Exploring Pancreatic Metabolism and Malignancy [1st ed. 2019] 978-981-32-9392-2, 978-981-32-9393-9

Table of contents :
Front Matter ....Pages i-xii
Biology of Pancreas and Possible Diseases (Gowru Srivani, Begum Dariya, Batoul Farran, Afroz Alam, Ganji Purnachandra Nagaraju)....Pages 1-25
Pancreatitis: Clinical Aspects of Inflammatory Phenotypes (Nyshadham S. N. Chaitanya, Aramati BM Reddy)....Pages 27-33
Diabetes and Pancreatic Cancer: A Bidirectional Relationship Perspective (Manoj Kumar Gupta, Vemula Sarojamma, Ramakrishna Vadde)....Pages 35-51
Metabolic Adaptations in Diabetes Mellitus and Cancer (Anil Kumar Pasupulati, Nageswara Rao Dunna, Srikanth Talluri)....Pages 53-69
Role of Mitochondria in Pancreatic Metabolism, Diabetes, and Cancer (Noble Kumar Talari, Ushodaya Mattam, Naresh Babu V. Sepuri)....Pages 71-94
Targeting Mitochondrial Enzymes in Pancreatic Cancer (Gowru Srivani, Begum Dariya, Afroz Alam, Ganji Purnachandra Nagaraju)....Pages 95-110
Diabetes with Pancreatic Ductal Adenocarcinoma (Gowru Srivani, Begum Dariya, Afroz Alam, Ganji Purnachandra Nagaraju)....Pages 111-131
Role of Inflammatory Cytokines in the Initiation and Progression of Pancreatic Cancer (Madanraj Appiya Santharam, Vignesh Dhandapani)....Pages 133-156
Perspectives and Molecular Understanding of Pancreatic Cancer Stem Cells (L. Saikrishna, Prameswari Kasa, Saimila Momin, L. V. K. S. Bhaskar)....Pages 157-172
The Role of Hypoxia Inducible Factor-1α in Pancreatic Cancer and Diabetes Mellitus (Saimila Momin, Ganji Purnachandra Nagaraju)....Pages 173-181
Role of Heat Shock Protein 90 in Diabetes and Pancreatic Cancer Management (Pinninti Santosh Sushma, Saimila Momin, Gowru Srivani)....Pages 183-195
Insulin Resistance Is a Common Core Tethered to Diabetes and Pancreatic Cancer Risk (Henu Kumar Verma, L. V. K. S. Bhaskar)....Pages 197-213
Immunotherapy for Diabetogenic Pancreatitis and Pancreatic Cancer: An Update (Sathish Kumar Mungamuri, Anil Kumar Pasupulati, Vijay Aditya Mavuduru)....Pages 215-236
Exosomes: Mediators and Therapeutic Targets of Diabetes and Pancreatic Cancer ( Deepak KGK, Rama Rao Malla)....Pages 237-251
Methods and Models in Exploring Pancreatic Functions (Rama Rao Malla, Seema Kumari, Krishna Chaitanya Amajala, Deepak KGK, Shailender Gugalavath, Prasuja Rokkam)....Pages 253-268

Citation preview

Ganji Purnachandra Nagaraju  Aramati BM Reddy Editors

Exploring Pancreatic Metabolism and Malignancy

Exploring Pancreatic Metabolism and Malignancy

Ganji Purnachandra Nagaraju Aramati BM Reddy Editors

Exploring Pancreatic Metabolism and Malignancy

Editors Ganji Purnachandra Nagaraju Department of Hematology and Medical Oncology Emory University School of Medicine Atlanta, GA, USA

Aramati BM Reddy Department of Animal Biology University of Hyderabad Hyderabad, Telangana, India

ISBN 978-981-32-9392-2 ISBN 978-981-32-9393-9 https://doi.org/10.1007/978-981-32-9393-9

(eBook)

