EGFR family in breast cancer. Upon ligand binding and

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activation, EGFR physically interacts with and activates Src [14]. Thereafter, EGFR can be phosphorylated on multiple sites by Src, most notably Tyr-845, resulting in stimulation of cell growth, angiogenesis, and survival pathways [4, 14, 73, 74]. Phosphorylation of Tyr-845 can activate STAT5 and Cox II signaling pathways promoting EGF-induced cell proliferation and cell survival, respectively [74–76]. Moreover, Src can regulate lateral activation of the EGFR by extracellular stimuli other than EGF, such as steroids, cytokines, extracellular matrix (ECM) proteins, and ionizing radiation [74, 77, 78]. As a result, these studies demonstrated that Src provides the interaction between EGFR and nonrelated membrane receptors/intracellular signaling molecules through phosphorylation of Tyr-845, resulting in the increase of EGFR activity. In addition, Src has an important role in the receptor tyrosine kinase-mediated responses through the activation of the growth factor receptor-bound protein 2 (Grb-2) and PI3K/Akt pathways. Src with phosphorylated FAK creates a binding site for the Grb2-Sos complex that leads to the activation of Grb2/Ras/MAPK cascade [24, 58] and recruits the p85 regulatory subunit of PI3K that leads to the stimulation of PI3K/Akt pathways [24]. After Ras/MAPK pathway activation through stimulation of Src tyrosine kinases and integrin signaling, it causes changes in gene expression and activation of transcription factors, which are responsible for tumorigenicity and expression of matrix disrupting proteases, such as metalloproteases MMP2 and MMP9 [58, 79]. Src can also promote directly the activation of PI3K by binding PI3K to the Src SH3 domain or by mediating the phosphorylation of PI3K and protects cells from death during tumor growth, invasion, and metastasis [80]. Moreover, Src can inhibit PTEN, which suppresses PI3K by hydrolyzing PIP3 to PIP2, inducing PI3K pathway [81]. Recently, a correlation between increased signaling in the PI3K pathway and resistance to Src inhibitor drugs in cancer cells has been shown [82].

4  SRC Signaling in Cancer and Tumor Microenvironment

4.5 Src-Induced Cell Adhesion and Migration Adhesion and migration are closely related events involving cytoskeletal rearrangement and molecular interactions during invasion and metastasis of cancer cells [83]. Src acts primarily as a negative regulator of Rho-dependent cell-matrix adhesions, actin bundle formation, and adhesive force as well as controls the biochemical events occurring during migration of the cell. The assembly/disassembly of both cell-matrix adhesions and actin-based cytoskeletal structures are required during migration and each of these events, which are controlled by the Rho family GTPase members such as Rho, Rac, and Cdc42 and integrin [84]. Rac and Cdc42 activity promotes the formation of new adhesions and cytoskeleton dynamics, whereas Rho function stimulates the stabilization of adhesions, the assembly of actomyosin fibers, and the contractility [24, 85]. Thus, cells having the activated Src show poor adhesion to ECM and increase migration in cancer [83, 86]. Integrins that are located on the surfaces of both tumor cells and all types of stromal cells play critical roles in cancer progression and interactions with the tumor microenvironment. It has long been known that integrin-mediated signaling effectors in cancer cells include the Src kinases [87, 88]. Important functions of Src include the links between ECM, integrins, and cytoskeleton, inducing adhesion turnover and remodeling the actin cytoskeleton [89]. After ECM ligand binding, the cytoplasmic tail of the β3 integrin directly interacts with the SH3 domain of Src. Later, activated Src can enhance the activation of Vav proteins and Tiam, inducing the stimulation of actin-driven activity at the site of integrin engagement [84]. The phosphorylation of β1-integrin causes inhibition of Rho-mediated cytoskeletal contractility, leading to transformed phenotype during migration. The interaction between the β3 integrin and SH3 domain of Src can trigger the stimulation of STAT3 and FAK signaling to promote tumor growth [84, 90]. Also, the PI3K/Akt

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pathway is involved in the integrin signaling through Src [91]. The activation of integrindependent PI3K at adhesion sites plays a central role in migration and causes PIP3 accumulate in cells where it can recruit signaling proteins from the cytoplasm to initiate actin polymerization, spreading of cancer cells [92]. Focal adhesion kinase (FAK), which is a major mediator of integrin signaling, is another kinase molecule capable of binding to the Src-SH2 domain and activating the Src kinase [83, 93]. The elevated Src activity and increased FAK expression, which are commonly detected in cancer cells, enhance the migratory potential of tumor cells and facilitate invasion and metastasis [89]. FAK acts both as a signaling molecule and a scaffold to recruit Src and the Src substrates to integrin engagement sites [83, 93, 94]. FAK is able to bind to SH2/SH3 domains of Src and several other signaling and cytoskeletal molecules [95]. After Src/FAK complex activation, phosphorylated FAK-associated Src substrates including Cas, p190RhoGAP, Paxillin, and Tensins promote the reorganization of the cytoskeleton and cell migration [85]. CRK-associated substrate (Cas) is an adaptor protein that plays a role in integrin-mediated cell adhesion and able to bind a number of signaling molecules including Src and FAK [94, 96]. With integrin ligation, Src promotes the phosphorylation of Cas, which serves to recruit Crk protein that interacts with dedicator of cytokinesis (DOCK) 180 protein, leading to activation of Rac [24, 97]. Therefore, the local stimulation of Rac at the site of ECM-integrin contact results in localized activity and formation of a cytoplasmic extension, the first step of the migratory cycle [96]. Moreover, Rac can activate c-Jun N-terminal kinases (JNK), which leads to transcriptional activation and increased expression of matrix metalloproteinases (MMP)-2 and MMP-9 and then causes proteolysis and invasion [94, 98]. The activated JNK can also induce migration by effecting serine phosphorylation of Paxillin [85]. Paxillin, which was first identified as a phosphotyrosine protein in cells transformed by Src,

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is one of the key substrates within focal adhesionrelated molecules, coordinating cytoskeleton organization, adhesion turnover, and cell migration [99, 100]. Paxillin binding to FAK and Src tyrosine phosphorylation site creates docking sites for SH2 domain-containing signaling molecules at cell matrix. Phosphorylated Paxillin may bind to Crk and thereby activates the Crk/ DOCK180 pathway, resulting in Rac stimulation like Cas [96, 101]. Also, by replacing both downstream and upstream of FAK, Paxillin can enhance the recruitment of FAK at adhesion sites and stimulate their turnover [102]. FAK-mediated phosphorylation of p190RhoGAP, which is associated with the inhibition of RhoA during cell spreading on fibronectin, forms a complex with p120RasGAP [103]. Phosphorylated Paxillin binds to p120RasGAP, which diminishes the interaction of p120RasGAP with p190RhoGAP at the membrane. This increases the amount of p190RhoGAP available for interacting with Rho and results in the suppression of localized RhoA activity and facilitates adhesion and motility [104]. Besides, the recruitment of paxillin to the FAK-Src complex can further modulate cell motility. Paxillin also leads to an increased ERK/ MAPK activity that is required for activation of protease calpain, proteolytic cleavage, and disruption of adhesions [85, 94, 105]. Tensin is one of the members of integrin-dependent adhesion proteins and can serve as Src substrate. The SH2 domains of Tensin are responsible for promigratory and proangiogenic functions by binding Cas and FAK in cancer cells [47]. As a result, the FAK-Src complex coordinates the strength of cell-cell adhesions and cell-ECM interactions, which also promotes collective cell migration in cancer [106, 107].

4.6 Role of Src in Invasion of Cancer Cells Cancer invasion is initiated and maintained by signaling pathways that control the turnover of cell-matrix/cell-cell junctions and cytoskeletal

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dynamics in tumor cells. This is also an essential prerequisite for the development of distant metastasis [24, 49]. The mesenchymal-type invasion is one of the most important mechanisms of tumor cell invasion and involves the combined effects of migration and ECM breakdown caused by matrix-degrading proteases [108, 109]. Src induces the upregulation of MMP2, and then TNF-α induces expression of MMP-9 by Srcmediated EGFR/PI3K/Akt-dependent manner [105, 110]. However, small heat shock protein (HSP) 27 increases the upregulation of MMP-9 in breast cancer cells and promotes cell invasion by inhibition of Yes tyrosine kinase [111]. Src also enhances the expression of MMP-1 and MMP-2 by regulating the ERKs/PEA3/STAT [112] and ERK/Sp1 signaling, respectively [113]. Moreover, Src can inhibit the endocytosis and increase the cell surface expression of membrane-type (MT) 1-MMP, which is responsible for the activation of MMP2 [114]. Overall, these studies demonstrated that Src kinases regulate MMPs expression and activation and thus modulate the course of cancer cell, invasion, metastasis, and progression.

4.7 Role of Src in Epithelial-toMesenchymal Transition In order to invade and metastasize to distant tissues, cancer cells transform themselves through the epithelial-to-mesenchymal transition (EMT) and modulate the microenvironment, inducing angiogenesis and invasion [115]. Recent studies indicated that Src promotes EMT phenotype which has profound effects on tumor invasion and metastasis [57]. During EMT, Src activity promotes disruption of cell-cell junctions and invasion through the phosphorylation of the E-cadherin-β-catenin complex, resulting in functional loss of E-cadherin that maintains epithelial connections to neighboring cells [24]. This event releases β-catenin in the cytoplasm, leading to its accumulation in nucleus and stimulation of β-catenin-dependent transcriptional activities

4  SRC Signaling in Cancer and Tumor Microenvironment

involved in EMT and invasion, such as Myc, Snail, cyclin D1, vimentin, and matrix-degrading proteases [109, 116]. Src can also cause the degradation of E-cadherin by promoting its phosphorylation, endocytic internalization, and lysosomal targeting [117, 118]. Thus, epithelial tumor cells migrate into vascular structures and invade the parenchyma of distant organs, causing metastasis [119]. Src has been shown to phosphorylate caspase-8 at multiple tyrosine sites, altering its function from proapoptotic to promigratory function in tumors [24]. Phosphorylation of caspase-8 at Tyr-465 by Src prevents its cleavage and suppresses apoptosis. Then, the inactive caspase-8 allows the phosphorylation of Tyr397 by binding to the SH2 domain of Src, further activating Src by promoting Tyr-419 phosphorylation on Src. Thus, caspase-8 can act both as a substrate and an activator of Src [120]. Also, Src-mediated phosphorylation of caspase-8 enables its interaction with PI3K, thereby stimulating cell migration through subsequent activation of Rac, ERK, and calpain [121]. The interaction between Src and caspase-8 plays an important role on these critical functions by integrating survival and migratory inputs in the tumor environment.

4.8

Src Activation in Hypoxic TME

Hypoxia is a common characteristic of the tumor microenvironment that can promote cellular functions associated with tumor progression and dissemination including invasion, migration, and survival [122]. Recent studies have shown a tight association between Src and hypoxia through hypoxia-inducible factor-1α (HIF-1α), which is a molecular marker for hypoxia. HIF-1α independently activates Src, which may function as the downstream and upstream effector of HIF-1, to promote metastases [123]. Src also mediates hypoxia-induced VEGF production in a number of cell types, contributing to the control of tumor angiogenesis. The Src/VEGF pathway can be stimulated by hypoxia and promotes angiogene-

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sis for the growth of both primary tumor and distant metastases [122–124].

4.9 Therapeutic Targeting Src in Cancer Patients Due to the well-established role of Src in multiple signaling pathways that regulate cell proliferation, survival, invasion, metastasis, and angiogenesis [24, 88, 125] and positive correlation between poor clinical outcome in patients, Src has been viewed as a promising drug target for various cancers [16, 126]. In fact, Src family kinases have been investigated in the cancer treatments for three decades, and extensive work has been performed on the development of Src inhibitors [127]. Four orally effective Src/multikinase inhibitors called bosutinib, dasatinib, ponatinib, and vandetanib are FDA approved for the treatment of various malignancies (Table  4.1). Bosutinib and ponatinib are approved for the treatment of leukemias (CML and ALL) and are currently in clinical trials for the treatment of breast cancer and glioblastoma and various solid tumors, respectively. Dasatinib is approved for the treatment of CML and underwent numerous clinical trials for various solid tumors and all. Mostly, FDA approved these drugs on hematologic malignancies, but not directed against solid tumors. Vandetanib that was initially developed as a VEGFR2 inhibitor is approved for the treatment of medullary thyroid carcinoma and is in clinical trials for numerous solid tumors (www.brimr.org/PKI/PKIs.htm) [16, 47]. Although these inhibitors are generally well tolerated with restricted toxicity and good efficacy in hematological malignant diseases, the efficacy in clinical trials in solid tumors has been modest [135]. The reason for the failure of SRc kinase inhibitors in solid tumors is currently unclear. Additionally, AZD0530 and AZD0424 are currently undergoing clinical trials for various malignancies (www.clinicaltrials.gov). It is reported that the combination of inhibitors of Srcfamily kinases with other kinase inhibitors or chemotherapy may show synergistic effects or more improved efficacy [135].

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66 Table 4.1  Selected effective Src/multikinase small molecule inhibitors Drug (year approved) Dasatinib (2006)

Structure (PubChem CID)

Vandetanib (2011)

Bosutinib (2012)

Known targets/disease BCR-Abl, EGFR, Src, Lck, Yes, Fyn, Kit, EphA2, PDGFRβ Ph+ CMLb, Ph+ ALL, breast, colorectal, endometrial, head and neck, ovarian, and small cell lung cancers, glioblastoma, melanoma, and NSCLC RET, EGFRs, VEGFRs, Brk, Tie2, EphRs, and Src family kinases Medullary thyroid cancer, breast, head and neck, kidney cancers, NSCLC, and several other solid tumors

References [15, 128]

BCR-Abl, Src, Lyn, and Hck

[15, 132, 133]

[129–131]

Ph+ CMLb, Ph+ ALLb, breast cancer, glioblastoma

Ponatinib (2012)

4.10 Concluding Remarks Since its discovery in 1976, Src kinase has been shown to play a pivotal role in promoting tumor growth and progression. Due to its clinical significance and association with poor patient outcome and shorter survival, several Src inhibitors have been developed and approved by FDA for treatment of various cancers. Despite being a highly complex and dynamic milieu, TME plays a significant role in induction and activation of Src by multiple factors, including growth factors and chemokines and a cross-talk between cancer cells and TME, leading to cell proliferation, survival, tumor growth, invasion/metastasis, angio-

BCR-Abl, BCR-Abl T3151, VEGFR, PDGFR, FGFR, EphR, Src family kinases, Kit, RET, Tie2, Flt3 Ph+ CMLb, Ph+ ALLb, endometrial, GIST, hepatic biliary, small cell lung, and thyroid cancers

[15, 134]

genesis, drug resistance, and progression. Further understanding the molecular mechanisms mediated by Src and effects of combinatory therapies with SRC inhibitors in the clinical setting will provide further benefits to treatment of patients and more effective targeted therapies.

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5

Purinergic Signaling Within the Tumor Microenvironment Dobrin Draganov and Peter P. Lee

Abstract

Keywords

Accumulating studies have clearly ­demonstrated high concentrations of extracellular ATP (eATP) within the tumor microenvironment (TME). Implications of these findings are multifold as ATP-mediated purinergic signaling has been shown to mediate a variety of cancer-related processes, including cell migration, resistance to cytotoxic therapy, and immune regulation. Broad roles of ATP within the tumor microenvironment are linked to the abundance of ATP-regulated purinergic receptors on cancer and stromal and various immune cell types, as well as on the importance of ATP release and signaling in the regulation of multiple cellular processes. ATP release and downstream purinergic signaling are emerging as a central regulator of tumor growth and an important target for therapeutic intervention. In this chapter, we summarize the major roles of purinergic signaling in the tumor microenvironment with a specific focus on its critical roles in the induction of immunogenic cancer cell death and immune modulation.