# Springer Nature Singapore Pte Ltd. 2019 This work is subject to copyright. All rights are reserved 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Notably, roughly 50% of patients with newly diagnosed pancreatic cancer have an existing diabetes diagnosis. Despite several studies evaluating the association between pancreatic adenocarcinoma and diabetes mellitus type 2, the direction of the relationship is not clear. Some studies report that diabetic patients have a two-fold increased risk of developing pancreatic cancer, while others suggest that increased risk of developing cancer exists in newly diagnosed diabetics and progressively decreases with a prolonged course of diabetes mellitus type 2. Several meta-analyses aiming to analyze this relationship have reached various conclusions; in general, they conclude that patients with diabetes mellitus type 2 are likely to develop pancreatic cancer 24–36 months following diagnosis. This relationship, diabetes causing PC development, has been thoroughly evaluated. On the other hand, diabetes development following PC diagnosis is thought to be a result of tumor infiltration, ductal obstruction, and endocrine glandular destruction. PC’s release of mediators affects insulin secretion and action. Therefore, such patients typically demonstrate insulin resistance and hyperinsulinemia. Further studies must be done to elucidate this bidirectional relationship. In this book, we aim to discuss methods and models for exploring pancreatic functions, pancreatic biology, other pancreatic diseases, the role of various organelles, therapeutic options for diabetes mellitus type 2 and pancreatic cancer, and pancreatic functions. The pancreas is a complex organ with multiple functions; it functions as an exocrine and endocrine gland and plays a role in many important pathways. A thorough understanding of pancreas anatomy and pancreatic cancer biology is key for developing new, more effective medications for pancreatic diseases including diabetes mellitus type 2 and pancreatitis, which have been shown to contribute to the development of pancreatic cancer. Their relationship is explored in this book. Mitochondria and exosomes are also important mediators of diabetes and pancreatic cancer. As organelles play a key role in the development of cancer, they may be targeted in future pancreatic cancer therapy. In this book, we will discuss current and innovative therapeutic options for diabetes and pancreatic cancer. Atlanta, GA, USA Hyderabad, Telangana, India

Ganji Purnachandra Nagaraju Aramati BM Reddy

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Acknowledgment

This book is dedicated to our families, teachers, contributors, and friends.

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Contents

1

Biology of Pancreas and Possible Diseases . . . . . . . . . . . . . . . . . . . . Gowru Srivani, Begum Dariya, Batoul Farran, Afroz Alam, and Ganji Purnachandra Nagaraju

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2

Pancreatitis: Clinical Aspects of Inflammatory Phenotypes . . . . . . . Nyshadham S. N. Chaitanya and Aramati BM Reddy

27

3

Diabetes and Pancreatic Cancer: A Bidirectional Relationship Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manoj Kumar Gupta, Vemula Sarojamma, and Ramakrishna Vadde

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Metabolic Adaptations in Diabetes Mellitus and Cancer . . . . . . . . . Anil Kumar Pasupulati, Nageswara Rao Dunna, and Srikanth Talluri

5

Role of Mitochondria in Pancreatic Metabolism, Diabetes, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Noble Kumar Talari, Ushodaya Mattam, and Naresh Babu V. Sepuri

35 53

71

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Targeting Mitochondrial Enzymes in Pancreatic Cancer . . . . . . . . . Gowru Srivani, Begum Dariya, Afroz Alam, and Ganji Purnachandra Nagaraju

95

7

Diabetes with Pancreatic Ductal Adenocarcinoma . . . . . . . . . . . . . . 111 Gowru Srivani, Begum Dariya, Afroz Alam, and Ganji Purnachandra Nagaraju

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Role of Inflammatory Cytokines in the Initiation and Progression of Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Madanraj Appiya Santharam and Vignesh Dhandapani

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Perspectives and Molecular Understanding of Pancreatic Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 L. Saikrishna, Prameswari Kasa, Saimila Momin, and L. V. K. S. Bhaskar