Extracellular ATP (eATP) · Tumor ­microenvironment (TME) · Purinergic signaling · P2X/P2Y receptors · Immunogenic cell death (ICD) · Autophagy · Cancer immunotherapy

D. Draganov Calidi Biotherapeutics, San Diego, CA, USA P. P. Lee (*) Department of Immuno-Oncology, City of Hope Comprehensive Cancer Center, Duarte, CA, USA e-mail: [email protected]

5.1

 igh eATP as a New Hallmark H of Cancer

The tumor microenvironment (TME) has a major impact on the response of cancer cells to both chemotherapy/radiation and immune-based therapies. These include (1) hypoxia driving activation of the HIF-1α/VEGF/β-catenin axis that has been associated with activation of stem cells and development of resistance to therapy [1, 2], (2) the Warburg effect characterized by aerobic glycolysis associated with acidification of the tumor microenvironment, and (3) accumulation of high concentrations of extracellular ATP and adenosine, whose roles on tumor growth and metastasis are becoming increasingly evident [3, 4]. ATP release has been implicated in both resistance to chemotherapeutic drugs and induction of immunogenic cell death (ICD) since the balance between ATP and immunosuppressive adenosine is critical for the regulation of antitumor immune

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_5

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responses [5, 6]. In addition to immunogenic cell 100 nM, concentration of ATP within the tumor death, ATP release also triggers P2X-/P2Y-­ microenvironment is often in the range of 0.1– dependent purinergic signaling cascades mediat- 0.5  mM, approaching the concentration of ATP ing a plethora of biological functions in the tumor within the cytosol (5–10 mM) [7]. Maintenance microenvironment and beyond (Table  5.1). To of such high levels of extracellular ATP cannot be depict a complete and comprehensive picture of attributed entirely to tissue damage and accidenthe complex and often contradicting functions of tal necrotic cell death (which can result in plasma extracellular ATP in cancer, we will begin to membrane rapture and release of cytosolic ATP). unravel and contrast the distinct roles of puriner- Instead, ATP release is a highly regulated mechagic signaling on the induction of immunogenic nism associated with exocytosis of ATP stored cell death and the various ATP-dependent mecha- within various cytosolic vesicles or transient and nisms promoting tumor growth and immunologi- reversible plasma membrane permeabilization – cal evasion (Fig.  5.1). The critical balance these are physiological processes that can be between these dual antitumor and tumor-­ uncoupled from cell death [8, 9]. Nonselective promoting functions of purinergic signaling pores formed by Pannexin-1 and Connexins are within a tumor will determine its progression and believed to be the primary source of ATP release provides opportunities for novel therapeutic into the tumor microenvironment. These versatile intervention. channels respond to a variety of signals including ATP has long been thought to accumulate ion flux, mechanical and osmotic shock, and within the tumor microenvironment, but experi- ATP-gated purinergic receptors. This regulated mental validation and direct measurement of manner of ATP release is possible due to the fact extracellular ATP (eATP) levels was not possible that opening of Pannexin-1 channels is inhibited until the pioneering work of Pellegatti et al. using by eATP, providing a negative feedback mechaplasma membrane luciferase reporter [3, 4]. As nism. At the same time, this also represents an demonstrated by the group of Di Virgilio and oth- opportunity for various feed-forward amplificaers, while eATP in normal tissues rarely exceeds tion loops upon coupling with different P2X and Table 5.1  Downstream effects of eATP Immunostimulatory

Immunosuppressive

Metabolic

Functional/ECM modulation

Recruit inflammatory cells Chemotaxis via P2X/P2Y receptors Promote DC maturation, function Concentration dependent cancer cell death via P2X7R Release of HMGB1, NLRP3 inflammasomes, and inflammatory cytokines T cell activation and amplification of TCR signaling CD39/73 mediated breakdown to adenosine Increase chemotaxis of Treg, MDSC ATP degradation by Tregs Promotes MDSC immunosupression Augmented mitochndrial respiration and oxidative phosphorylation Stablizining HIF1a and b-catenin for hypoxia adaptation Increase GLUT1 and other glycolytic enzymes Uptake by cancer cells as energy source Promote MMP9, cathepsin B release Increase ROS from NADPH oxidase and mitochondria Intracellular Ca2+ −> activation of PI3K, AKT, ERK, MAPK pathways Promote stemness, survival, resistance to therapy, EMT Increase motility, migration, invasion, metastasis Promote VEGF production and angiogenesis Increase autophagy/autophagosome/microvesicle ATP release

5  Purinergic Signaling Within the Tumor Microenvironment

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Fig. 5.1  High extracellular level of ATP (eATP) represents a hallmark of the tumor microenvironment and plays multiple roles in the modulation of tumor growth and survival

Fig. 5.2  Sources and fates of eATP within the tumor microenvironment. Accumulation of high eATP in the tumor microenvironment is a function of the complex balance between the rates of cellular ATP release and consumption

P2Y purinergic receptors driving ATP-induced ATP release [10–14]. These mechanisms further argue in favor of a highly regulated and generally non-lytic source of eATP in the tumor microenvironment (Fig. 5.2).

specific immune responses is critically dependent on the release of intracellular ATP as a prototypic member of the danger-associated molecular patterns (DAMPs) family. In contrast to microbial pathogen-associated molecular patterns (PAMPs), DAMPs are associated with sterile inflammation and sensing of ubiquitous cellular 5.2 The Role of ATP in ICD components, whose release normally occurs only upon conditions of stress, damage, and cell death. ATP has emerged as a key mediator in the induc- Extracellular release of the central cellular energy tion of immunogenic cell death (ICD) within the metabolite ATP has therefore evolved as a natural tumor microenvironment [15–20]. The ability of signal for cellular distress and drives multiple dying cancer cells to prime and promote tumor-­ downstream signaling cascades to promote cell

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activation and tissue homeostasis. Accumulating evidence have shown that long-term clinical responses in some patients after chemotherapy involve host anticancer immune responses associated with the induction of immunogenic cell death (ICD) by several chemotherapeutic agents, including doxorubicin and oxaliplatin [15, 21, 22]. ICD is characterized by the exposure and release of specific DAMPs with often critical and nonredundant functions [16]. Multiple DAMPs have already been implicated in the induction of ICD and subsequent potentiation of protective tumor-specific immunity, including the release of intracellular ATP and HMGB1, surface exposure of key intracellular chaperons such as calreticulin and various heat shock proteins, as well as the secretion of type I interferons [15]. These pro-inflammatory and immunostimulatory effects associated with immunogenic cell death are not restricted to cancer. ATP and HMGB1 mediate immunostimulatory functions in cardiac infarction and brain stroke, where ischemia/reperfusion (I/R) injury is associated with massive inflammatory responses and necrotic cell death through P2X7-dependent purinergic signaling and NLRP3-/caspase-1-dependent pyroptosis [23]. Pyroptosis is an important

defense mechanism that might have evolved to protect myeloid and epithelial cells against certain intracellular parasites including viruses and bacteria (e.g., Salmonella and Listeria) [24–28]. Components of activated inflammasomes can be secreted and recaptured by other myeloid cells, providing a potent amplification step stimulating protective immunity [29–31]. Caspase-1 has also been reported to regulate nonclassical secretion of nuclear HMGB1 and serves as a link between HMGB1 and purinergic signaling [32]. ATP and HMGB1 function as prototypic danger signals that can promote immune-mediated destruction as well as inflammatory-reparative responses [33, 34]. The interplay between ATP and HMGB1 appears to be essential for tumor growth, angiogenesis, and metastasis. Hypoxia and maintenance of elevated levels of extracellular ATP and HMGB1 are characteristics shared between the tumor microenvironment and sites of I/R injury. However, it is still unclear how tumors manage to effectively utilize the tumor-promoting functions of the ATP/HMGB1/caspase-1 system while successfully evading immune-mediated destruction – this is a paradox that will be revisited and discussed in more depth later in this review (Fig. 5.3).

Fig. 5.3  The role of eATP in immunogenic cell death. Acute ATP-dependent purinergic signaling in the TME drives P2X7-mediated cellular cytotoxicity and release of

multiple cellular DAMPs including HMGB1, CALR/ HSP, IFNs, and pro-inflammatory cytokines

5  Purinergic Signaling Within the Tumor Microenvironment

In addition to its diverse functions within the tumor or other damaged and hypoxic microenvironments, the ATP/HMGB1/caspase-1 axis also plays a central role in the activation and effector functions of multiple immune cell populations. Interplay between P2X1, P2X4, and P2X7 purinergic receptors and their coupling to Pannexin-­ 1-­mediated release of cytosolic ATP creates a feed-forward loop providing essential TCR signal amplification that enables proper activation and effector functions of both conventional and nonconventional T-cell subsets [35–39]. Release of ATP and subsequent engagement of purinergic receptors commonly expressed on myeloid cells additionally drive the maturation and activation of various phagocytic and antigen-presenting cells in response to classical adjuvants and various crystalline structures [40–44], including dendritic cells as the primary antigen-presenting cell type [45, 46]. Interplay between P2X7 signaling and activation of the NLRP3 inflammasome in DCs drives capsase-1-mediated processing and nonclassical secretion of the pro-inflammatory cytokine IL-1b [47, 48]. Pathogen infections are also known to trigger activation of the ATP/ P2X7/NLRP3 inflammasome axis in myeloid cells [49]. Moreover, P2X7-dependent purinergic signaling operating through K+ efflux and NADPH oxidase-generated ROS and NLRP3/ caspase-1/inflammasome activation appears to be functional in multiple hematopoietic as well as epithelial cell types [50, 51], where it represents an important cellular defense mechanism restricting infection by a variety of pathogens, including viruses, bacteria, mycobacteria, protozoa, and fungi [52, 53]. This primary defense mechanism of the ATP/P2X7/NLRP3 inflammasome axis, while linked to the potent bactericidal NADPH oxidase-mediated respiratory burst in the phagosomes of activated neutrophils, is also associated with activation of caspase-1, IL-1b/IL-18 release, and the highly inflammatory pyroptotic form of cell death [23]. These effects potentially involve a similar ATP release-mediated feed-forward loop converging on the P2X7 and NLRP3/caspase-­1 inflammasome as seen in the context of immunological synapse formation and T-cell activation [54–57].

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Release of extracellular ATP in the context of immunogenic tumor cell death plays a critical role in the recruitment of immune cells. Extracellular ATP levels can function as a chemokine gradient both directly by driving the migration of immune cells over short distances and indirectly through amplification of other chemokine signals, especially over longer distances. ATP release via Pannexin-1 and Connexin 43 hemichannels and autocrine purinergic signaling have been demonstrated to play a central role in the migration of neutrophils and macrophages in response to various chemotactic signals including formyl peptide, IL-8, and complement/anaphylatoxin C5a [58]. A specific role for P2Y2 and P2Y12 receptors in the ATP-mediated amplification of chemotactic signals has been shown. Other reports, however, indicate that ATP itself released from dying/apoptotic cells or sites of inflammation and tissue damage/distress can directly guide the recruitment of immune cells through P2X4, P2Y2, and P2Y12 receptors [59– 62]. This function of ATP in mediating chemotaxis and recruitment of immune cells is consistent with its role as a prototypic DAMP that is characteristically released during various inflammatory processes and tissue damages. Sustained Ca2+ flux resulting in mitochondrial overload/dysfunction and engagement of NADPH oxidase driving ROS production also link ATP release/purinergic signaling with activation of the unfolded protein response (UPR), autophagy, and subsequent surface exposure of other key ICD-associated DAMPs such as calreticulin and heat shock proteins. Importantly, autophagy has been demonstrated to be necessary for extracellular ATP release during ICD, a phenomenon that may be associated with accumulation of large amounts of ATP in autophagosomes, driven by acidification and the H+ gradient created by vacuolar V-ATPase proton pumps and counteracted by the vesicular nucleotide transporter (VNUT) or alternatively by adenylate kinase synthesis of ATP from ADP in the lumen of the phagosomes [7, 8, 63]. Recent data indicate that ATP release from cancer cells undergoing ICD, including the activity of cytotoxic chemotherapeutic drugs or radiation, is mediated

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by the ATP/P2X4/P2X7-gated Pannexin-1 channels and may involve caspase-dependent C-terminus cleavage and activation of Pannexin-1, which appears to be required for autolysosome exocytosis [19, 64–68]. While ATP release may serve the role of a potent “find me signal,” surface exposure of calreticulin and heat shock proteins enables engagement of their cognate receptors on antigen-presenting cells, thus also providing an adequate “eat me signal” by means of efficient delivery, processing, and cross-­ presentation of tumor-associated antigens. These multifaceted effects of extracellular ATP release and purinergic signaling appear to be crucial for the ability of dying tumor cells to prime potent tumor-specific immune responses by facilitating the recruitment of antigen-presenting cells, tumor antigen acquisition, and proper antigen presentation (see Figs. 5.1 and 5.3). One of the biggest paradoxes in immuno-­ oncology is that despite all of the potent pro-­ inflammatory and immunostimulatory functions of purinergic signaling in cancer and beyond, high extracellular ATP is one of the hallmarks of the tumor microenvironment [3, 4]. This paradox becomes even more puzzling considering that cancer cells of diverse origin are known to be very sensitive to high concentrations of extracellular ATP [53]. This is likely due to the expression of high levels of P2X7 receptors that, if stimulated, can trigger irreversible pore formation, membrane permeabilization, mitochondrial collapse (MTP), and direct cytotoxicity  – yet paradoxically has been correlated with tumor survival, progression, and metastasis [69–71]. This may be linked to nontoxic, low-level ATP trophic signaling and potentially constitutive activation of the inflammasome pathway in cancer [72, 73] (Figs. 5.4 and 5.5). P2X7 signaling has been implicated in inducing direct cytotoxicity in tumor cells in the ­context of exposure to high concentrations of extracellular ATP, usually in the mM range – this reflects the much lower affinity of the P2X7 receptors for ATP compared to all other purinergic receptors. Threshold for activation of P2X7 receptors in the tumor microenvironment is much higher, explaining why ATP-mediated cytotoxic-

D. Draganov and P. P. Lee

ity is a rather rare event that can be uncoupled from the largely trophic and pro-activation effects of lower ATP concentrations. P2X7 receptor operates as a trimeric receptor that functions as an ATP-gated ion channel allowing the influx of extracellular Na + and Ca2+ as well as the efflux of cytosolic K+. Overstimulation of the P2X7 receptor is associated with permanent opening of a still poorly understood large-pore in the plasma membrane and irreversible membrane permeabilization to solutes up to 900 Daltons, ultimately driving a mixed apoptotic and necrotic form of cell death, including the extremely inflammatory pyroptosis [74]. These cytotoxic effects of purinergic signaling are not limited to cancer cells but can also be observed in other P2X4-/P2X7-­ expressing immune cell populations, including T cells and macrophages [75–77]. It has been argued that the mysterious P2X7-dependent pore is an intrinsic property of the P2X7 receptor itself or alternatively it can be attributed to the recruitment of other known ATP channels, Pannexin-1 and Connexins being the most likely candidates [7, 23, 78–80]. The connection between P2X7 receptors and Pannexin-1 is particularly clear in the context of pyroptosis and chemotherapy-­ induced apoptosis of cancer cells, being consistent with the known caspase-3/7/11-mediated cleavage of the cytoplasmic inhibitory C-terminal tail of Pannexin-1 resulting in irreversible opening of the macropore and permanent membrane permeabilization [23, 65]. However, given that Pannexin-1 channels respond to multiple signals including mechanical forces, volume or hypotonicity/hypertonicity changes, and cytosolic Ca2+ or K+ fluxes, the link between P2X7 and Pannexin-1, direct or indirect, may involve noncytotoxic P2X4/P2X7 signaling, generation of ROS, and inflammasome activation [36, 81, 82]. While these signaling cascades normally produce only controlled and defensive ATP release and transient membrane permeabilization, overstimulation of this pathway can be coupled to potent cellular cytotoxicity. Consequently, treatment of cancer cells with high doses of ATP/P2X7 agonists or allosteric modulators of the P2X4/P2X7/ Pannexin-1 axis produces massive and highly inflammatory cancer cell death, as demonstrated

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Fig. 5.4  The role of eATP in promoting tumor growth. Tonic ATP-dependent purinergic signaling in the TME drives multiple trophic and tumor-supportive functions

Fig. 5.5  Regulated breakdown of eATP and subversion of its immunostimulatory effects

by us and others [53, 73]. Thus, overstimulation of purinergic signaling is deleterious for cancer cell survival and confers higher sensitivity to potential therapeutic interventions.

5.3

The High eATP Paradox

Overall, purinergic signaling is a characteristic feature of the tumor microenvironment and appears to be favored, well-tolerated, and even beneficial for cancer growth. These findings suggest that purinergic signaling is selectively

a­ ctivated in the tumor microenvironment where it typically supports rather than antagonizes tumor growth. Accumulating data also indicate that tumors have developed multiple strategies to utilize the trophic and tumor-promoting functions of purinergic signaling while employing various mechanisms to evade its immunostimulatory and direct cytotoxic effects. Exogenous ATP controls cellular and tissue defense/repair processes via signaling through P1, P2X, and P2Y purinergic receptors. Even the potentially cytotoxic P2X7 signaling has recently been associated with tumor growth and

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­metastasis [69, 83–88]. High extracellular ATP levels also occur in vivo at sites of trauma, ischemia, or stroke and are associated with massive inflammatory responses and cell death (e.g., in excitable cells such as neurons). Thus, ATP can function as a prototypical danger signal that activates a potent immune response but can also promote cancer progression – two diametrically opposed functions. ATP release and downstream purinergic signaling appear to play a complex and dual role within the tumor microenvironment. Specifically, tumor growth and survival appear to critically depend on optimal extracellular ATP levels that balance tumor-promoting and cytotoxic functions. The extremely high levels of extracellular ATP in the tumor microenvironment are clear evidence for the overall beneficial and trophic role of ATP-driven purinergic signaling [69]. The tumor-promoting functions of purinergic signaling in cancer encompass its ability to support tumor proliferation, survival, autophagy and energy metabolism, migration, angiogenesis, as well as a variety of tumor immune-evasion mechanisms.

adox as to why P2X7 overstimulation is cytotoxic in some but not all P2X7 high-expressing cancer cells has recently been addressed by Gilbert et  al., who demonstrated tumor-specific expression of a new conformational form of the P2X7 receptor termed non-pore functional nfP2X7 [91]. This nfP2X7, while having intact ion channel functions and ability to mediate trophic ion fluxes, is defective in its ability to drive macropore formation associated with the cytotoxic properties of the P2X7 receptor. Moreover, nfP2X7 expression appears to be upregulated by high extracellular ATP in the tumor microenvironment, thus reflecting tumor cell adaptation to sustained purinergic signaling and allowing cancer cells to take advantage of the trophic functions of the P2X7 signaling even in environments where ATP levels are sufficiently high to cause irreversible macropore opening and cancer cell death.

5.4

Purinergic signaling and P2X7 receptors in particular have been implicated in the control of key metabolic pathways that are essential for cancer cell growth in the specific tumor microenvironment with low glucose, hypoxia, and acidic characteristics. Seminal reports by Adinolfi and others have shed light on the potential of basal purinergic signaling to increase mitochondrial function, oxidative phosphorylation, and ATP output, as well as to provide direct pro-survival and antiapoptotic signals [92–94]. Purinergic signaling and acquisition of extracellular ATP by cancer cells can compensate for insufficient energy supply to support growth, survival, and drug resistance [95]. These pro-energy metabolism effects of purinergic signaling, however, may be inadequate in the hypoxic tumor microenvironment, where insufficient oxygen supply makes mitochondrial oxidative phosphorylation unable to satisfy the needs of rapidly proliferating cancer cells. Subsequent studies have shown that the P2X7 receptor is also a key regulator of

The Pro-survival and Proliferative Functions of Purinergic Signaling

In spite of their cytotoxic potential, P2X7 receptors are commonly overexpressed in various types of solid and hematological malignancies, suggesting a predominantly tumor-promoting and trophic functions [7, 53, 69]. Early findings by Vazquez-Cuevas et al. demonstrated that purinergic signaling in ovarian cancer cells treated with P2X7 agonists stimulated cell proliferation rather than cell death [71]. These trophic effects of purinergic signaling appeared to be mediated by autocrine ATP release, cytosolic Ca2+ flux, as well as stimulation of pAKT and pERK. Moreover, inhibition of ATP/P2X7 signaling resulted in loss of cell viability/proliferative potential. Analogous engagement of the pro-proliferative PI3K/AKT, ERK, and PKC pathways by the P2X7 receptors was also described for astrocytes and glioma cells [89, 90]. The long-standing unresolved par-

5.5

Purinergic Signaling Regulates Energy Metabolism

5  Purinergic Signaling Within the Tumor Microenvironment

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aerobic glycolysis. Intriguingly, the P2X7-­ ity of ATP release hemichannels such as mediated necrotic/pyroptotic cell death is typi- Pannexin-1 and Connexin hemichannels [100, cally observed in non-cancer acidic and hypoxic 101]. Tumor-promoting inflammation, however, environments like various forms of ischemia/ can be equally effective at driving the migration reperfusion injury [87]. Depletion of cellular ATP of both cancer and various immune inflammatory has been linked to MPTP-induced necrotic rather cells, reflecting a complex interplay between ATP than apoptotic cell death in the context of toxins, release, purinergic signaling, Ca2+ flux, ROS chemotherapeutic agents, or oncolytic viruses generation, and cytoskeletal reorganization, [96–98] and appears to be tightly linked to Ca2+ which can be uncoupled from the similar underoverload, CaMKII-mediated MPTP, and PARP-­ lying mechanisms described in the context of mediated depletion of ATP and NAD+− patho- ICD [19, 70, 102, 103]. A significant role of logical events that are normally restrained by inflammasome activation in promoting cancer autophagy and caspase-3-mediated cleavage of cell growth and metastatic potential has been prePARP during the default apoptotic pathway to viously suggested by us and recently confirmed properly execute the default immunologically by Lee et  al. in tumor-associated macrophages silent apoptotic cell death. Notwithstanding these [73, 104]. It remains to be established if constitupotential cytotoxic functions of overstimulated tive ROS-dependent NLRP3 inflammasome/caspurinergic signaling, basal P2X7 signaling con- pase-­ 1/HMGB1 activity in cancer cells is a ditions in the tumor microenvironment appear to driving factor for tumor invasion and metastasis favor tumor metabolism and survival. Recent [33, 72, 105, 106]. Recent data indicate that autoreports identified P2X7 receptors as key regula- crine purinergic signaling mediated by P2X7 tors of the PI3K/GSK3b/VEGF and AMPK-­ receptors is directly involved in TGFb-driven PRAS40-­mTOR signaling networks, promoting migration of cancer cells. More specifically, the AKT/HIF-1/VEGF axis while downregulat- TGFb was found to stimulate the release of eATP ing GSK3b [86, 99]. From these reports and oth- from cancer cells through exocytosis of ATP-rich ers, it is becoming clear that P2X7-dependent vesicles. Interestingly, this process was depensignaling improves the efficiency of glycolysis dent on activity of the vesicular nucleotide transby stabilizing HIF1a, upregulating GLUT1-­ porter (VNUT) and appears similar to ATP mediated uptake of glycose, and augmenting the release from autophagosomes/lysosomes as expression of key glycolytic enzymes, thus described earlier in the context of ICD [70, 103]. allowing cancer cells to maintain ATP homeosta- P2X7-dependent stimulation of AKT signaling sis and survive in the low glucose, hypoxic, and can also drive the migration and invasion of lactate-rich tumor microenvironment. breast and prostate cancer cells [107, 108]. The ability to promote cancer cell migration and invasion is not limited to the P2X7 receptor. Similar 5.6 Purinergic Signaling to their role in neutrophil migration, P2Y2R and P2Y11R are able to promote migration of breast Promotes Cancer Cell cancer cells through activation of the MEK-­ Migration and Invasion ERK1/2 pathway and cytoskeletal reorganization The tumor microenvironment is often associated [109]. Similar effects of P2Y2R and P2Y11R with unresolved chronic inflammation and signaling have been shown in prostate and heparecruitment of myeloid cells, including tumor-­ tocellular carcinoma cells, where purinergic sigassociated neutrophils (TANs), macrophages naling seems to play a role in the induction of (TAMs), and myeloid-derived suppressor cells epithelial-to-mesenchymal transition (EMT) (MDSCs). The role of purinergic signaling in the [110–113]. recruitment and immunosuppressive functions of The link between cancer and thrombosis has these cells is well established and can be directly been well established, potentially reflecting the linked to the ATP-rich environment and the activ- role of P2X7  in platelet activation [114].

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Circulating cancer cells are often coated by ATP-­ rich platelets into metastatic emboli that protect them from immune attack and mediate their trans-endothelial migration and metastasis by releasing their ATP content to trigger P2Y2R signaling in endothelial cells, thus promoting loosening of the gap junctions and cancer cell extravasation [115]. In addition, there are reports demonstrating that P2X7 signaling drives the release of matrix metalloproteinase 9 (MMP-9), which mediates degradation of the extracellular matrix, thus enabling both the recruitment of immune cells and invasion or metastasis of cancer cells [116]. Overall, accumulating data demonstrate that purinergic signaling may be involved in both the short- and long-distance metastatic potential of cancer cells, which highlights another important tumor-promoting function of high extracellular ATP concentrations in the tumor microenvironment.

5.7

Purinergic Signaling Promotes Angiogenesis

Purinergic signaling also appears to be intricately involved in tumor angiogenesis [117]. Recently, it was demonstrated that P2X7 signaling promotes VEGF production through modulation of the PI3K/AKT/GSK3b and HIF1a/VEGF pathways [86], as well as the GSK3b/b-catenin axis which has been known to induce VEGF production and signaling in endothelial cells [118]. Moreover, the coordinated activity of P2X7, Ca2+, and ROS, which has been previously described as a pathway driving the migration of TAMs and TANs, appears to also upregulate VEGF production in myeloid cells [102]. The hypoxic environments associated with tumor growth and sites of I/R injury following stroke or other pathological conditions are also known to engage, activate, and recruit vascular cells, such as pericytes. Pericytes have been implicated as a key factor in both normal and tumoral angiogenesis [119]. The interplay between endothelial cells and pericytes may be critical in promoting restorative/regenerative processes following injury as well as in driving tumor growth and

angiogenesis. Importantly, high eATP and purinergic signaling appear to attract and stimulate the migration and recruitment of human pericytes. This represents an important step in vessel normalization and stabilization in  vivo, thus potentially providing critical support for angiogenesis and tumor growth [120]. The emerging interplay between pericytes and cancer stem cells further emphasizes the importance of ATP-driven migration and recruitment pathways for tumor angiogenesis, invasion, and metastasis [121].

5.8

Regulation of ATP Degradation

As described above, tumor growth and survival are critically dependent on an optimal balance between the pro-survival and cytotoxic functions of purinergic signaling, particularly those mediated by P2X7. Apart from being directly cytotoxic, high extracellular ATP concentrations can break local immunosuppression and promote inflammation and immune-mediated rejection. To properly understand how cancer cells successfully utilize the trophic functions of purinergic signaling while avoiding direct cytotoxicity or engaging antitumor immunity, it is important to note that ATP levels in the tumor microenvironment are subject to tight regulation via controlled release from cancer cells and a plethora of ATP-­ degrading enzymes, such as the extracellular ATPases CD39 and CD73, which sequentially degrade ATP to immunosuppressive adenosine. While the P2X7 receptor is a primary mediator of cellular cytotoxicity, it has the lowest affinity for ATP and thus requires very high concentrations of extracellular ATP within the range of 100–500 uM in order to drive permanent opening of the macropore and irreversible membrane permeabilization resulting in cell death. Such concentrations of eATP are often reached and even exceeded within the tumor microenvironment, but activation of the macropore seems restricted by the selective tumor-specific overexpression of the nfP2X7 form of the receptor. This allows eATP to drive exclusively trophic and tumor-­ promoting signaling by various P2X receptors,

5  Purinergic Signaling Within the Tumor Microenvironment

including P2X7, without compromising tumor cell survival. In addition, ATP/P2X7 signaling appears to promote the immunosuppressive functions of MDSC, thus simultaneously subverting the immunostimulatory functions of ATP [101]. Degradation products of ATP and other similar nucleotides, including ADP, AMP, UTP, and UDP, have less specificity for the P2X receptors and appear to engage various P2Y receptors with predominantly trophic rather than cytotoxic functions (Fig. 5.5) [3, 122]. Adenosine, the end product of extracellular ATP degradation, is one of the primary mediators of immunosuppression within the tumor microenvironment and may be considered an immune checkpoint [123]. Adenosine triggers signaling from four different adenosine G-protein-coupled receptors (GPCR)  – A1R, A2AR, A2BR, and A3R – with A2AR and A2BR mediating most of the immunosuppressive functions of adenosine via upregulation of cAMP and PKA.  Adenosine exerts its immunosuppressive functions at multiple levels, including T- and NK-cell suppression, inhibition of dendritic cell maturation and function [45], and stimulation of the immunosuppressive functions of Tregs, TAMs, and MDSCs [124].

5.9

Therapeutic Directions

The central but multifaceted role of the P2X4/ P2X7/Pannexin-1/NLRP3/caspase-1/IL-1b axis in the maintenance of the tumor microenvironment represents an important therapeutic target. P2X7 receptors are often overexpressed in cancer and associated with tumor growth, aggressiveness, and resistance to therapy. The role of purinergic signaling in cancer is complex and reflects the existence of multiple feed-forward and feedback mechanisms. The feed-forward mechanisms are particularly interesting because of their potential to function as molecular switches [32, 125]. Potential cytotoxicity of high-dose ATP is associated with the overactivation of the low affinity P2X7 receptors [126], which can be further linked to reactive oxygen species (ROS), mitochondria/ER status, and their homeostatic control by autophagy. Autophagy is a key player

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in the decision between survival and cell death, and it has been argued that despite being mostly a defense mechanism, excessive autophagy can result in cancer cell death involving the overactivation of Na+/K+ ATPase pumps [127]. A recent report showed that pharmacological stimulation of purinergic signaling can induce pyroptosis in cancer cells [26]. Given the dual role of purinergic signaling and P2X7 receptors in particular, both agonist and antagonist approaches have been developed in the clinic and already reviewed elsewhere [3, 74, 88]. The sensitivity of tumors to ATP and agonistic analogues or antagonist of trophic purinergic signaling has been extensively investigated, but the therapeutic window for such systemic approaches may be limited by toxicity and lack of tumor specificity. To circumvent this issue, we have proposed to modulate purinergic signaling with agents that enhance the sensitivity of the P2X4/P2X7/Pannexin-1 complex to ATP, such as the antiparasitic “wonder drug” ivermectin [73, 128, 129]. Ivermectin is an FDA-approved antiparasitic agent that has been reported by us and others to modulate the activity of the P2X4/ P2X7/Pannexin-1/NLRP3/caspase-1/IL-1b axis in cancer cells. Such an approach capitalizes on the high levels of extracellular ATP characteristic of the tumor microenvironment, thus minimizing systemic toxicity issues. Allosteric modulators also have the potential to overcome resistance due to the tumor-specific expression of the nfP2X7. The important role of purinergic signaling in the regulation of cancer immunity has been further validated by the recent findings that the ATP-­ degrading ectoenzyme CD39 is a marker of tumor-specific but exhausted T cells [130]. It has also become evident that the adenosine-­degrading enzyme ADA, which is known to bind surface CD26, identifies an interesting population of CD26high CD4 T cells that appear to be able to home to and persist within the tumor microenvironment, reflecting their higher stemness and migratory potential [131]. Indeed, blocking CD39 enzymatic activity with an anti-CD39 antibody can facilitate immune cell infiltration into T-cell-poor tumors and rescue anti-PD-1 resistance [132]. This activity was shown to be

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­ ediated by ATP’s signaling of the P2X7 recepm tor and consequent stimulation of the inflammasome. Altogether, modulation of purinergic signaling and ATP metabolism represents promising yet largely untapped opportunities in cancer therapy.

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6

TGFβ Signaling in the Tumor Microenvironment Cassandra Ringuette Goulet and Frédéric Pouliot

Abstract

Transforming growth factor beta (TGFβ) is a pleiotropic growth factor. Under normal physiological conditions, TGFβ maintains homeostasis in mammalian tissues by restraining the growth of cells and stimulating apoptosis. However, the role of TGFβ signaling in the carcinogenesis is complex. TGFβ acts as a tumor suppressor in the early stages of disease and as a tumor promoter in its later stages where cancer cells have been relieved from TGFβ growth controls. Overproduction of TGFβ by cancer cells lead to a local fibrotic and immune-suppressive microenvironment that fosters tumor growth and correlates with invasive and metastatic behavior of the cancer cells.

C. R. Goulet Oncology Division, CHU de Québec Research Center, Quebec, QC, Canada Department of Surgery, Faculty of Medicine, Laval University, Quebec, QC, Canada F. Pouliot (*) Oncology Division, CHU de Québec Research Center, Quebec, QC, Canada Department of Surgery, Faculty of Medicine, Laval University, Quebec, QC, Canada Department of surgery, CHU de Québec Research Center – Laval University, Quebec City, QC, Canada e-mail: [email protected]

Here, we present an overview of the complex biology of the TGFβ family, and we discuss the roles of TGFβ signaling in carcinogenesis and how this knowledge is being leveraged to develop TGFβ inhibition therapies against the tumor. Keywords

TGFβ · TGFβR · Tumor · Microenvironment · Epithelial-mesenchymal transition · Invasion · Metastasis · Metastatic niche · Angiogenesis · Cancer-associated fibroblast · Immune cells · Extracellular matrix · Matrix remodeling · Targeted therapy · TGFβ pathway inhibitors

6.1

Introduction

The tumor microenvironment is composed of diverse types of cells, including infiltrating immune cells, blood and lymphatic vascular networks, and carcinoma-associated fibroblasts (CAFs) [1]. The crosstalk between these cells plays not only a supportive but also a crucial role in tumor development and progression. Tumor cells use paracrine signaling pathways to modify the surrounding stroma, which in turn, secretes additional growth factors, cytokines, and chemokines to promote tumor growth [2].

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_6

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The transforming growth factor (TGFβ) signaling pathway is one of the most important signaling pathways in tumor microenvironment. Many cell types have the ability to respond to TGFβ, and this cytokine is involved in the regulation of most cellular and molecular processes during both development and disease processes. TGFβ plays a dual role in the carcinogenesis. In early phases, TGFβ acts as a tumor suppressor by activation of growth arrest and apoptosis. However, in advanced tumor, cancer cells evade suppressive effects of TGFβ and use TGFβ regulatory functions to prone tumor progression. TGFβ signaling is an important regulator of tumor initiation and progression, with clear roles in processes supporting cancer cell invasion, epithelial-­ to-mesenchymal transition (EMT), immune response inhibition, angiogenesis, stromal activation, and metastatic niche formation. TGFβ expression is correlated with TNM stages, recurrence, progression, presence of metastases, and short overall survival in various solid malignancies [3–7]. Thus, understanding the critical roles of TGFβ within the tumor microenvironment may provide new targets for design of therapeutics against cancer (Fig. 6.1).

6.2

 GFβ Structure T and Bioavailability

TGFβ superfamily comprises 33 members separated into TGFβ and bone morphogenetic protein (BMP) subfamilies with few distant outliers based on structural and functional criteria. The TGFβ subfamily includes TGFβ, activins, nodal, myostatin, and growth differentiation factors (GDF) while the BMP subfamily comprises BMPs, several GDFs, and the anti-Müllerian hormone. In most cases, these cytokines are secreted as a dimeric molecule stabilized by a disulfide bond. Of these secreted cytokines, three different isoforms of TGFβs have been identified in mammals: TGFβ1, TGFβ2, and TGFβ3. The most abundant isoform is TGFβ1, a 44  kDa protein, expressed in all tissues. TGFβ is secreted in a latent form, called either small or large latent TGFβ. These cytokines are

encoded as large precursor proteins, which undergo multiple posttranslational modifications. One of them is the cleavage of the precursor protein into the N-terminal region, called the ­ latency-­ associated peptide (LAP), and the C-terminal region, corresponding to the mature TGFβ protein. LAP interacts with TGFβ via noncovalent bonds to form a dimer (small latent TGFβ), which hides critical contact sites of the cytokine with its receptors. The LAP-TGFβ dimer is either secreted or covalently linked by disulfide bonds to latent TGFβ binding proteins (LTBP) forming a 240 kDa large latent complex (LLC). LLC is secreted to the extracellular matrix (ECM) network playing a key role in TGFβ storage for future activation by binding fibrillin proteins [8, 9]. To mediate its biological functions, TGFβ must be released from LAP, a tightly regulated process achieved through several mechanisms including enzymatic proteolysis, allosteric interactions, and mechanical dissociation. As an example, cleavage by extracellular serine proteases, such as plasmin, cathepsin D, and matrix metalloproteases (MMPs), release active TGFβ from its latent form. Another mechanism involves the ECM protein thrombospondin 1, which binds to a conserved sequence in LAP, disrupting LAP-­ TGFβ interactions to expose the receptor binding sites [10, 11]. The best-understood mechanism is the activation of TGFβ by mechanical tensions, which is an acute process that depends on both contractile forces of cells and ECM remodeling [12, 13]. The binding of integrin alpha-V to an Arg-Gly-Asp (RGD) motif sequence in the prodomain and tension exerted on this domain induce the unfolding of LAP and liberation of active TGFβ [14]. Once activated, TGFβ induces signaling via cell surface receptors to the nucleus. TGFβ receptors (TGFβR or TβR) encoded in human genome are grouped into seven type I (TβRI) and five type II (TβRII) transmembrane serine/threonine protein kinase receptors. All TGFβ ligands differ in their binding affinity to TβRII.  For example, TGFβ2 binds TβRII 100–1000-fold more weakly than TGFβ1 and TGFβ3 [15]. Cell responsiveness to TGFβ2 has been shown to be dependent

6  TGFβ Signaling in the Tumor Microenvironment

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Fig. 6.1 TGFβ signaling in tumor microenvironment. Early in tumorigenesis, TGFβ functions as a tumor suppressor and helps maintain tissue homeostasis by controlling the growth of normal epithelium through the SMAD-dependent induction of cell cycle arrest. Loss of growth inhibitory responsiveness in cancer cells by mutations in the TGFβ pathway confers a selective advantage to this subpopulation leading to its expansion. Tumor cells that have overcome the growth inhibitory effects of active TGFβ signaling can undergo epithelial-mesenchymal transition acquiring motile and invasive properties. In tumor microenvironment, TGFβ plays a critical role in tumor progression. Tumor-derived TGFβ activates stromal fibroblasts into CAFs, which in turn, secrete higher

amounts of growth factors, such as TGFβ, and extracellular matrix (ECM) components. CAFs are also involved in mechanical remodeling of the surrounding ECM, which is a critical step for cancer progression from a primary tumor to metastatic disease. TGFβ creates an immunosuppressive microenvironment by suppressing T-cell cytotoxic function and promoting Treg differentiation allowing tumor cells to escape immune clearance. TGFβ can increase the permeability of capillaries and facilitate the trans-endothelial passage of metastatic cells. In metastatic niche, TGFβ promotes the establishment of metastatic cancer by mediating homing and transforming the microenvironment to facilitate adhesion, mesenchymal-to-­ epithelial transition (MET), angiogenesis, and osteolysis

of betaglycan, a transmembrane proteoglycan with both chondroitin sulfate and heparan sulfate polysaccharide chains of 250–350  kDa, also known as TβRIII. Betaglycan is involved in the regulation of TGFβ2 signaling by acting as a co-­ receptor that selectively binds the cytokine via its core protein to stabilize the interaction with TβRI

and TβRII [16]. Betaglycan knockout cells show reduced sensitivity to TGFβ2 resulting in low SMAD2 nuclear translocation and reduced growth suppression, but not in response to TGFβ1 and TGFβ3 [17]. Moreover, the extracellular domain of betaglycan can be released by proteolytic cleavage in vivo where it can act as a recep-

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tor antagonist by sequestering TGFβ [18]. Thus, depending on mechanisms, betaglycan can promote or suppress TGFβ signaling.