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The Role of Hypoxia Inducible Factor-1α in Pancreatic Cancer and Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Saimila Momin and Ganji Purnachandra Nagaraju

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Role of Heat Shock Protein 90 in Diabetes and Pancreatic Cancer Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Pinninti Santosh Sushma, Saimila Momin, and Gowru Srivani

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Insulin Resistance Is a Common Core Tethered to Diabetes and Pancreatic Cancer Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Henu Kumar Verma and L. V. K. S. Bhaskar

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Immunotherapy for Diabetogenic Pancreatitis and Pancreatic Cancer: An Update . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Sathish Kumar Mungamuri, Anil Kumar Pasupulati, and Vijay Aditya Mavuduru

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Exosomes: Mediators and Therapeutic Targets of Diabetes and Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Deepak KGK and Rama Rao Malla

15

Methods and Models in Exploring Pancreatic Functions . . . . . . . . . 253 Rama Rao Malla, Seema Kumari, Krishna Chaitanya Amajala, Deepak KGK, Shailender Gugalavath, and Prasuja Rokkam

About the Editors

Ganji Purnachandra Nagaraju is a faculty member at the Department of Hematology and Medical Oncology at Emory University School of Medicine. He obtained his M.Sc. and Ph.D., both in biotechnology from Sri Venkateswara University in Tirupati, Andhra Pradesh, India. He received his D.Sc. from Berhampur University in Berhampur, Odisha, India. His research focuses on translational research in gastrointestinal malignancies. He has published over 70 research papers in highly respected international journals and has presented more than 50 abstracts at various national and international conferences. He is author and editor of several. He is an editorial board member of several internationally recognized academic journals, and is an associate member of the Discovery and Developmental Therapeutics research program at Winship Cancer Institute. He is a member of the Association of Scientists of Indian Origin in America (ASIOA), the Society for Integrative and Comparative Biology (SICB), the Science Advisory Board, the RNA Society and the American Association of Cancer Research (AACR). Aramati BM Reddy is an Assistant Professor at the School of Life Sciences, University of Hyderabad, India. He obtained his M.Sc. in Biotechnology from SV University, Tirupati, India, and Ph.D. from L.V. Prasad Eye Institute, University of Hyderabad, India. He received postdoctoral training at the University of Pennsylvania and University of Texas Medical Branch, Galveston, in epigenetics, inflammation, and metabolic disorders. His research interests focus on understanding cell signaling and gene regulation in the pathophysiology of metabolic malignancies and glaucoma. His lab is xi

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About the Editors

also working on understanding the role of several candidate genes and their significance for the outflow pathway of the aqueous humor of the anterior segment of the eye and glaucoma pathology. He has published 30 research papers in peer-reviewed journals and four book chapters. In addition, he has co-edited three special issues for Hindawi publishers in the field of translational and health sciences. He is a life member for several societies and is currently an executive board member of the Indian Society of Cell Biology.

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Biology of Pancreas and Possible Diseases Gowru Srivani, Begum Dariya, Batoul Farran, Afroz Alam, and Ganji Purnachandra Nagaraju

Abstract

Pancreas is one of the small, flattened organs in the gastrointestinal tract associated with dynamic functions. Most of the food is digested before entering into the other organs and then is converted into energy to regulate the metabolic systems. However, digestion mainly involves the hydrolysis of carbohydrates, lipids, amino acids, proteins, and fatty acids. Releasing enzymes and hormones, which are crucial for regulating the glucose homeostasis and energy production, is the main function of both the endocrine and exocrine pancreas. Disorders of the pancreas can affect the exocrine and endocrine functions, thus resulting in devastating diseases, such as acute and chronic pancreatitis, diabetes, and pancreatic cancer. The main predisposing factors for the development and progression of these disorders are still unclear; therefore, it is important to understand the mechanisms that regulate pancreas homeostasis. In this present chapter, we discuss the biology of the pancreas and its functions and the development of disorders and pathological conditions. We also examine how this accumulating knowledge is guiding the discovery of new diagnostic and therapeutic approaches to cure or prevent disease. Keywords

Pancreas · Exocrine · Endocrine · Pancreatitis · Diabetes and pancreatic cancer

G. Srivani · B. Dariya · A. Alam Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India B. Farran · G. P. Nagaraju (*) Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_1

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Abbreviations 4EBP1 Akt AMPK ASC Atg1 Atg5 Atg6 ATP CD CDK4I CDKN2A COSMIC DAMPs DNA EGFR ERK FOXO FTIs G1 phase GAD65 GAD67 GTP H HIF-1α HMGB1 HSP70 ICA-512 IDF IL-33 IL-1 IL-10 IL-18 IL-6 IL-8 IL-β JNK MAPK MEK MEN1 mTOR NETS NF-Kb

4E-Binding protein Protein kinase B Adenosine monophosphate-activated protein kinase Apoptosis-associated speck-like protein Autophagy-related 1 Autophagy-related 5 Autophagy-related 6 Adenosine triphosphate Cluster of differentiation Cyclin-dependent kinase 4 inhibitor Cyclin-dependent kinase inhibitor 2A Catalogue of Somatic Mutations in Cancer Damage-associated molecular patterns Deoxyribonucleic acid Epidermal growth factor receptor Extracellular signal-regulated kinases Forkhead box protein Farnesyltransferase inhibitors Gap 1 phase Glutamic acid decarboxylase 65 Glutamic acid decarboxylase 67 Guanosine-5’-triphosphate Histidine Hypoxia-inducible factor-1α High-mobility group box protein 1 Heat-shock protein 70 Islet cell antigen 512 International Diabetes Foundation Interleukin-33 Interleukin-1 Interleukin-10 Interleukin-18 Interleukin-6 Interleukin-8 Interleukin-β c-Jun amino N-terminal kinase Mitogen-activated protein kinase Mitogen-activated protein kinase Multiple endocrine neoplasia Mammalian target of rapamycin Neuroendocrine tumors Nuclear factor kappa B

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NLRP3 P16 p38 PAMPs PanIN Ph domain PI3K PIP3 PKC PLC PNEC R RAS Rictor RNA ROS S phase SMAD4 SPINK1 TLR TNF TNF-α Tp53 Ulk1 gene VEGF VEGFR VHL

3

Nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing-3 Protein 16 Protein 38 Pathogen-associated molecular patterns Pancreatic intraepithelial neoplasia Pleckstrin homology Phosphatidylinositol 3-kinase Phosphatidylinositol 3, 4, 5-triphosphate Protein kinase C Phospholipase C Pancreatic necrosis Arginine Reticular activating system Rapamycin-insensitive companion of mTOR Ribonucleic acid Reactive oxygen species Synthesis phase Small worm phenotype and Drosophila mothers against decapentaplegic 4 Serine protease inhibitor Kazal type 1 gene Toll like receptors Tumor necrosis factor Tumor necrosis factor-α Tumor protein 53 Unc-51 like autophagy activating kinase Vascular endothelial growth factor Vascular endothelial growth factor receptor von Hippel–Lindau

1.1

Introduction

1.1.1

Pancreas Location and Anatomy

The pancreas is a glandular and digestive retroperitoneal organ, positioned at the point of the second lumber vertebra, and lies behind the stomach in the abdomen. It is closely related with the duodenum as both organs share a common blood supply. The pancreas structurally consists of the following regions: 1-. Head – A rounded and large portion of the head is placed on the abdomen’s right side and adjacent to the C loop of the duodenum, which receives the pancreatic fluid delivered by the pancreatic duct.

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2-. Neck – It is the smallest portion of the pancreas and is directly situated in the superior mesenteric vessels and portal vein. 3-. Body – It is wrapped behind the stomach and is surrounded by the splenic artery and vein. 4-. Tail – The last part of the pancreas and continuation of the body. Both are extended at the level of the thoracic vertebra [1].