6.3

TGFβ Signaling Pathways

The TGFβ signaling is well-conserved pathways that emerged with multicellular organisms and is known to be important in many developmental and cellular processes in a wide variety of animals [19]. Initially, TGFβ binds to a single TβRII, which brings together two TβRI and two TβRII receptors into a heterotetrameric complex. The ligand-mediated assembly of these two receptor types triggers the phosphorylation, and thereby the activation of TβRI by TβRII and initiates downstream signaling pathways through their serine/threonine activity. The signal is transduced in either canonical (SMAD-dependent) or non-­ canonical (SMAD-independent) signaling pathways.

6.3.1 Canonical TGFβ Signaling Signal transmission from the receptors to the nucleus is provided predominantly through the SMAD protein family, which is called canonical TGFβ signaling. This family is divided into three different functional groups: receptor-regulated SMAD (SMAD2 and SMAD3), co-SMAD (SMAD4), and SMAD inhibitors (SMAD6 and SMAD7). Once active, TβRI phosphorylates SMAD2 and SMAD3 on two serine residues, which allows the recruitment of SMAD4. This SMAD2/3/4 complex migrates to the nucleus and binds to DNA, either directly or in combination with other proteins, where it acts as a transcription factor to activate or repress hundreds of target genes [20]. Interactions with co-activators and co-repressors determine the transcriptional effect. SMAD7 is involved in the regulation of TGFβ signaling by competitively inhibiting SMAD2/3 binding to TβRI [21]. In addition, SMAD7 can recruit two E3 ubiquitin ligases, Smurf1 and Smurf2, to the activated TβRI, which leads to its degradation by the proteasome [22,

23]. SMAD7 can also interfere with functional SMAD-DNA complex formation by directly binding to DNA via its MH2 domain and therefore blocks TGFβ-mediated cell responses [24].

6.3.2 Non-canonical TGFβ Signaling Non-canonical TGFβ signaling involves the activation of PI3K-Akt, Rho GTPase, ERK, and MAPK pathways, but the mechanisms involved remain to be elucidated. Activation of non-­ canonical TGFβ signaling pathways plays an important role in the cell migration, proliferation, and invasion, ECM protein synthesis, apoptosis, and cellular differentiation. For example, the PI3K-Akt pathway contributes to TGFβ-induced cell apoptosis and EMT process. Breast cancer cells produce autocrine TGFβ that activate the PI3K-Akt and ERK pathways to drive their motility and invasiveness [25]. The small GTPases of the Rho/Rac family are also important mediators of TGFβ cytoskeletal organization involved in cell motility and EMT [26]. Moreover, the activation of JNK and p38 MAPK signaling through the adaptor proteins TRAFs and TAK1 has been shown to promote TGFβ-induced EMT and invasion in various cancer cells in  vitro [27, 28]. However, studies using TβRI mutants defective in SMADs activation, but retain kinase activity, have shown that SMADs are essential to some TGFβ-mediated responses. Thus, TβRI mutants are sufficient to promote apoptosis but not sufficient to induce EMT [29]. These results are consistent with studies showing that crosstalk between canonical and non-canonical signaling is involved in the control of cell migration, invasion, and EMT [30].

6.4

Roles of TGFβ in Tumor Microenvironment

In tumor microenvironment, TGFβ is produced by the tumor cell itself or other cells including stromal, immune, and vascular cells. TGFβ has a dual role in tumorigenesis depending on the stage of tumor development, functioning as a tumor

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suppressor in pre-malignant cells and as a tumor promoter in malignant cells [31]. Although this dichotomous function has been extensively studied, the mechanism by which this switch occurs has yet to be elucidated. Dysfunctional TβR may account for a major mechanism of tumor cell resistance to the TGFβ suppressive effects. Dysregulation of TGFβ signaling pathway is a frequent event in solid malignancies through deletion or inactivating mutations of the TβRs or SMADs effectors. TβR deletions or mutations are found in 12–25% of solid cancers, while loss of expression have been seen in 13–50% of solid cancers [32, 33]. Cancer cells with an inactivated TβR or subverted SMAD pathway lost their TGFβ’s tumor-suppressive effects, leading to sustained proliferation. Moreover, cancer cells have the potential to turn TGFβ into stimulus for cancer progression supporting cellular migration and invasion, metastatic dissemination, chemoresistance, and immune evasion.

proto-oncogene c-MYC expression [40]. c-MYC repress the transcription of CDK inhibitor p15; as a consequence, TGFβ inhibition of c-MYC results in cell proliferation inhibition [37]. Although TGFβ-induced apoptosis is a well-­ known process, the underlying biological mechanisms are still poorly understood. TβR are not directly coupled to apoptosis activating pathways. Thus, studies have shown that TGFβ-­ induced apoptosis is mediated by p38 MAPK signaling [41, 42]. Moreover, pre-malignant cells that harbor oncogenic mutations, such as KRAS and HRAS mutations, are sensitized to TGFβ-­ induced apoptosis [35, 43]. The TGFβ signaling pathway can also inhibit tumor growth by activating autophagy. TGFβ stimulates the accumulation of autophagosomes as well as autophagy-related genes (ATGs) and Beclin autophagy markers into cancer cells, which potentiates TGFβ-mediated induction of proapoptotic genes, Bim and Bmf [44–47].

6.4.1 T  GFβ Tumor-Suppressive Effects

6.4.2 Inactivation of the TGFβ Signaling Pathway During Tumor Progression

The primary functions of TGFβ are to limit proliferation and induce complete differentiation of epithelial cell, which create a barrier to cancer initiation and progression [34]. TGFβ functions as a tumor suppressor that blocks tumor growth by inhibiting cell cycle progression in cancer cells and stimulating apoptosis in pre-malignant cells. Consistent with its central role in maintaining cellular homeostasis, evidence from gene ablation studies has shown that loss of TGFβ signaling components is sufficient for tumor initiation [35]. Cell division cycle is regulated by the action of cyclin-dependent kinase (CDK). TGFβ inhibits cell proliferation by reducing CDK activity, which is required for exit from the G1 phase and progression into the S phase of the cell cycle, resulting in impeded G1/S phase cell cycle progression [36]. Thus, TGFβ induces the expression of several CDK inhibitors, including p15, p16, p19, p21, p27, and p57, which contribute to growth arrest and senescence response [37–39]. Moreover, TGFβ suppresses the expression of

Loss of TGFβ growth inhibitory effect is a hallmark of human cancers. Cancer cells could escape the inhibitory effects of TGFβ through various mechanisms, including genetic alterations in central components of the TGFβ signaling pathway, gene silencing through hypermethylation and inhibition of TGFβ signaling effectors [48, 49]. Mutations induce loss of sensitivity to TGFβ and provide a strong selective advantage over benign tumor [50, 51]. Thus, head and neck, breast, bladder, prostate, and ovary adenocarcinomas harbor mutations in TβRI in 6–30% of cases [52]. The most frequent mutations are found in TβRII due to a mutational hot spot in coding sequence containing an area of ten base-pair polyadenine repeats [53]. Head and neck, brain, breast, stomach, colorectal, and ovary adenocarcinomas contain mutations or deletions in TβRII genes in 12–28% of cases [54–57]. Central player in signal transduction, SMAD genes mutations occur in 4–60% of cases

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Par6  in a SMAD-independent manner, which also leads to the dissolution of cell junction complexes [75–77]. Thus, blocking TGFβ signaling efficiently prevents cancer cells from undergoing EMT in vivo [78]. Acquisition of migratory properties by the cancer cells is a direct consequence of EMT. TGFβ promotes the detachment of the cancer cells from the primary tumor and their dissemination throughout the stroma via multiple signaling pathways including activation of ADAM17 [79], modulation of paxillin and the serine/threonine protein phosphatase PP-1 [80], suppression of reelin expression [81], activation 6.4.3 Pro-invasive and Metastatic of ERK/MAPK signaling pathway [82], and inhiFunctions of TGFβ bition of the expression of lncRNA-NEF [83]. In a distinct mechanism, lamellipodia protrusions, Compensatory TGFβ overexpression that follows produced by motile cells, are managed by Rho loss of sensitivity to TGFβ correlates with high-­ family GTPase, which are themselves coordigrade tumor, increased invasiveness, disease pro- nated by TGFβ [26]. TGFβ-regulated microRgression, and a poorer prognosis [65–67]. NAs also play a crucial role in the mediation of Increased expression of TGFβ has been reported cancer cell migration [84]. For example, TGFβ-­ in almost all solid cancers [68]. Cancer cells silenced miR-584 induces actin rearrangement themselves respond to and are affected by the and breast cancer cell migration, while TGFβ-­ increased TGFβ levels. Thus, cancer cells that induced miR-130b promotes migration of evade TGFβ tumor suppression and still remain colorectal cancer cells [85–87]. responsive to TGFβ pro-tumoral effects acquire In addition to its pro-migratory role, TGFβ invasive phenotype that favors tumor progression also contributes to the cancer cell tissue invaand culminates in distant metastasis [69, 70]. sion, which allows the systemic dissemination However, how TGFβ tumor-suppressive effects of selected clones of primary tumor cells in might be substituted by pro-invasive and meta- order to establish metastasis at a distant organ static responses in cancer cells is still unknown. [69]. Thus, TGFβ has been correlated with The dynamic process of metastasis to distant metastasis of lymph nodes and distant organs in organs contains several steps including epithelial-­ several types of cancer [5, 88–90]. However, the mesenchymal transition (EMT), cell migration, mechanisms that activate and sustain pro-metaand invasion. The EMT involves the dissolution static TGFβ signaling remain incompletely of cell-to-cell and cell-to-ECM adhesions and understood. Recently, evidences point out the adoption of the invasive and migratory behavior role of TGFβ-­regulated microRNA in metastasis that is characteristic of mesenchymal cells. Many process [91–94]. Interestingly, TGFβ has been pathways have been implicated in EMT, includ- shown to induce angiopoietin-like 4 (ANGPTL4) ing TGFβ signaling pathway [71]. TGFβ-­ expression in cancer cells, a cytokine known to stimulated SMADs upregulate Snail, Slug, Twist, disrupt vascular endothelial cell-cell junctions, and Id2 gene expression which suppresses the increase the permeability of capillaries, and transcription of E-cadherin and, consequently, facilitate the trans-endothelial passage of tumor leads to the dissociation of desmosomes and cells [70]. Consequently, blocking TGFβ signaldepletion of one of the most important cellular ing with inhibitors of TβRI or TβRII in cancer adhesion complexes [72–74]. In addition, TGFβ cell lines decreased the ability of these cells to disturbs cell polarity by phosphorylation of generate metastases in xenograft mouse tumor of colon, uterus, pancreas, stomach, lung, breast, head and neck, and prostate cancers [58–60]. Loss of tumor suppressor function can also occur through epigenomic modifications. For example, methylation of TβR was found to be associated with resistance to TGFβ induced signaling [61– 63]. A novel mechanism of TGFβ resistance involves the oncogenic tyrosine kinase receptor ALK and SMAD4 effector. Phosphorylation of SMAD4 by ALK prevents its binding to DNA and fails to stimulate TGFβ tumor suppressing responses [64].

6  TGFβ Signaling in the Tumor Microenvironment

model [95, 96]. Moreover, by increasing the expression of MMPs and plasmin, TGFβ contributes to the ECM remodeling, allowing cancer cells to cross through the ECM to reach distant metastatic sites [97]. Indeed, studies showed that TGFβ strongly induced MMP2 and MMP9 expression in a SMAD-dependent manner [98, 99]. In line with this, knockdown of TGFβ expression in tumor cells negatively affected MMP9 gene expression and significantly decreased the progression of tumors in  vivo [100]. This increased MMP expression and ECM degradation which, in turn, contributes to the release of stored TGFβ from the ECM and the MMP-mediated TGFβ activation, acting as a positive regulatory loop [101]. Moreover, TGFβ activates LOXL2 transcription through SMAD signaling, which then promotes metastasis by increasing tissue stiffness and enhancing recruitment of bone marrow-derived cells to the metastatic site [102]. Inversely, combined inhibition of LOXL2 and TβRI activities in vivo reverses the pathological collagen accumulation leading to fibrosis [103]. TGFβ can foster metastasis to specific tissues by acting on the target metastatic microenvironment in order to allow a permissive milieu for the establishment of metastatic cancer. It has been described that in breast, melanoma, and prostate cancers, TGFβ signaling is essential for the formation of metastases and inhibition of TGFβ signaling impedes the development of bone metastases [94, 104–108]. Mechanistically, TGFβ has been shown to be involved in the expression of multiple genes including CXCR4, MMP1, IL11, JAG1, and PTHRP that promote bone metastases by mediating homing to the bone tissue or transforming the microenvironment to facilitate invasion, angiogenesis, and osteolysis [109–112]. As an example, TGFβ enhances metastatic cells secretion of interleukine (IL)-11, which disrupts the bone remodeling process by altering bone resorption and bone formation [110, 113]. Activated osteoclasts start damaging the bone tissue, which releases the local TGFβ, leading to a loop that supports metastatic cell growth and survival [114, 115].

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6.4.4 T  GFβ and Tumor Immune Responses Under physiological conditions, TGFβ has a key role in the regulation of the immune system by inhibiting many immune cell types activation and functions [116]. Consequently, TGFβ mutant mice present spontaneously activated T cells and die early after birth of multiorgan exacerbated inflammation [117, 118]. It is well established that in tumor microenvironment, despite the presence of tumor-specific antigens and immune cells, aggressive tumors have the ability to avoid immune-mediated damage as TGFβ antagonizes the effective immunity responses, in order to promote tumor growth and metastasis. Thus, TβRII mutant mice harboring tumor develop anti-­ tumoral response and then regulatory T cells (Tregs) leading to complete loss of cancer growth [119]. In advanced malignancies, TGFβ have been largely recognized for its ability to suppress the host’s T-cell immunosurveillance through various mechanisms. First, TGFβ acts directly on cytotoxic T lymphocyte functions during tumor evasion of immune surveillance by specifically inhibiting the expression of five cytolytic factors known as perforin, granzyme A, granzyme B, Fas ligand, and interferon γ. Mechanistically, TGFβ-­ stimulated SMADs together with the transcription factor ATF1 bind to the promoter regions of these genes to repress their expression [120]. Inversely, inhibition of SMAD-dependent signaling restores expression of these genes in tumor-­ specific cytotoxic T cells (CD8+) in vivo, leading to tumor clearance. Second, TGFβ inhibits proliferation of T lymphocytes in a SMAD3-dependent manner by suppressing the expression of IL-2 and its receptor, a cytokine that stimulates T helper (CD4+) cell proliferation [121]. Third, by stimulating apoptosis in both B and T lymphocytes, TGFβ induces tumor microenvironment immunosuppression, preventing the activation of infiltrated immune cells in the tumor and allowing the tumor to escape host immunosurveillance [122, 123]. Finally, TGFβ increases generation of Tregs by inducing FOXP3 expression in naïve CD4+ T cells in a SMAD-dependent manner

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[124]. These immunosuppressive effects of TGFβ have been shown by the inhibition of T cell-­ specific TGFβ signaling, which restores T cell-­ mediated immune response. Indeed, blockade of TGFβ signaling in mice allows anti-tumor immune response able to eliminate tumor cells [119]. Thus, the increased concentration of released TGFβ in the tumor microenvironment dramatically contributes to the tumor progression process, by promoting immunosuppression and allowing the tumor to escape host immunosurveillance.