1.2

Functions of Pancreas

The pancreas plays a vital role in the production of digestive enzymes as well as glucose homeostasis and is also capable of both endocrine and exocrine functions. The endocrine function of the pancreas is mediated by a cluster of tiny cells distributed on the center known as the islets of Langerhans, which consist of four kinds of cells: the α-cells, β-cells, δ-cells, and γ-cells. These cells secrete insulin, somatostatin, glucagon, and pancreatic polypeptides, respectively. The α-cells and β-cells play a crucial part in maintaining carbohydrate metabolism, lipids, proteins, and fats as well as glucose levels in the blood (Table 1.1). The exocrine function of the pancreas is to secrete pancreatic juices, which are constricted with digestive enzymes such as bicarbonates, proteases, lipases, and amylases that help in the breakdown of food (Table 1.2) [1].

1.3

Diseases of the Pancreas

The devastating nature of pancreatic diseases is mainly due to metabolic imbalances such as congenital diseases and endocrine functional disorders, which can cause diabetes mellitus and pancreatic cancer (PC), one of the deadliest cancers worldwide. They can also be caused by the dysregulation of the exocrine pancreas, resulting in conditions such as pancreatitis, exocrine pancreatic insufficiency, and pancreatic adenocarcinoma.

1.4

Pancreatitis

Pancreatitis is defined as an inflammatory damage of the pancreas caused by excess alcohol consumption, smoking, certain genetic alterations, toxins, bile duct blocked by the gallstones, and sequential activation of pancreatic proenzymes, which are secreted by acinar cells such as proelastase, prophospholipase A2, procolipase, and chymotrypsinogen. These proenzymes are stored in the form of zymogens. There are two forms of pancreatitis: acute and chronic pancreatitis. Acute pancreatitis – In majority of the cases, acute pancreatitis inflammation is driven by several etiological factors (Fig. 1.1) that damage the tissue. Many of these factors are very rare, while others play a significant role in causing acute pancreatitis. Although 40% of the cases are biliary and 30% are alcoholic, the remaining

Insulin (51 amino acid peptide)

Somatostatin (14 amino acid peptide)

Pancreatic polypeptide (36 amino acid peptide)

δ-Cells

γ-Cells

Hormones Glucagon (29 amino acid peptide)

β-Cells

Islet cells α-Cells

PDX1 and PAX4

NKX6.1, NKX2.2, MAFB, PDX1, and PAX6

Transcriptional factors MAFB and PAX6

1

10

70

Composition (%) 20

Table 1.1 Endocrine pancreas – islet of Langerhans cells (Janet L. Fun) Function Glucagon activates gluconeogenesis (glucose synthesis) and glycogenolysis (glycogen storing), actively participates in fatty acid oxidation and ketogenesis (generates ketone bodies) when glucose is not available in the brain (Richard N. Mitchell 2014) Glucose homeostasis and metabolism; it promotes the synthesis of glucose transporter-4 (GLUT-4) in the adipose tissue, cardiac muscle, and skeletal muscle Acts as autocrine function and shows the paracrine function on islet cells producing hormones (suppresses the glucagon and insulin release) in the gastrointestinal tract Somatostatin slows down the nutrient absorption through suppression of gut motility and suppression of exocrine pancreatic function Controls both the endocrine and exocrine functions

Gastrointestinal tract

Brain, pituitary, pancreas, and gastrointestinal tract – mainly gut

Liver, muscle, fat

Targeted tissue Liver

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Table 1.2 Exocrine functions Secretion enzymes (inactive forms) Trypsinogen