6.4.5 T  GFβ and Cancer-Associated Fibroblasts CAF is the most abundant cell type of the tumor microenvironment. They are activated fibroblasts that share several similarities with fibroblasts found in fibrotic tissues or during the healing phase of a wound [125]. In contrast to the healing of a wound, but similarly to the fibrotic state of a tissue, CAFs are not permanently activated until they reach an irreversible level, notably via epigenetic regulation [126–128]. Their presence in a tumor is associated with poor prognosis in several types of cancer [129–131]. The different cellular origins and factor composition of the tumor microenvironment shape the phenotype of CAF and contribute to their heterogeneity. Although they appear to originate from several cell types, including smooth muscle cells, pericytes, mesenchymal stem cells (MSCs), adipocytes, and epithelial cells, local fibroblasts are widely considered to be the main source of CAF [132, 133]. Despite the fact that CAFs are largely associated with increased tumor progression, few studies have investigated the mechanisms involved in their activation. Resident fibroblasts would progressively acquire the CAF phenotype under the influence of various soluble factors secreted by the cancer cells. For example, exposure of normal fibroblasts to cancer cell-conditioned media induced differentiation of these cells into CAF [134, 135]. The exogenous signals leading to the activation of fibroblasts in the tumor

C. R. Goulet and F. Pouliot

­ icroenvironment are numerous and probably m distinct for different types of tumors. Currently, TGFβ is the most studied factor in tumor microenvironment fibroblast activation [136–138]. Indeed, in vitro, CAF phenotypes can be induced by supplementing the culture medium of normal fibroblasts with TGFβ [139]. In co-culture, the secretion of TGFβ by colon, breast, and skin cancer cells has been shown to stimulate the activation of fibroblasts into CAF [140, 141]. Recently, it was shown that cancer cells trigger activation of fibroblasts into CAF by exosomal TGFβ transfer from cancer cell to fibroblasts, which activates the SMAD signaling pathway [135]. Interestingly, TGFβ response signaling in CAF could predict disease relapse in cancers [142]. A hallmark of activated fibroblasts is their increased metabolism leading to sustained secretion of cytokines and ECM proteins. CAFs have been shown to secrete significant levels of TGFβ, which act as autocrine-signaling loops to maintain the CAF phenotype [143, 144]. Previous studies have demonstrated that elevated expression of stromal-derived TGFβ is associated with poor prognosis and is predictive of disease recurrence while low stromal TGFβ signaling correlated with improved survival [142, 145, 146]. In the tumor microenvironment, CAF-derived TGFβ plays a key paracrine role in controlling epithelial carcinogenesis. More specifically, TGFβ secreted by CAF promotes the EMT process by weakening intercellular epithelial adhesion [147]. Moreover, CAF-derived TGFβ stimulates EMT in the adjacent cancer cells in various types of cancer [148, 149]. Indeed, TGFβ secreted by CAF induces EMT by stimulating RNA expression, such as the long non-coding RNA ZEBNAT in bladder cancer or the ZEB1 mRNA in breast cancer [150, 151]. TGFβs produced by CAFs have been identified to be one of the factors associated with the aggressiveness of gastric carcinoma cells, as it increases the motility of cancer cells, including migration and invasion ability [148, 151, 152]. In these studies, pharmacological inhibition of the TGFβ pathway reversed the effects of CAF on cancer cells. Furthermore, CAF-derived TGFβ stimulates metastasis process in the adjacent cancer cells in various types

6  TGFβ Signaling in the Tumor Microenvironment

of cancer by providing favorable microenvironment [150, 153]. As an example, TGFβ secreted by CAF promotes breast cancer metastasis by inducing the expression of the lncRNA HOTAIR [154]. TGFβ enhances metastasis by promoting the co-dissemination of cancer cells and CAFs to the metastatic niche and by upregulating the expression of genes involved in cell-cell attachment, which sustains cancer cell survival during metastasis dissemination [155]. Beyond this effect, CAFs have also been involved in the modulation of the anti-tumor immune response by impairing the maturation of tumor dendritic cells and promoting Treg differentiation [156, 157]. Finally, CAF-secreted TGFβ promotes tumor cells to form capillary-like structure in vitro and significantly enhanced vascular mimicry formation in mouse xenografts by inducing VE-cadherin, MMP2, and laminin5γ2 expression in tumor cells [158].

6.5

Future Directions: Targeting TGFβ in Cancer

As outlined above, TGFβ tumor-suppressive effects are often lost in advanced tumors, while tumor pro-invasive functions prevail. The subsequent increased secretion of TGFβ in tumor microenvironment affects cancer cell growth and stimulates migration, invasion, and angiogenesis, contributes to CAF induction, and causes local immunosuppression, further contributing to tumor progression and metastasis. Moreover, since TGFβ expression correlates with poor prognosis and its transduction pathways are specific to the cytokine, TGFβ and its signaling pathway offer an attractive opportunity for targeted therapy. In this regard, many efforts have been made to target TGFβ in cancer; as such, the efficacy of this strategy is investigated in clinical trials. Over the years, several approaches blocking TGFβ signaling pathways have been developed, and some of them were evaluated in clinical trials [159]. Such inhibitors include (1) kinase inhibitors or aptamers, which interfere with the function of the downstream TGFβ signaling proteins, such as SMADs proteins; (2) monoclonal

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a­ ntibodies or soluble TGFβ decoy receptors, consisting of soluble extracellular domains of TGFβ receptors, which block TGFβ and its interaction with its receptor; and (3) antisense molecules which directly inhibit TGFβ synthesis by interfering with its mRNA. The most extensively studied compound is the TβRI inhibitor galunisertib, also known as LY2157299, which has been shown to inhibit growth of lung and breast cancer cell in  vitro [160]. In animal studies, galunisertib has significantly suppressed tumor growth in several types of cancers [161, 162]. Interestingly, strong synergy has been found when combining galunisertib and immune checkpoint inhibitors, such as anti­PD1 [163–166]. These pre-clinical studies led to phase I and II trials in advanced cancers where galunisertib, as monotherapy or in combination, showed significant therapeutic effects [167–172]. Currently, phases I, I/II, and II trials are ongoing in carcinosarcoma of the uterus or ovary, metastatic pancreatic cancer, advanced non-small cell lung cancer, recurrent hepatocellular carcinoma, advanced resistant TGFβ-activated colorectal cancer, and metastatic castration-resistant prostate cancer (NCT03206177, NCT02734160, NCT03470350, NCT02452008, NCT02423343). Another interesting approach is therapies based on antibodies that block systemic TGFβ. The fresolimumab (1D11) anti-pan-TGFβ antibody has shown promising anti-tumor responses in several pre-clinical studies, whereas its combination with radiotherapy potentiates its effects resulting in greater efficacy [173, 174]. Completed safety and efficacy phase I trials have shown that fresolimumab was well tolerated in patients [175, 176]. Results from phase II studies report that patients with metastatic breast cancer receiving fresolimumab in combination with focal radiotherapy have longer median overall survival [177]. The antisense molecule AP 12009 developed for advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma has demonstrated a good clinical potential in pre-clinical studies and a longer overall survival in treated patients in phase I/II trial [178–180].

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Although some molecules have shown encouraging and promising results in clinic, anti-TGFβ therapies still have very important challenges to overcome [159]. Indeed, so far, results of the clinical trials targeting TGFβ pathway have not been conclusive. Only one compound has completed a phase III trial, the Belagenpneumatucel–L vaccine, but it showed no clinical efficacy. The complex nature of TGFβ pathway in tumor regulation renders TGFβ-targeted therapy a challenge to develop safe and effective interventions. As this cytokine exerts crucial functions in many physiological processes, one of the major concerns in TGFβ-targeted therapy is the off-target toxic side effect. Because, blocking the TGFβ signaling has potentially deleterious effects in normal tissues, careful tumor-specific targeting will be mandatory. Moreover, the biphasic role of TGFβ in cancer constitutes a key challenge in the development of TGFβ-directed therapy. Indeed, the fact that TGFβ is tumor-suppressive during early tumor development emphasizes the necessity to identify patients in which the pro-­ tumorigenic effects of the TGFβ signaling pathway are predominant as inhibiting TGFβ in the wrong subpopulation could be damaging. These concerns highlight the need to better understand the role of TGFβ signaling in the tumorigenic process to define a therapeutic road map for each patient. Finally, TGFβ is an interesting druggable target in a number of cancers that requires further clinical validation but, if confirmed, will offer strong evidence supporting the personalized therapy approach based on TGFβ pathway.

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7

Wnt Signaling in the Tumor Microenvironment Yongsheng Ruan, Heather Ogana, Eunji Gang, Hye Na Kim, and Yong-Mi Kim

Abstract

Keywords

Dysregulated Wnt signaling plays a central role in initiation, progression, and metastasis in many types of human cancers. Cancer development and resistance to conventional cancer therapies are highly associated with the tumor microenvironment (TME), which is composed of numerous stable non-cancer cells, including immune cells, extracellular matrix (ECM), fibroblasts, endothelial cells (ECs), and stromal cells. Recently, increasing evidence suggests that the relationship between Wnt signaling and the TME promotes the proliferation and maintenance of tumor cells, including leukemia. Here, we review the Wnt pathway, the role of Wnt signaling in different components of the TME, and therapeutic strategies for targeting Wnt signaling.

Wnt · β-Catenin · Cancer · Tumor microenvironment · Cancer stem cell · Immune cell · Immune tolerance · Immune evasion · Extracellular matrix · Fibroblast · Endothelial cell · Stromal cell · Therapy

Y. Ruan Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA

7.1

Introduction

The Wnt signaling pathway is a critical regulator of embryogenesis development, tissue homeostasis, stemness control, wound repair, and malignancy [1]. Recently, it was shown that aberrant Wnt signaling is involved in the pathogenesis of cancer, including immune evasion and immunomodulation [2, 3]. The microenvironment of tumor cells is composed of tumor cells, immune cells, and stromal cells, which contribute to drug resistance and survival of the tumor [4]. Here, we review the relationship between aberrant Wnt signaling and the tumor microenvironment (TME) and summarize the potential therapeutic targeting strategies.

Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou, China H. Ogana · E. Gang · H. N. Kim · Y.-M. Kim (*) Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA e-mail: [email protected]

7.2

Wnt Signaling Pathway

The Wnt signaling pathway has been extensively studied and reviewed [1, 5, 6]. In general, there are three pathways: the canonical Wnt pathway,

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_7

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the non-canonical Wnt-planar cell polarity (PCP) pathway, and the non-canonical Wnt-calcium (Ca2+) pathway.

7.2.1 The Canonical Pathway The canonical pathway relies on cytoplasmic β-catenin stabilization [7]. However, β-catenin is a highly unstable protein. In the absence of Wnt ligands, β-catenin is part of the β-catenin destruction complex composed of adenomatous polyposis coli (APC), AXIN, casein kinase 1α (CK1α), and glycogen synthase kinase 3 (GSK-3), which catalyzes the phosphorylation of β-catenin at its N terminus and tags ubiquitin protein ligase β-Trcp onto β-catenin. After poly-­ubiquitination, β-catenin is then degraded by proteasomes [8]. The Wnt ligand binds to the Frizzled (FZD) receptor, which is a G protein-­coupled receptor (GPCR) with seven-­transmembrane domains [9] and the co-receptor low density lipoprotein receptor-related protein 5/6(LRP5/6) [10]. Next, disheveled segment polarity protein (DVL) is recruited intracellularly as a platform for AXIN to interact with the cytoplasmic domain of LRP5/6. This interaction disassembles the destruction complex, thus resulting in the stabilization of β-catenin. Ultimately, β-catenin accumulates and translocates to the nucleus, where it can bind to the transcription factor T-cell factor/ lymphoid enhancer factor (TCF/LEF) and recruit the transcriptional Kat3 co-activator p300 and/or CREB-binding protein (CBP) to transcribe Wnt target genes [11]. In contrast, TCF is in an inactive state after it binds to the repressor Groucho. The genes activated by Wnt have important functions for many processes in oncogenesis and development, such as self-­renewal, differentiation, proliferation, and metastasis [12, 13] (Fig. 7.1).

cell polarity (PCP) pathway, the Wnt ligand binds to the Frizzled receptor and activates the small GTPases RhoA and Ras-related C3 botulinum toxin substrate 1 (RAC1) via activation of DVL. RhoA upregulates Rho kinase, while activated RAC1 enhances c-Jun N-terminal kinase (JNK) expression, inducing the expression of downstream target genes [5]. The non-canonical Wnt-Ca2+ pathway is initiated by G protein-­ mediated phospholipase C (PLC) activation, which induces the influx of calcium. Calcium acts as a second messenger and further activates downstream proteins such as calmodulin-­ dependent protein kinase II (CAMKII) and protein kinase C (PKC), resulting in cell migration [14]. Wnt signaling contributes to the stabilization of proteins other than β-catenin to maintain intracellular functions through these alternative pathways [15].

7.3

 nt Signaling and Cancer W Stem Cells

The self-renewal potential of cancer cells is characterized by the presence of cancer stem cells (CSCs) in solid tumors [16]. There is adequate evidence that Wnt signaling plays a vital role in the maintenance and progression of CSCs [17]. Leukemia stem cells (LSCs) share similar properties with CSCs, and leukemogenesis is closely related to aberrant Wnt signaling in both leukemic cells and stromal cells in the bone marrow microenvironment [18, 19]. One of the hallmarks of CSCs is the sustainment of long telomeres through high expression of telomerase reverse transcriptase (TERT) [20]. β-Catenin directly augments TERT expression through promoter binding [21]. Recent studies have shown that GSK3β, AXIN binding to tankyrase, and SOX family transcription factors play key roles in the maintenance of CSC traits in breast cancers through the Wnt signaling pathway [22–24]. One target gene of Wnt is leucine-rich repeat-­ 7.2.2 The Non-canonical Pathways containing G protein-coupled receptor 5 (LGR5), The non-canonical Wnt pathways coexist with a bona fide marker of adult stem cells in the gasthe canonical pathway and are β-catenin-­ trointestinal tract that acts to enhance Wnt/β independent [6]. In the non-canonical Wnt-planar catenin signaling [25]. In colorectal cancer,

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Fig. 7.1  The canonical Wnt pathway. (a) Inactivation of Wnt signaling. (b) Activation of Wnt signaling. LRP lipoprotein receptor-related protein, DVL disheveled segment polarity protein, CK1α casein kinase 1α, GSK-3 glycogen

synthase kinase 3, β-cat β-catenin, APC adenomatous polyposis coli, TCF/LEF T-cell factor/lymphoid enhancer factor, CBP CREB-binding protein

LGR5 has recently been found to also be a marker of colorectal CSCs [26]. Furthermore, intestinal farnesoid X receptor (FXR) function is antagonized by bile acids and induces proliferation and DNA damage in LGR5-positive CSCs [27]. Colorectal cancer cell stemness was found to be enhanced by miR-372/373, a cluster of stem cell-­ specific microRNAs transactivated by the Wnt pathway, via repression of differentiation genes [28]. In addition, LGR5 has a vital oncogenic role in cervical cancer by upregulating Wnt signaling and promoting cervical CSC traits [29]. In non-small cell lung carcinoma, RIF1 and serine-­ arginine protein kinase 1 (SRPK1) promote tumor growth and CSC-like properties as positive regulators of Wnt/β-catenin signaling [30, 31]. Activation of the RNA-binding motif on the Y

chromosome (RBMY), which is present in only male hepatocellular carcinoma, results in the augmentation of CSC traits via Wnt-3a stimulation [32]. In summary, Wnt signaling affects several downstream targets that are important for the maintenance of CSC function.

7.4

 nt Signaling and Immune W Cells

Many vital advancements regarding the relationship between tumors and the immune system have been made over the past two decades. The immune system is known to play a role not only in tumor suppression but also in cancer progression [33]. An extensive immunogenomic analysis of more

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than 10,000 tumors of 33 cancer types from The Cancer Genome Atlas (TCGA) revealed six immune subtypes—wound healing, IFN-­gamma dominant, inflammatory, lymphocyte-­ depleted, immunologically quiet, and TGF-beta dominant— characterized by differences in TME signatures [34]. Wnt signaling not only affects the differentiation of T-cells [35–39] but also influences other immune cells, such as natural killer (NK) cells [40, 41] and dendritic cells (DC) [42–44].

7.4.1 W  nt Signaling in Immune Tolerance Some lymphocytes within the TME that infiltrate solid tumors regulate the immune tolerance of tumors. For example, β-catenin in DCs serves as a key mediator in promoting both CD4+ and CD8+ T-cell tolerance. One possible reason is that DCs can be modulated by denileukin diftitox (DD), a diphtheria toxin fragment-IL2 fusion protein that can upregulate the immune tolerance-­associated β-catenin pathway [45]. The inhibition of interactions between Wnt and its cognate co-­receptor LRP5/6 and Frizzled suggested that LRP5 and LRP6 in DCs play a critical role in immune tolerance [46]. Another study demonstrated that β-catenin/mTOR/IL-10 signaling impairs the ability of DCs to cross-prime CD8+ T-cell immunity [43]. A recent study showed that WNT5a released from melanoma cells resulted in paracrine WNT5a-β-catenin signaling in DCs, leading to an increase in the immunoregulatory enzyme indoleamine 2,3-dioxygenase-1 (IDO), which plays a vital role in tumor-mediated immune tolerization [47]. On the other hand, in colon cancer, activation of β-catenin resulted in the production of T helper 17 (TH17) cell-­mediated inflammation that promoted cancer function [48].