Activator Enterokinase

Enzyme Trypsin

Chymotrypsinogen

Trypsin

Chymotrypsin

Proprotease E

Protease E

Procarboxypeptidase A Procarboxypeptidase B

Carboxypeptidase A Carboxypeptidase B

Proelastase 2

Elastase 2

Procolipase

Colipase

Prophospholipase A2

Phospholipids

Ribonuclease Deoxyribonuclease Pancreatic lipase

RNA DNA Triglycerides

Function Breakdown of peptide bonds in aromatic amino acids Catalysis: the hydrolysis of peptide bonds and ester bonds; cleaving peptide bonds at carboxyl side chain of aromatic amino acids Breakdown of proteins, lipids, and carbohydrates Breakdown of carboxy terminal amino acids with aromatic rings Breakdown of carboxy terminal amino acids with basic side chain Breakdown of bonds at carboxyl side of the aliphatic amino acids Forming the active site for pancreatic lipase Catalysis: the hydrolysis of the fatty acyl bond to release fatty acids and lysophospholipids; precursor molecule for eicosanoides Formation of nucleotides Formation of nucleotides Cleaving the dietary fatty acids into fatty acids and glycerol

conditions are associated with other factors. Initially, these factors alter the physiology of the pancreas, resulting in the activation of pathological contents including the autodigestion of enzymes in acinar cells, which are responsible for inflammation [2]. In acute pancreatitis, pancreatic necrosis (PNEC) is a major complication leading to morbidity in a short time span. In all cases of acute pancreatitis, alcohol abuse contributes to 35% of the conditions and is the major risk factor. In fact, chronic alcohol exposure can result in the development of PNEC. Patients suffer from subclinical and multiple clinical disorders during the sequential development of chronic pancreatitis. In fact, chronic alcohol ingestion induces the release of the protein-rich pancreatic fluid, which develops the insoluble protein plugs that cause difficulties in the pancreatic ductules [2]. Hypocalcemia (increased levels of serum calcium) promotes the intraglandular activation action of zymogens, caused by the deposition of calcium (calcium salts) in the pancreatic ducts, and releases fatty acids, leading to hyperparathyroidism [3]. Hereditary pancreatitis occurs in both acute pancreatitis and chronic pancreatitis and is associated with germline mutations in the PRESS1 gene (cationic trypsinogen

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Gall stones

Alcohol, Smoking

Hyperglycemia Hyper calcaemia Hypothermia

Acute Pancreatitis

ERCP

Drugs

Hyperlipidemia

Gene alterations Toxins

Microbiome infections – cocksackie, measles, mycoplasma, mumps, helicobacter pylori, hepatitis and epstein bar virus

Ischaemia

Fig. 1.1 Etiological factors for acute pancreatitis; the main risk factors for the development of acute pancreatitis are gallstones, hyperglycemia, hypercalcemia, toxins, drugs gene alterations, and ischemia. Other contributing factors include microbiome infections – measles, mycoplasma, mumps, helicobacter pylori, hepatitis, and Epstein–Barr virus

gene), namely, point mutations. For instance, a single guanine to adenine (G to A) transition was detected in the 3rd (third) exon of the PRESS1 gene, resulting in an R– H (arginine to histidine) substitution termed R117H. Another mutation results from a single A–T transition identified at amino acid 21, leading to an asparagine to isoleucine substitution termed N211. These mutations inactivate the trypsinogen and trypsin activity. The resulting mutated trypsin abnormally activates the other proenzymes, causing the development of acute pancreatitis [4]. Additionally, mutations in the gene serine protease inhibitor Kazal type 1 (SPINK1), a trypsin inhibitor, can lead to the development of pancreatitis [5]. Obesity has been shown to contribute to acute pancreatitis as well as chronic pancreatitis. In fact, an elevated level of obesity increases the leptin, which is responsible for pro-inflammatory conditions, and decreases the anti-inflammatory agent adiponectin [6]. Furthermore, obesity enhances the number of CD8+ T-cells in macrophages and downregulates T-regulatory cells, further elevating the levels of inflammatory intermediaries such as IL-1β, IL-8, IL-6, and tumor necrosis factor-α (TNF-α) which activate the inflammasome and can lead to inflammation [7]. Moreover, through activating the Akt–mTOR signaling pathway, obesity suppresses the autophagy genes Atg5, Atg6/Beclin1, and Ulk1/Atg1, which increases the severity of pancreatitis [8]. Chronic pancreatitis causes the permanent loss of the pancreas due to irreversible damage, ductal dilation, fibrosis, and damage of acinar cell and islets tissue [4]. The development of acute and chronic pancreatitis is triggered by inflammation driven by the acinar cell damage due to the activation of intrapancreatic trypsinogen, although