7.4.2 W  nt Signaling in Immune Evasion Active canonical Wnt signaling in immune cells in the TME is a crucial cause of resistance to cancer immunotherapies called immune checkpoint

inhibitors [2]. Effective recognition of tumor-­ associated antigens and thus eradication of cancer by cytotoxic T lymphocytes (CTLs) can be inhibited via cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death 1 (PD1) and programmed death-ligand 1 (PD-L1) pathway [49]. In melanoma, T-cell exclusion and resistance to anti-PD-L1/anti-­CTLA-­4 monoclonal antibody therapy were mediated via tumorintrinsic active β-catenin signaling [50]. One of the key factors was GSK-3, which could potentially decrease CD8+ T-cell function in cancer therapy via the upregulation of PD-1 expression [51]. Moreover, in a prospective hepatocellular carcinoma genotyping clinical study, active Wnt/βcatenin signaling was associated with lower disease control rates (DCRs), shorter median progression-free survival (PFS), and shorter median overall survival (OS), even though the 31 patients were treated with immune checkpoint inhibitors [52]. New evidence was found that not only the T-cell-inflamed phenotype but also non-T-cellinflamed tumors correlated with the efficacy of immune checkpoint blockade via tumor-intrinsic Wnt/β-catenin signaling activation [53]. Furthermore, stroma-­derived Dickkopf-1 (Dkk1) targeted β-catenin in myeloid-derived suppressor cells (MDSCs), thus exerting immune suppressive effects during tumor progression [54].

7.5

Wnt Signaling and Extracellular Matrix

The extracellular matrix (ECM) is a strikingly dynamic structure that undergoes mechanical remodeling by most tumor cells, and this process is critical for cancer progression from a primary tumor to metastatic disease [55, 56]. A stiff ECM activates the integrin/focal adhesion kinase (FAK) pathway, which increases the expression of members of the Wnt/β-catenin pathway and in turn enhances the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance [57]. Interestingly, the expression levels of B-cell lymphoma 2 (Bcl-­ 2)-associated X protein (Bax), procaspase-3 and procaspase-9, matrix metalloproteinase 1

7  Wnt Signaling in the Tumor Microenvironment

(MMP1), MMP3, MMP13, WNT3a, WNT5a, WNT7a, and β-catenin were significantly decreased by resveratrol (RES) in osteoarthritis chondrocytes [58]. This finding indicates that sirtuin 1 (Sirt1), which can be upregulated by RES, may regulate ECM degradation in RES-treated osteoarthritis chondrocytes through the Wnt/β-­catenin signaling pathway [58]. In cervical cancer, the ECM protein collagen triple helix repeat containing 1 (CTHRC1) is regulated by E6/E7, which are early genes of the high-risk mucosal human papillomavirus type [59]. Through activation of the non-canonical Wnt/PCP signaling pathway, the E6/E7-p53-POU2F1-CTHRC1 axis ultimately promoted cervical cancer cell invasion and metastasis [59]. In a urinary bladder cancer study, increased WNT7a expression was associated with metastasis and poor prognosis. Mechanistically, WNT7a-mediated MMP10 activation is mediated by the canonical Wnt/β-­catenin pathway [60]. In breast cancer, deposition of type I collagen in the ECM was shown to play an important role in metastasis because Wnt signaling caused an increase in the expression of MRTF-A, which was critical for regulating the type I collagen gene COL1A1  in breast cancer cells [61]. Moreover, MRTF-A integrated signals from the Rho-ROCK-actin and Wnt/β-catenin pathways to regulate migration-related genes, including MYL9, CYR61, and lncRNA HOTAIR, which in turn stimulate breast cancer cell ­migration [62]. In glioma, MYH10 gene silencing resulted in reduced expression of MTA-1, MPP-­2, MMP-9, and vimentin and increased expression of TIMP-2, E-cadherin, and collagen 1 through inhibition of the Wnt/β-catenin pathway [63]. HEmT-DCN/sLRP6, an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor, eradicated excessive ECM accumulation in pancreatic cancer via inhibiting the Wnt/β-catenin signaling pathway [64]. Previously, another study demonstrated a similar effect using an ECM-degrading and Wnt signal-­ disrupting oncolytic adenovirus (oAd/DCN/LRP) [65]. Wnt/ β-catenin-activated Ewing sarcoma cells upregulated the secretion of ECM proteins such as structural collagens, matricellular proteins and tenascin C (TNC) [66]. Hence, ECM is secreted

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excessively and remodeled by cancer cells via the Wnt/β-catenin pathway in the TME.

7.6

Wnt Signaling and Fibroblasts

Cancer-associated fibroblasts (CAFs) are important stromal cell components in the TME that play critical roles in tumor initiation, progression, and metastasis [55, 67, 68]. There have been a tremendous number of studies that identify how Wnt signaling pathways are involved in the formation and regulation of CAFs in different tumor types. In contrast to its expression in cancer cells as a Wnt antagonist to suppress the progression of various cancers [69–71], the stromal expression of Dickkopf-3 (DKK3), an HSF1 effector, regulated the pro-tumorigenic behavior of CAFs via Wnt signaling and activation of the Hippo pathway transducers YAP/TAZ [72]. CAFs also promoted the stemness, metastasis, and chemoresistance of colorectal cancer cells by secreting exosomes to increase the levels of miR-92a-3p, activating the Wnt/β-catenin pathway and inhibiting mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1 [73]. Furthermore, CAFs upregulated T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1), one of the Wnt signalingassociated genes, resulting in chemoresistance in colorectal cancer [74]. Moreover, the desmosomal protein plakophilin-2, encoded by the PKP2 gene, is a Wnt/β-catenin target gene and acts to inhibit this pathway, suggesting that PKP2 plays a role in the negative feedback control of Wnt/β-catenin signaling in normal and colon CAFs [75]. It was found that high levels of WNT2, which are associated with a poor prognosis in colorectal cancer, binds to its putative receptor FZD8 and activates autocrine canonical Wnt signaling in CAFs, which results in promoting colorectal cancer progression, invasion, and metastasis [76]. In head and neck squamous cell carcinoma, CAF-derived POSTN is an upstream ligand of protein tyrosine kinase 7 (PTK7) and promotes the CSC-like phenotype via PTK7-Wnt/β-catenin signaling, thus inducing proliferation and invasion [77]. Deletion of hypoxiainducible transcription factor (HIF-1) was

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associated with Wnt/β-catenin inactivation, significantly inhibiting tumor progression and decreasing CAF production in the TME [78, 79]. It has been reported that overexpression of phospholipase D2 (PLD2) in colon tumors induces senescence in neighboring fibroblasts and leads to a senescence-associated secretory phenotype (SASP), contributing to tumor development via Wnt pathway activation in colon cancer [80]. A three-dimensional multi-culture tumor-CAF spheroid phenotypic screening platform was developed to profile 1024 candidate genes for CAF-intrinsic anti-spheroid activity, and CAF-­ derived products, such as Wnt and the G protein coupled-receptor OGR1, were found to be potential therapeutic targets for colorectal cancer [81]. The activation of Wnt/β-catenin and Hgf/Met signaling significantly mediates interactions between CSCs and CAFs in mammary gland tumors [82]. In epithelial ovarian cancer, STAT4 overexpression induced normal omental fibroblasts to obtain CAF-like features via tumor-­derived WNT7a [83]. On the other hand, WNT7a-mediated fibroblast activation was dependent on not only canonical Wnt signaling but also TGF-β receptor signaling in breast cancer [84]. Interestingly, aged fibroblasts can secrete a Wnt antagonist, sFRP2, which activates a multi-step signaling cascade that results in m ­ elanoma metastasis and therapy resistance [85]. Paracrine WNT10b from p85α-deficient fibroblasts regulated breast cancer tumorigenesis and progression via TME remodeling and epithelial-­to-­mesenchymal transition induced by the canonical Wnt pathway [86]. Additionally, in breast cancer, the effect of oxidative stress induced by oxidized ataxia-telangiectasia mutated protein kinase (ATM) on aberrant CAF proliferation is regulated through the ERK, PI3K-AKT, and Wnt signaling pathways [87].

7.7

Wnt Signaling and Endothelial Cells

A vascular network is imperative in tumor development and metastasis, and endothelial cells (ECs) in the TME are highly responsive to cues to promote the angiogenesis of tumor-infiltrating

blood vessels. First, differentiated ECs undergo phenotypic transition to mesenchymal cells through a complex process called endothelial-­ mesenchymal transition (EndMT) [88]. TGF-β and Snail transcription factors are two important stimulators of EndMT via the Notch and Wnt signaling pathways utilizing Frizzled-2 (FZD2), FZD9, and Wnt5B in the induction process [89]. A similar finding for oral squamous cell carcinoma indicates that Wnt5B functions in EndMT and regulates the protein expression of Snail and Slug through activation of the canonical and non-­ canonical Wnt signaling pathways [90]. Second, many advances in tumor angiogenesis via Wnt signaling have been reported. Regulators of Wnt signaling, including FZD7 [71] and R-spondin3, play important roles in vascular endothelial cells [91]. Increased non-canonical WNT5a in squamous cell lung carcinoma inhibits endothelial cell growth and motility [92], implicating its potential as a therapeutic target of angiogenesis-­related diseases [93]. In malignant glioma, increased WNT7 expression in Olig2+ oligodendrocyte precursorlike cells (OPLCs) allowed for the invasion of glioma cells in the vasculature, and inhibition of Wnt blocked invasion and enhanced the response to temozolomide therapy [94]. Microparticles (MPs) released from ovarian cancer cells mediate the activation of Wnt/β-­catenin, in which RAC1 and AKT are responsible for phosphorylation and nuclear translocation in ECs for neo-angiogenesis [95]. Ribosomal protein s15a (RPS15A) promoted angiogenesis in primary hepatocellular carcinoma (HCC) by enhancing Wnt/β-catenininduced FGF18 expression, suggesting that the RPS15A/FGF18 pathway may be a target for antiangiogenic therapy of HCC [96]. Importantly, it has been demonstrated that FZD5—a Wnt/FZD family member—is a receptor for secreted frizzled-­related protein 2 (SFRP2) and mediates SFRP2-­ induced angiogenesis through the calcineurin/nuclear factor of activated T-cell cytoplasmic 3 (NFATc3) pathway in ECs [97]. Furthermore, hypoxic colorectal cancer cells were shown to secrete exosomes enriched with WNT4 in a manner dependent on hypoxia-­inducible factor α (HIF1α) and promoted the proliferation and migration of ECs through increased β-catenin

7  Wnt Signaling in the Tumor Microenvironment

nuclear translocation [98]. Interestingly, HIF-1α activation via the Wnt/β-catenin pathway promoted vasculogenesis and angiogenesis, even in normoxic conditions, due to lactate release by glioma cells [99]. Other important mediators, such as vascular endothelial growth factor-A (VEGF-A) [100] and pro-inflammatory factor TNF-α [101], were associated with activation of the Wnt/β-catenin signal pathway in ECs. On the other hand, loss of Norrin (an atypical Wnt)/ FZD4-mediated signaling in ECs created a tumorpermissive microenvironment at the earliest, preneoplastic stages of medulloblastoma [102]. In addition, in colorectal cancer, Norrin/FZD4 plays a vital role in the regulation of angiogenesis [103].

7.8

 nt and Mesenchymal Stem W Cells

The stroma of the TME is comprised of a heterogeneous population of connective tissue cells, such as fibroblasts, epithelial cells, and mesenchymal stem cells (MSCs), also referred to as mesenchymal stromal cells. The bone marrow microenvironment also contains MSCs that make up the bone marrow niche. Within the niche, MSCs function to regulate and maintain hematopoietic stem cells (HSCs) and give rise to the majority of bone marrow stromal cell lineages, including chondrocytes, osteoblasts, fibroblasts, adipocytes, endothelial cells, and myocytes [104]. Wnt signaling is important for the proliferation and differentiation of MSCs, but its exact role has not yet been elucidated. Huang et al. found that in the chondrogenic differentiation of MSCs, the Wnt signaling modulator BIO activated canonical Wnt signaling and genes important for stemness and proliferation, while the PFK compound inhibited signalinginduced chondrogenic differentiation and β-catenin translocation into the nucleus [105]. Another study showed that upon inhibiting GSK-3, the kinase that mediates the phosphorylation and degradation of β-catenin, the Wnt/βcatenin pathway was upregulated, and chondrogenic differentiation was increased [106, 107]. These results indicate that the Wnt

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pathway is critical for MSC developmental function with regard to proliferation and differentiation. In terms of the role of Wnt signaling in MSCs and cancer, it has been found that in many cancers, the Ror2 receptor tyrosine kinase can act as a receptor for WNT5a to mediate β-catenin-independent non-canonical Wnt signaling. The constitutive WNT5a-Ror2 signaling in MSCs promotes the proliferation and aggressiveness of gastric cancer cells by enhancing the expression of CXCL16, thereby activating the CXCL16-CXCR6 axis in a cell-autonomous manner [108]. One study used qPCR to demonstrate that MSCs had detectable mRNA expression of all 16 Wnt ligands, with WNT5a, WNT3, WNT10a, and WNT7b expression being the highest [109]. The authors found that co-culture of acute lymphoblastic leukemia (ALL) with MSCs provided a protective effect to the ALL cells due to changes in MYC, LEF1, CCNDBP1, and GSK3b levels, which led to activation of the Wnt pathway and promoted leukemic cell proliferation in ALL [109]. In acute myeloid leukemia (AML), it has been suggested that AML-MSCs have canonical Wnt signaling pathway dysregulation, in which there is a decrease in β-catenin/TCF-LEF complex formation and a decrease in bone morphogenetic protein 4 (BMP4) expression [110]. MSC-derived WNT5a inhibits proliferation and promotes the differentiation of HL60 cells, an AML cell line, via activation of the non-canonical Wnt signaling pathway due to significantly increased expression of Ror2 and calcium/calmodulindependent protein kinase II (CaMKII) and decreased expression of β-catenin and cyclin D1 [111]. Healthy donor hematopoietic stem/progenitor cells (HSPCs) co-cultured with MSCs derived from Fanconi anemia patients with AML showed reduced secretion of prostaglandins (PGs) by mesenchymal inhibition of COX2, which led to decreased expression of NR4A transcription factors and β-catenin, thus revealing a novel COX2/PG/NR4A/Wnt signaling axis [112]. In summary, Wnt signaling is implicated in MSC function in cancer development and progression and can significantly remodel the TME to promote oncogenesis.

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7.9

Targeting Wnt Signaling

New agents that target Wnt signaling have emerged in research. For example, porcupine (PORCN) inhibitors (e.g., LGK974 or WNT974, IWP-L6, CGX1321, ETC1922159, and RXC004), AXIN1 activators (e.g., XAV939 and niclosamide), Dickkopf1 (DKK1) antibodies (e.g., DKN-01 and CKAP4), FZD receptor inhibitors (e.g., OMP18R5 and OMP54F28), CBP/β-­catenin inhibitors (e.g., PRI-724 and ICG-001), and a WNT5A mimic (Foxy-5) have all been studied [2, 113]. Table 7.1 lists the current Wnt modulators used for cancer therapy in clinical trials from the US National Library of Medicine database (https://www.clinicaltrials.gov/ accessed on June 23, 2019).

7.9.1 PORCN Inhibitors PORCN inhibitors block the secretion of Wnt by inhibiting palmitoylation in the TME.  The efficiency of LGK974 was determined in different tumors, such as squamous cell carcinoma [114], colorectal cancer [115], prostate cancer [116], and chronic myeloid leukemia (CML) [117]. However, LGK974 has low solubility and high toxicity in different tissues, and a potent cyclodextrin:LGK974 complex was investigated in lung cancer [118]. WNT974  in combination with a tyrosine kinase inhibitor (TKI) enhanced the targeting effect of CML stem and progenitor cells [117]. IWP-L6 blocked the Wnt-LRP5/6 pathway and delayed tumor growth via the promotion of a strong tumor-specific T-cell response by regulating DC function [46].

7.9.2 AXIN1 Activators AXIN1 activators play a role in the induction of β-catenin degradation. Niclosamide, an FDA-­ approved anthelmintic drug, eradicated cancer stemness, and elicited therapeutic effects on colorectal cancer via disruption of the LEF1/ DCLK1-B axis [119]. However, due to the low

solubility, bioavailability, and systemic exposure of niclosamide, the effects of niclosamide-­ conjugated polypeptide nanoparticles were studied in colon cancer [120]. Repression of miR-31-5p suppressed the proliferation, invasion, and tumorigenesis of osteosarcoma cells via promoting AXIN1 [121].

7.9.3 DKK1 Antibodies Although DKK1 is a secreted inhibitor of β-catenin-dependent Wnt signaling, DKK1 appears to increase tumor growth and metastasis in preclinical models due to activation of β-catenin-independent Wnt signaling and mediation of the immunosuppressive TME and immune evasion [122]. A monoclonal antibody (mAb) against cytoskeleton-associated protein 4 (CKAP4), a novel DKK1 receptor mAb, suppressed xenograft tumor formation and extended the survival of pancreatic ductal adenocarcinoma mice [123]. Moreover, Dickkopf-related protein 2 (DKK2), an antagonist of Wnt/β-catenin signaling, suppressed tumor cell migration by reversing EndMT and downregulating stem cell markers in breast cancer [124].

7.9.4 FZD Receptor Inhibitors Wnt receptors have a decoy function to prevent Wnt binding to FZD receptors. A phase 1b dose escalation study of ipafricept (OMP54F28), a recombinant protein that inhibits Wnt signaling, in platinum-sensitive ovarian cancer was recently released, and the results indicated that bone toxicity at effective doses limits its use in ovarian cancer [125].