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Alcohol, smoking Gallstones Protein Kinase C Hyper glycemia Hyper calcaemia

NLR3 Inflammasome Inflammation

Activates NF-κB

Activates the STAT3 Genetic Mutations PRESS1, SPINK1

DAMPS &PAMPS TLR3 /TLR4

ROS Produces IL6,IL10,TNFα

Fibrosis

Fat accumulation Activates oncogens Kras

Acute Pancreatitis Chronic Pancreatitis Diabetes Fig. 1.2 Underlying mechanism of pancreatic diseases. Inflammation plays a central role in initiating the changes in the cellular microenvironment and thus results in the progression of diseases from acute pancreatitis to chronic pancreatitis, diabetes, and pancreatic cancer. Risk factors (alcohol, smoking) initiate the inflammation, and mutation of the genes activates the PRESS1 and SPINK1 genes; inflammatory mediators IL-6 and IL-10 cause the fibrosis and fat accumulation, activate the oncogene KRAS, and promote the pancreatic cancer

acinar cells sense tissue damage through several intracellular molecules released to the extracellular surface, which provoke damage-associated molecular patterns and pathogen-associated molecular patterns (DAMPs and PAMPs). However, a number of DAMPs and PAMPs are triggered by inflammation via adenosine triphosphate (ATP); high-mobility group box protein 1 (HMGB1); ROS (reactive oxygen species); heat-shock protein (HSP70); toll like receptors (TLR)-4; TLR-9; saturated fatty acids; innate immune molecules including IL-6, IL-10, IL-18, IL-β, IL-1, and IL-33; tumor necrosis factor; and protease cathepsin B, shown to play a significant role in experimental acute and chronic pancreatitis. These pathways stimulate NF-κB activation and express the NLRP3 inflammasome, which contains the NLRP3 (nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domaincontaining-3) protein complex and procaspase 1 and ASC. Figure 1.2 (Apoptosisassociated speck-like protein) [9, 10] A long-term standing of chronic pancreatitis is the development of diabetes mellitus. However, diabetes occurs due to the inflammation that destroys the islet cells such as β- and α-cells, undigested fat accumulation, fibrosis in the pancreas,

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deficiency in vitamins (A, D, E, and K), and inflammatory cell infiltration that can damage the pancreatic structure and function [11].