7.9.5 CBP/β-Catenin Inhibitors CBP/β-catenin inhibitors disrupt the interaction between CBP and β-catenin. One inhibitor, ICG-­ 001, partially reversed EndMT and attenuated cancer stemness [126]. In addition, ICG-001 suppressed pancreatic cancer growth and signifi-

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Table 7.1  Clinical trials of Wnt modulators in cancer Mechanism PORCN inhibitor

Agent WNT974/ LGK974

CGX1321 RXC004 ETC-1922159 Wnt-5a protein

Foxy-5

DKK1 antibody

DKN-01

Wnt signaling pathway inhibitor

CBP/beta-catenin antagonist

SM08502

Colon cancer Metastatic breast cancer Colorectal cancer Prostate cancer Hepatocellular carcinoma Endometrial cancer Uterine cancer Ovarian cancer Esophageal neoplasms Adenocarcinoma of the gastroesophageal junction Gastroesophageal cancer Squamous cell carcinoma Gastric adenocarcinoma Solid tumors

XNW7201

Advanced solid tumors

PRI-724

Colorectal adenocarcinoma Stage IVA colorectal cancer Stage IVB colorectal cancer Acute myeloid leukemia Chronic myeloid leukemia Acute myeloid leukemia Chronic myelomonocytic leukemia Myelodysplastic syndrome myelofibrosis Colon cancer Colon cancer Cancer

CWP232291

AXIN1 activator Wnt signaling modulator

Disease Squamous cell carcinoma Head and neck cancer Metastatic colorectal cancer Pancreatic cancer BRAF mutant colorectal cancer Melanoma Triple-negative breast cancer Head and neck squamous cell cancer Cervical squamous cell cancer Esophageal squamous cell cancer Lung squamous cell cancer Solid tumors GI cancer Cancer Solid tumor Solid tumors

Niclosamide Resveratrol

cantly prolonged survival in an in vivo pancreatic ductal adenocarcinoma xenograft mouse model [127]. ICG-001 significantly prolonged the survival of NOD/SCID mice engrafted with drug-­

Status Withdrawn

Identifier NCT02649530

Completed Recruiting

NCT02278133 NCT01351103

Recruiting

NCT02675946

Not yet recruiting Active, not recruiting Recruiting Completed

NCT03447470

NCT03883802 NCT02020291

Recruiting Recruiting

NCT03645980 NCT03395080

Recruiting

NCT02013154

Recruiting

NCT03355066

Not yet recruiting Withdrawn

NCT03901950 NCT02413853

Completed

NCT01606579

Completed

NCT01398462

Recruiting Completed

NCT02687009 NCT00256334

NCT02521844

resistant primary ALL [128] and inhibited colony formation in sorted CD34+ CML progenitors in combination with imatinib mesylate [129]. Moreover, inhibition by the CBP/β-catenin

Fig. 7.2 Wnt signaling in the tumor microenvironment (TME). ECs endothelial cells, EndMT endothelial-­ mesenchymal transition, CSCs cancer stem cells, LGR5 leucine-­rich repeat-containing G protein-coupled receptor 5, ECM extracellular matrix, CAF cancer-associated fibroblast,

TIAM1 T-lymphoma invasion and metastasis-inducing protein-­ 1, FAO fatty acid oxidation, CTLA-4 cytotoxic T lymphocyte-­ associated protein 4, PD-1 programmed cell death-1, CTL cytotoxic T lymphocyte, MSC mesenchymal stem cell, DC dendritic cell, MDSC myeloid-derived suppressor cell

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antagonist C-82/PRI-724 and the use of an FLT3 normal cells and restricts the administration of TKI decreased Wnt/β-catenin signaling in FLT3-­ potent Wnt signaling inhibitors due to off-target mutant AML [130]. toxicity. In the future, dual targeting of Wnt signaling and the TME may efficiently eradicate cancer cells. Taken together, Wnt signaling 7.9.6 WNT5A Mimic tightly correlates with the TME, and targeting the Wnt signaling pathway in the TME is a promisIn addition, WNT5A upregulated the expression ing tumor therapeutic strategy. and activity of the indoleamine 2,3-­dioxygenase-1 (IDO) enzyme in local DCs and induced immunotolerance [131]. WNT5A is a β-catenin-­ References independent ligand that has been shown to induce tumor suppression [132]. Foxy-5, a WNT5A-­ 1. Nusse R, Clevers H (2017) Wnt/beta-catenin signaling, disease, and emerging therapeutic modalities. mimicking peptide that has been recently used in Cell 169(6):985–999 clinical trials, significantly inhibited the initial 2. Galluzzi L et  al (2019) WNT signaling in cancer immunosurveillance. Trends Cell Biol 29(1):44–65 metastatic dissemination of prostate cancer cells with absent or low WNT5A expression [133]. In 3. Goldsberry WN et al (2019) A review of the role of Wnt in cancer immunomodulation. Cancers (Basel) human colonic cancer, the number of colonic 11(6) CSCs was decreased by Foxy-5 [134]. 4. Wu T, Dai Y (2017) Tumor microenvironment and

7.10 Concluding Remarks Herein, we systemically reviewed the complex relationship between Wnt signaling and many factors in the TME (Fig. 7.2). As discussed, most research has focused on canonical Wnt signaling, which is based on the Wnt/β-catenin interaction. Wnt signaling also plays an important role in sustaining the self-renewal potential of CSCs as well as LSCs and subsequently enhances CSC promotion. The immune cells in the TME mediate the immune tolerance and immune evasion of cancer cells via Wnt signaling. In addition, Wnt signaling regulates ECM remodeling and causes overproduction of extracellular proteins in the TME to protect cancer cells from eradication. The transformation of stromal normal fibroblasts into the CAF phenotype has been implicated in promoting primary tumor growth and progression to metastatic disease. ECs in the TME induce tumor angiogenesis through Wnt signaling activation. There are several potential Wnt signaling modulator targets, including but not limited to PORCN inhibitors, AXIN1 activators, Dickkopf1 antibodies, Wnt receptor decoys, CBP/β-catenin inhibitors, and a WNT5A mimic. However, Wnt signaling also plays a crucial homeostatic role in

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8

Lysophospholipid Signalling and the Tumour Microenvironment Wayne Ng and Andrew Morokoff

Abstract

Homeostasis is the key to survival. This is as true for the tumour cell as it is for the normal host cell. Tumour cells and normal host cells constantly interact with each other, and the balance of these interactions results in the prevailing homeostatic conditions. The interactions between the milieu of signalling molecules and their effects on the host and tumour cells are known as the tumour microenvironment. The predominant balance of effects within the tumour microenvironment will determine if the tumour cells can evade the host’s responses to survive and grow or if the tumour cells will be eradicated. Lysophospholipids (LPLs) are a group of lipid signalling molecules which exert their effects via autocrinic and paracrinic mechaW. Ng (*) Department of Neurosurgery, Gold Coast University Hospital, Hospital Boulevard, Southport, QLD, Australia School of Medicine, Griffith University, Gold Coast Campus, Parkwood, QLD, Australia A. Morokoff Centre for Medical Research, Department of Surgery, Royal Melbourne Hospital, Parkville, VIC, Australia Department of Surgery, Royal Melbourne Hospital Campus, University of Melbourne, Parkville, VIC, Australia e-mail: [email protected]

nisms. Therefore, LPLs are being explored to determine if they are potentially key signalling molecules within the tumour microenvironment. The effects of LPLs within the tumour microenvironment include modulating cell proliferation, cell survival, cell motility, angiogenesis and the immune system. These are all important activities that affect the balance of host-tumour cell interactions. This chapter expands on these functions and also the role that LPLs could play as a potential treatment target in the future. Keywords

Lysophospholipids · Autotaxin · Lysophosphatidic acid · Sphingosine 1-­phosphate · Homeostasis · Tumour microenvironment · Angiogenesis · Invasion · Cell proliferation · Immune modulation

Abbreviations 4PBPA 4-Pentadecylbenzylphosphonic acid ATX Autotaxin COX-2 Cyclooxygenase 2 CRP C-reactive protein ECM Extracellular matrix Edg Endothelial differentiation gene EGF Epidermal growth factor

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7_8

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EGFR EGF receptor G2A G2 accumulation GBM Glioblastoma GPCR G-protein-coupled receptor hESC Human embryonic stem cell HIF1α Hypoxia-inducible factor 1 alpha HRE Hypoxia-responsive elements IL Interleukin LPA Lysophosphatidic acid LPAR LPA receptor LPC Lysophosphatidylcholine LPL Lysophospholipids LT Leukotriene MAPK Mitogen-activated protein kinase MEK MAPK/ERK kinase MHC Major histocompatibility complex MMP Matrix metalloprotease MT1 Membrane type 1 mTOR Mammalian target of rapamycin NHERF2  Sodium-hydrogen exchange regulatory factor 2 OGR1 Ovarian cancer G-protein-coupled receptor OS Overall survival PAF Platelet-activating factor PDGF Platelet-derived growth factor PFS Progression-free survival PG Prostaglandin PI3K Phosphoinositol-3-kinase S1P Sphingosine 1-phosphate S1PR S1P receptor SCC Squamous cell carcinoma SPC Sphingosylphosphorylcholine SphK Sphingosine kinase SPNS2 S1P transporter spinster homologue 2 TDAG8 T-cell death-associated gene 8 TRIP6 Thyroid receptor-interacting protein 6 VEGF Vascular endothelial growth factor VEGFR VEGF receptor YAP Yes-associated protein

8.1

Introduction

Homeostasis is the key to survival. It is the constant strive to obtain a survivable balance in the face of ever-changing environmental conditions.

The cellular microenvironment exposes cells to biomechanical, reductive-oxidative, chemical, biochemical, electrical, molecular, genetic, epigenetic, inflammatory, immune and nutritional stresses. Cells have developed many mechanisms to respond to these stresses, but in more complex multi-cellular organisms, they have evolved to respond as functional units known as tissues or organs. By resisting or embracing these stresses as a functional unit, cells can develop efficiencies that enable them to contribute not only to their individual survival but also to that of the collective. Almost in contradistinction, from its moment of conception, the tumour cell (at least in part) separates from its functional unit and now faces some of these same realities of homeostasis in isolation. But, now the tumour cell must also contend with interactions presented by the host. In essence, the tumour, in part, represents a form of tissue xenograft, and part of its approach to survival must be to evade the host’s immune system. Therefore, it must employ strategies to survive in the surrounding tumour microenvironment, including the challenges presented by the host. If the tumour cell can survive this combination of challenges, then it is able to thrive. The mechanisms employed by tumour cells to enhance their chances of survival in order to thrive are myriad. They include mutations causing altered regulation of genes that allow the tumour cells to alter their shape, enhance motility, promote angiogenesis, enhance survival, proliferate, resist stresses including hypoxia and oxidative stress and evade immune surveillance [1–5]. All of these tumour cell properties are in response to or are an attempt to modulate the tumour cell’s microenvironment and the milieu of signalling molecules contained within. The resulting balance of the effects of the tumour cell properties will ultimately decide the tumour cell’s fate. It is likely that cell survival pathways such as PI3K/ mTOR/Akt and inhibition of apoptotic pathways initially facilitate survival of the tumour cell. Alterations in MHC expression also help tumour cells evade immune surveillance and therefore T-cell-mediated eradication [6]. Modulation of immune cell trafficking also allows tumour cells to evade eradication by the host immune system. Subsequently, tumour cells alter their cell shape, through actin-myosin or Rho-dependent mecha-

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nisms, which allow the tumour cell to become discohesive and thus isolate itself from its tissue of origin. This in turn can allow the tumour cell to proliferate by disengaging contact inhibition of growth. Simultaneously, dysregulation of cell growth and proliferation pathways such as EGFR, MEK, MAPK and YAP can also help promote proliferation of tumour cells. Induction of VEGF pathways activates angiogenesis, which in addition to induction of mechanisms resisting hypoxic stresses helps facilitate further tumour growth. Further, the discohesive tumour cell can now become motile, allowing it to migrate to a more favourable environment, collocate to other tumour cells or allow it to invade or metastasise (Fig. 8.1). Whether intentional or not, the tumour cells now find themselves within a functional unit whose primary aims are to survive and proliferate. Much

of this is achieved through near-field communications such as via autocrinic and paracrinic mechanisms. Many of the above pathways are similar to those modulated in inflammatory conditions. Much like cancer research, many of these discoveries have involved characterisation of proteins and their effects. However, an area of cancer research gaining momentum involves another major component of cell metabolism and synthesis: lipids. Steroids are examples of lipids that have a well-documented role in mediation of inflammatory (and oncogenic) pathways but are not the subject of discussion here. Instead, we will discuss the role of another class of bioactive signalling lipids, which, since their early descriptions, have emerged to be a major class of signalling lipids known as LPLs [7, 8].

Fig. 8.1 Overview of tumour-host interactions within the tumour microenvironment. Without contact inhibition tumour cells become discohesive and undergo changes in cell shape, and concurrent activation of cell proliferation pathways enables tumour growth. Angiogenesis delivers nutrients to allow tumour growth,

and along with lymphangiogenesis, both provide conduits for metastasis. Metalloproteases along with invasive tumour cell phenotypes enable invasion. Naïve circulating immune cells undergo trafficking into the tissue stroma and if activated can participate in the host/ immune response

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8.1.1 Bioactive Lysophospholipids

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Index

A Acquired resistance, 10 Active canonical Wnt signaling, 110 Acute lymphoblastic leukemia (ALL), 113 Acute myeloid leukemia (AML), 113, 117 Adaptive immune responses, 176 androgen deprivation, 178 DCs, 178 influenza vaccination, 178 T-cell proliferation, 177 Th17 cells, 176 Th2 phenotype, 177 Adenosine, 150–152, 154 Adenosine deaminase, 146, 147 Adenosine signaling acute injury phase, 146 biological actions, 147 catabolic pathway, 147 CD73, 146, 149 endothelial cells, 147 extracellular levels, 146 in vitro experiments, 154 lymphocytes, 152 macrophage, 150 MDSCs, 151 metabolite, 146 molecular and functional characteristics, 147 neoplasia, 146 neoplastic disease, 146 pericytes, 149 phagocytes, 150 purine metabolism, 146 receptor-independent effects, 147 receptors, 147 role, 149 source, 147 TME, 149 Adenosine−AMP−AMPK pathway, 155 Adipocytes, 34, 36 A disintegrin and metalloproteinase 12 (ADAM12), 53 Androgen receptor (AR) signaling

adaptive immune responses, 176 androgens, 169 CAFs, 171 epithelial, 171 epithelial cells and fibroblasts, 172 expression and function, 170, 172 mAR protein, 170 microenvironmental changes, 172 murine and human prostate, 170 nonclassical, 172 physiological serum testosterone, 172 prostate development, 170 role, 170 stromal, 170, 172 T cells, 177 UGM, 170 Angiogenesis, 13, 90, 95, 97, 113 Eph/Ephrin system, 49–51 GBM phenotypes, 132 MMP-9, 131 neointimal formation, 132 PI3K inhibition, 132 S1P production, 132 VEGF inhibitors, 131 Angiogenic process, 149 Angiopoietin-like 4 (ANGPTL4), 94 Anticancer immunity, 176 Anticancer immunotherapies, 16 Antigen-presenting cells (APCs), 176 Antisense molecule AP 12009, 97 Anti-TGFβ therapies, 98 Anti-tumor immune response, 97 Apoptosis, 62, 65 Apoptotic process, 154, 155 Apoptotic-inducing factor (AIF), 155 ATP/HMGB1/caspase-1 axis, 77 A2B receptors, 157 Autophagosomes, 93 Autophagy, 77, 81, 83 Autophosphorylation, Tyr-419, 59 Autotaxin (ATX), 135 AXIN1 activators, 114

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1270, https://doi.org/10.1007/978-3-030-47189-7

185

Index

186 B Belagenpneumatucel–L vaccine, 98 β-catenin, 108, 110–113 β-catenin signal, 48 Betaglycan, 91 Bone marrow microenvironment, 113 Bone metastases, 95 Bone morphogenetic protein (BMP), 90 Bosutinib, 65 C CAF-derived TGFβ, 96 CAF phenotypes, 96 Calmodulin-dependent protein kinase II (CAMKII), 81, 108, 113 Cancer NRGs role (see Neuregulin (NRG) signaling) tumorigenesis, 4 Cancer-associated fibroblasts (CAFs), 37, 38, 96, 97, 111, 112, 171 Actin-binding protein filamin A, 173 dermal fibroblasts, 174 DNA synthesis, 172 immortalized prostate, 173 protective function, 173 Cancer cell plasticity, 37 HGF/c-Met signalling (see HGF/c-Met signalling) Cancer cells, 94 Cancer immunotherapies, 110 Cancer stem cell (CSC), 32, 34, 37–39, 108, 109 Eph/Ephrin system, 52 in hGBM, 52 Cancer therapy, 110, 114 Canonical pathway, 108 Canonical TGFβ signaling, 92 Canonical Wnt pathway, 112 Carcinogenesis, 90 CBP/β-catenin inhibitors, 114, 117 CD39 enzymatic activity, 157 CD73 knockout mice, 157 Cell apoptosis, 154 Cell motility ATX, 130 LPA4 and LPA5, 130 metastatic behaviour, 130 migration, 129 RhoA-ROCK-dependent cell motility, 130 Cell proliferation, 155 CD133-enriched U87 glioma cells, 129 and invasion, 131 murine ovarian cancer models, 129 signalling pathways, 129 survival pathways, 129 Cellular and molecular mechanisms, 158 Cellular crosstalk, 34–36 Chemokines, 36 Chemoresistance, 37 Chemotherapy, 10 Clinical trials, 115

Collagen triple helix repeat containing 1 (CTHRC1), 111 Colon cancer mouse model, 151 CRK-associated substrate (Cas), 63 c-Src kinase (Csk), 59–60 Cyclin-dependent kinase (CDK), 93, 156 Cytokine, 32, 33, 36 Cytolytic factors, 95 Cytotoxic T-cells, 36 Cytotoxic T lymphocytes (CTLs), 51, 53, 110 D Dasatinib, 65, 66 Dendritic cells (DC), 51, 77, 110, 150, 151, 178 Dickkopf-3 (DKK3), 111 Dickkopf-related protein 2 (DKK2), 114 Disheveled segment polarity protein (DVL), 108 DKK1 antibodies, 114 Doxorubicin, 10 Drug resistance, 32, 38 Dysregulated NRG signaling, 9 E Endocrine therapy, 12 Endothelial cells (ECs), 112, 113, 147 adenosine receptors, 148 CD39 activity, 148 male and female ARKO mice, 174 prostate cancer cells, 174 steroid-responsive tissues, 174 TME, 147 tumor angiogenesis, 149 tumor endothelial cells, 148 VSMCs, 174 Endothelial-mesenchymal transition (EndMT), 112, 114 Eph receptors, 45 Eph/ephrin system in angiogenesis, 49–51 in colorectal and breast cancer initiation, 47–49 in CSC, 52 expression in tumors, 47 in metastatic environment, 52–53 in tumor immunity cytokine production, 51 DCs, 51 endothelial recruitment, 51 EphA10, 52 EphA2, 51 PD-1, 51 EphA receptors, 45 Ephrin-A, 45 Ephrin-A1 expression, 47, 50 Ephrin-B, 45 Ephrins, 45–47 Epidermal growth factor receptor (EGFR), 61, 62 Epigenetic alterations, 5, 8 Epigenetic targeting, 14

Index Epithelial-mesenchymal transition (EMT), 8, 34, 38, 39, 64, 92, 94, 96 ErB2-targeted therapy, 11 ErbB family breast cancer cells, 8 in CRC, 7 dysregulated ErbB signaling, 7 ErbB2, 3 ErbB3, 3 ErbB3–PI3K–AKT signaling cascade, 10 homo-/hetero-dimerization, 6 miRNA-mediated epigenetic regulation, 15 mutations, 6 NRG genes, 2 NRG/ErbB3 signaling, 11 NRG-1/ERBB3 autocrine mechanism, 8 NRGs (see Neuregulin (NRG) signaling) in NSCLC, 6 overexpression, 5 receptor-related abnormalities, 9 RTKs, 3, 5 subfamily members, 3 transmembrane receptors, 3 ErbB2-targeted therapy, 12 ErbB3-targeted antibodies, 14 Extracellular ATP (eATP) and adenosine, 73 autophagy, 77 as chemokine gradient, 77 cytotoxic, 82 degradation, 83 high levels, 80 in ICD, 75, 76 immunogenic tumor cell death, 77 in normal tissues, 74 multifaceted effects, 78 nfP2X7 expression, 80 P2X7 signaling, 78 Pannexin-1 channels, 74 purinergic signaling and acquisition, 80 in TME, 74, 75, 78, 82 Extracellular matrix (ECM), 90–92, 94, 95, 110, 111 Extracellular vesicles (EVs), 154 EZN-3920, 14 F Farnesoid X receptor (FXR), 109 F-box only protein 32 (FBXO32), 172 Fibroblasts, 61, 62, 111, 112 Focal adhesion kinase (FAK), 61–64 FOS-related antigen-1 (FRA-1), 157 Frizzled (FZD) receptor, 108 Frizzled-2 (FZD2), 112 FZD receptor inhibitors, 114 G G protein-coupled receptor (GPCR), 108 Galunisertib, 97

187 Genome-wide analysis, 32 Gliomas, 7, 111 Growth factor receptor-bound protein 2 (Grb-2), 62 H Hematopoietic stem cells (HSCs), 113 Hematopoietic stem/progenitor cells (HSPCs), 113 Hepatocyte growth factor (HGF), 9, 12, 33, 61 Hepatocytes, 32 Hepatokines, 36 Heregulins (HRGs) expression, 15 HRG β1, 13 HRG-α, 7 (see also Neuregulin (NRG) signaling) and MM-121, 14 peptide growth factors, 2 signaling-induced VEGF expression, 13 HGF/c-Met signalling biological responses, 33 in brain disorders, 33 in cancer, 34 in cancer cell plasticity adaptive response to metabolic stress, 37 neo-angiogenesis, 39–40 to CSCs maintenance, 37–39 tumor-stroma crosstalk, 40 developments, 33 embryogenesis, 33 on glucose transport and metabolism, 33 HGF, 33 immunosuppressive role, 36 mechanisms, 33 organogenesis, 33 phosphorylated tyrosines, 32 proteolytic cleavage, 32 renewal processes, 33 restrictions, 33 survival signalling, 33 tumor inflammation and neo-angiogenesis, 34–36 in tumor-stroma crosstalk, 38 HIF-1α activation, 38 Histone deacetylases (HDACs), 14 Homeostasis, 124 Human glioblastoma multiforme (hGBM), 52 Human macrophages, 176 Human Src, 58 Hypoxia, 65, 73 Hypoxia-inducible transcription factor (HIF-1), 111 I ICG-001, 114 Immature myeloid cells (IMCs), 151 Immune cells, 89, 95 cancer progression, 109 immune evasion, 110 immune tolerance, 110 Immune checkpoint inhibitors, 110 Immune escape, 32, 40

Index

188 Immune evasion, 110, 114, 117 Immune modulation, 136 Immune therapy, 51 Immune tolerance, 110, 117 Immunogenic cell death (ICD) ATP release, 73, 77 ICD-associated DAMPs, 77 pro-inflammatory and immunostimulatory effects, 76 pyroptosis, 76 role of ATP, 75, 76 Immunoglobulin-like (IgG-like) domain, 2 Immunological adjuvants, 51 Indoleamine 2,3-dioxygenase-1 (IDO), 110 Inflammation, 34 in TME, 38 Inflammatory cell infiltration, 33 Inflammatory pathways, 36 Innate immune cells ARKO mice, 175 cancer patients, 175 human bone marrow, 174 macrophages, 176 MDSC, 175 neutrophils, 175 NK cell activity, 176 pro-inflammatory cytokines, 175 and sex hormones, 174 sexual dimorphism, 174 Integrin/focal adhesion kinase (FAK) pathway, 110 Integrins, 61, 63 Invasion, 58, 61–65, 92–97 Irreversible membrane permeabilization, 78 J Juxtacrine signaling, 46, 52 K Kinase Src (see Src kinase) Kinase inhibitors/aptamers, 97 L Large latent complex (LLC), 90 Latency-associated peptide (LAP), 90 Latent TGFβ binding proteins (LTBP), 90 Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), 108 Leukemia stem cells (LSCs), 108 LGK974, 114 LY2157299, 97 Lymphocytes, 152 A2A receptors, 153 CD73, 153 chemotherapeutic agents, 153 cytotoxic CD8+T cells, 152 solid tumors, 152 Tregs differentiation, 153

Lysophosphatidic acid (LPA) alkyl and acyl variants, 126 Edg family, 126 LPL signalling, 126 pathway in tumourigenesis, 127 Rho-GTPase-dependent, 130 signalling, 127–129 and S1P, 126 SPC, 129 Lysophospholipids angiogenesis, 131 apoptotic pathways, 128 ATX pathway, 126 autocrinic and paracrinic mechanisms, 125 autocrinic and paracrinic-signalling lipids, 126 bioactive, 126 bioactive signalling lipids, 125 bronchoalveolar lavage fluids (BALF), 133 cancer angiogenic mechanisms, 131 biological effects, 128 cell motility, 129 cell proliferation, 129 cell survival, 128 tumourigenesis, 127 cancer biomarkers, 137 cancer treatment, 136 CD4 T-lymphocytes, 133 cell motility, 129 cell proliferation, 129 cellular microenvironment, 124 clinical future, 136 Edg family, 126 Edg6, 126 homeostasis, 124 immune cell trafficking, 124 immune system, 133 invasion and metastasis, 134 LPA and S1P, 126 MAPK/ERK signalling pathways, 128 PI3K/Akt signalling, 128 signalling molecules, 124 steroids, 125 surviving host-tumour interactions, 128 tumourigenesis, 128 VEGF pathways, 125 VEGF transcription, 128 M Macrophage colony-stimulating factor (M-CSF), 150 Macrophages, 60 functions, 150 MAPK activity, 52 Matrix metalloproteinase 9 (MMP-9), 82 Matrix remodeling, 90, 91, 95 Melanoma differentiation-associated gene-9 (MDA-9), 50 Membrane permeabilization, 78 Mesenchymal stem cells (MSCs), 34–36, 113

Index Mesenchymal-type invasion, 64 Metabolic stress, 32, 37, 38 Metastasis, 1, 6, 8, 9, 13, 32, 39, 58, 59, 62–66, 94–97, 156 A2A receptors, 157 CD39 and CD73 enzymes, 158 ECM, 157 role, 157 tumor metastases, 157 Microbiota, 15 Microenvironment, 60, 64 Microparticles (MPs), 112 Migration, 58, 60–65 miRNA-mediated epigenetic regulation, 15 MiRNAs, 8, 15 Molecular signalling, 32 Monoclonal antibody (mAb), 114 Monocytes and macrophages, 175 Mononuclear phagocytes, 150 Motility, 62, 64 Murine ovarian cancer model, 132 Myeloid-derived suppressor cells (MDSCs), 50, 175 Myeloid-specific ARKO (MARKO), 176 Myristoylation, 58, 59 N Nanoparticles (NPs), 51 Natural killer (NK) cells, 51, 176 Neo-angiogenesis, 34, 36–39 Neoplasia, 146 Neuregulin (NRG) signaling acting models, 3 and biological consequences, 4 biological functions, 3 in cancer initiation and development breast cancer, 6 ErbB receptors, 5, 6 ErbB2 in gastric cancer, 7 lung cancer, 6–7 mammary gland, transgenic mice, 6 pancreatic cancer, 7 proliferative signaling, 5 in cancer metastasis, 8–9 cell–cell interactions, 2 ErbB family, 2 ErbB receptors, 4 ErbB subfamily, 3 genes, products and expression, 2, 5 microbiota, 15, 16 MiRNAs, 15 NRG-1, 2 peptide growth factors, 2 structures and distribution, isoforms, 2 therapeutic resistance in cancer acquired resistance, 10 chemotherapy, 10 endocrine therapy, 12 primary resistance, 10 targeted therapy, 10–12

189 therapeutic targeting, in cancer treatment, 13–15 in TME regulation, 12–13 NGF/Trk signaling, 13 Niclosamide, 114 Non-canonical pathways, 108 Non-canonical TGFβ signaling, 92 Non-canonical Wnt/PCP signaling pathway, 111 Nonreproductive tissues, 170 Non-small-cell lung cancer (NSCLC), 6, 9, 11, 15 Notch intracellular domain (NICD), 9 Notch signaling pathway, 9 NRG/ErbB3 signaling, 11 Nuclear factor of activated T-cell cytoplasmic 3 (NFATc3), 112 Nucleoside transporters, 147 O Oncogene, 58 Oncogenesis, 58 O2 supply, 49 Ovalbumin (OVA), 51 P P2X/P2Y purinergic receptors, 75, 77, 79, 83 Paclitaxel resistance, 10 Palmitoylation, 58 Paracrine biological functions, NRGs, 3 EGF/CSF-1 invasion loop, 9 interaction, 12 oncogene fusions, 9 signaling pathways, 2 Pathogen infections, 77 Paxillin, 63, 64 PD-L1 expression, 52, 53 Peptide vaccine-based immune therapy, 51 Pericytes, 39, 60, 82, 149 capillary diameter, 149 CD39 and CD73, 150 functions, 149 tumor microvessels, 149 Phosphatase and tensin homolog (PTEN), 11, 16 Phospholipase D2 (PLD2), 112 Plakophilin-2 (PKP2), 111 Platelet-derived growth factor (PDGF), 61, 62 Platelet-derived growth factor-B (PDGFB), 13 Polybasic amino acids, 58 Ponatinib, 65 Porcupine (PORCN) inhibitors, 114 Primary resistance, 10 Programmed cell death-1 (PD-1), 51 Proliferation, 58, 61, 62, 65, 66 Pro-migratory, 94 Prostate cancer (PCa), 170 Prostate cancer cell lines, 156 Prostate carcinogenesis, 171 Prostate intraepithelial neoplasia (PIN), 170 Protein kinase A (PKA), 152

Index

190 Protein tyrosine kinase 7 (PTK7), 111 Protein tyrosine phosphatases (PTP), 60 Purinergic pathway, 148 Purinergic signaling angiogenesis, 82 ATP/HMGB1/caspase-1 axis, 77 biological functions, in TME, 74 energy metabolism, regulation, 80–81 high eATP paradox, 79–80 immuno-oncology, 78 immunostimulatory and direct cytotoxic effects, 79 migration and invasion, cancer cell, 81–82 P2X7 signaling, 78–79 pro-survival and proliferative functions, 80 pyroptosis, 76 receptors, 77 regulation of cancer immunity, 83 regulation, ATP degradation, 82–83 tumor growth and survival, 82 Pyroptosis, 76 R Ras/Erk signaling pathway, 49 Ras/MAPK signaling, 47 Receptor tyrosine kinases (RTKs), 3, 5, 10 Resident fibroblasts, 96 Resveratrol (RES), 111 RhoA signaling pathway, 49 RhoA/ROCK signaling pathway, 49 Rho-dependent mechanisms, 124–125 Ribosomal protein s15a (RPS15A), 112 RNA-binding motif on the Y chromosome (RBMY), 109 Ror2 receptor tyrosine kinase, 113 Rous sarcoma virus (RSV), 58 RTK signaling, 8 S Secreted frizzled-related protein 2 (SFRP2), 112 Senescence-associated secretory phenotype (SASP), 112 Signal transducer and activator of transcription-3 (STAT-3), 62, 63 SMAD, 92 SMAD7, 92 Src family cellular events, 58 members, 57 signal transduction, 57 src gene c-Src, 58 RSV, 58 v-src, 58 Src inhibitors, 60 Src kinase activation in hypoxic TME, 65 activity, 58 adhesion and migration, 63 autophosphorylation at Tyr-419, 59 in cancer invasion, 64

in cancer treatments, 65–66 Cas, 63 in EMT, 64 enzymatic activity, 59 FAK, 63 growth factor signaling pathways, 61 integrins, 63 60-kDa protein, 58 molecular mechanisms, 60 N-terminal region, 58 paxillin, 64 recognition pockets, 59 regulation, Src activity, 59–60 SH1 domain, 59 SH3 functions, 59 SH4 domain, 59 Src homology domain, 58 Src/multikinase inhibitors, 65, 66 and Src-related proteins, 58 Tyr-416, 59 Src signaling network, 61 Stochastic genetic changes, 32 Stromal cell, 107, 108, 111, 113 Syntenin, 49–50 T Targeted therapy, 97, 98 NRG signaling, cancer, 10–12 Src in cancer patients, 65–66 T-cell factor (TCF), 108 T-cell factor/lymphoid enhancer factor (TCF/LEF), 108 Telomerase reverse transcriptase (TERT), 108 Temozolomide therapy, 112 TGFβ cytoskeletal organization, 92 TGFβ pathway inhibitors, 97 TGFβ receptors (TGFβR), 90 TGFβ-regulated microRNAs, 94 TGFβ signaling canonical TGFβ signaling, 92 cellular processes, 92 non-canonical TGFβ signaling, 92 tumor microenvironment, 90, 91 TGFβ signaling proteins, 97 TGFβ tumor-suppressive effects, 93 The Cancer Genome Atlas (TCGA), 110 Theory of cancer cells, 32 Therapeutic resistance, 10 Therapeutic resistance in cancer NRG signaling acquired resistance, 10 chemotherapy, 10 endocrine therapy, 12 primary resistance, 10 targeted therapy, 10–12 Therapeutic target purinergic signaling, 83–84 Thyroid cancer cell line, 154 T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1), 111

Index Transforming growth factor (TGFβ) bioavailability, 90, 92 CAF, 96, 97 cancer cells, 93 carcinogenesis, 90 dichotomous function, 93 metastatic functions, 94, 95 mutations, 93 pro-invasive, 94, 95 signaling pathway (see TGFβ signaling) structure, 90, 92 targeted therapy, 97, 98 tumor immune responses, 95, 96 tumor microenvironment, 92 tumor suppressor, 90 tumor suppressor function, 94 tumorigenesis, 92 tumor-suppressive effects, 93 TβRs, 93 Tumor angiogenesis, 13, 112 Tumor-associated antigen (TAA), 51 Tumor-associated macrophages (TAMs), 8, 9, 35, 36, 175 Tumor-associated natural killers (TANKs), 36 Tumor biology, 2, 9 Tumor cells, 91 Tumor heterogeneity, 32 Tumor immune responses, 95, 96 Tumor microenvironment (TME), 146 cross-communication network, 60 eATP role, 73–75 HGF and c-Met (see HGF/c-Met signalling) hypoxia, 73 in cancer treatment, 60 inflammation, 38 regulation, Src activity EGFR, 62 Grb-2, 62 growth factor signaling pathways, 61 PDGF, 62 PI3K/Akt, 62 STAT-3, 62 Src signaling network, 60 TGFβ (see Transforming growth factor (TGFβ)) Warburg effect, 73 Wnt (see Wnt signaling) Tumor plasticity, 32, 34 Tumor progression, 47, 49, 53 Tumor suppressor, 90 Tumorigenesis, 4–6, 13, 14, 60, 62

191 Type-1 pericytes, 60 Tyrosine kinase inhibitor (TKI), 114 TβRI inhibitor, 97 U Urogenital sinus mesenchyme (UGM), 170 V Vandetanib, 65 Vascular endothelial growth factor (VEGF), 7, 13, 61, 62, 65, 131 Vascular endothelial growth factor-A (VEGF-A), 113 Vascular smooth muscle cells (VSMCs), 174 Vesicular nucleotide transporter (VNUT), 77 W Warburg effect, 73 Wnt signaling AXIN1 activators, 114 CAFs, 111, 112 canonical pathway, 108 canonical Wnt pathway, 109 CBP/β-catenin inhibitors, 114, 117 clinical trials, 115 CSCs, 108, 109 DKK1 antibodies, 114 ECM, 110, 111 ECs, 112, 113 FZD receptor inhibitors, 114 immune cells cancer progression, 109 immune evasion, 110 immune tolerance, 110 microenvironment, tumor cells, 107 MSCs, 113 non-canonical pathways, 108 pathogenesis, cancer, 107 PORCN inhibitors, 114 TME, 116 WNT5A, 117 WNT5A, 117 WNT5a-Ror2 signaling, 113 X Xenografts, 14, 15, 49, 52, 97, 114, 172, 173, 179