1.5

Diabetes Mellitus

Diabetes mellitus is characterized by endocrine or metabolic disorders including anabolism and catabolism of carbohydrates, fatty acids, and lipid; protein dysregulation of regulatory networks responsible for storage and the mobilization of metabolic functions; glucose intolerance and hyperglycemia leading to improper insulin action; failure of insulin secretion and insulin resistance; or combined deficiency [12]. According to the International Diabetes Foundation (IDF), the number of people between the age group 20 and 79 years suffering from diabetes worldwide will raise from 415 million in 2015 to 642 million in 2040, suggesting that one in five people at least will suffer from diabetes [13]. Based on its mechanisms and clinical symptoms, diabetes mellitus is classified into two types: type 1 diabetes and type 2 diabetes [14]. Furthermore, based on etiology and clinical symptoms, diabetes mellitus can be further classified into four categories: type 1 diabetes, type 2 diabetes, gestational diabetes, and other specific types [15]. Type 1 diabetes is also termed as insulin-dependent, juvenile-onset, and T-cellmediated diabetes. The main causative factors for its development include inflammation of the pancreas, deficiency of insulin through autoimmune reaction leading to the β-cell destruction produced by the islets of exocrine pancreatic cells, and infiltration of leucocytes, i.e., natural killer cells (NK cells), B-cells, and CD8–Tcells [14, 16]. Type 1 diabetes displays mainly four symptoms, namely, polyphagia, glycosuria, polyuria, and polydipsia. Usually, type 1 diabetes occurs in children below 4 years of age which account for the highest incidental reports. Fifty percent of the patients are approximately 20 years old and present with fasting hyperglycemia that leads to ketoacidosis and weight loss and polyphagia among other conditions (American Diabetes Association 2014). Furthermore, about 80% of patients express elevated levels of antibodies of the islet cell antibodies including autoantibodies to insulin/proinsulin, GAD65 and GAD67 (glutamic acid decarboxylase), tyrosine phosphates IA-2 and IA-2β, and islet cell antigen (ICA)-512 [17]. Type 2 diabetes, also named as adult-onset diabetes and non-insulin-dependent or insulin-resistant diabetes, produces insulin, but the body doesn’t respond to it. Ninety to ninety-five percent of diabetes patients suffer from type 2 diabetes worldwide, particularly adults above 40 years of age. The main causative factors for type 2 diabetes are inflammation, dysregulation of amylin, obesity, hypertension, increased risk of hypoglycemia, and dyslipidemia [18, 19]. Low-grade systemic inflammation and elevated levels of pro-inflammatory cytokines, i.e., IL-6 and tumor necrosis factor-α (TNF-α), which are derived from adiponectin-derived factor, are classical features of type 2 diabetes and insulin

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resistance [20]. However, stimulation of the two signaling pathways, i.e., c-Jun amino N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) pathways, contributes to the development of insulin resistance and type 2 diabetes [21].

1.6

Gestational Diabetes

In pregnancy, various physiological changes occur, such as abnormal glucose levels which lead to imbalances in glucose homeostasis in the form of diabetes. About 10% of all pregnant women develop this condition, known as gestational diabetes, during the second trimester of the pregnancy [22]. Hyperinsulinemia and glucose intolerance are the hallmarks of gestational diabetes. However, enhanced insulin resistance is normally detected during pregnancy to ensure the availability of glucose for the fetus. This is mainly caused by the effects of hormones such as placental prolactin, cortisol, progesterone, placental lactogen, adiponectin, and leptin. These physiological alterations that occur during pregnancy lead to disrupted glucose tolerance, which can in turn result in gestational diabetes. Insulin contributes to various metabolic functions including increased cellular uptake of glucose, amino acids, fatty acids, and potassium ions. It is also involved in anabolic actions and enhances the cellular formation of lipids, glycogen, and proteins. Hence, these key physiological functions can be impaired if insulin action is reduced, [23], especially in the third trimester of pregnancy due to increased insulin resistance leading to decreased carbohydrate tolerance [24]. Additionally, evidence suggests that there might be an interaction between obesity, the activation of NF-κB, TNF-α, and the maternal glucose levels in gestational diabetes [25].

1.7

Pancreatic Cancer

Pancreatic cancer (PC) is the fourth most devastating cancer in the world with less than 5 years survival rate [26]. Although surgery remains the most frequent strategy for treating PC, most of the patients are diagnosed with advanced-stage disease due to the lack of proper diagnostic methods and are thus unfit for surgical treatment. Additionally, pancreatic tumors are very aggressive and develop resistance to targeted chemotherapeutic drugs [27]. Chronic pancreatitis is also one of the risk factors for pancreatic cancer, and almost 5% of chronic pancreatitis cases can develop into pancreatic cancer [28]. Both the exocrine and endocrine pancreas can develop cancer; however, most of the tumors formed by the dysregulation of acinar cells are exocrine in nature. Furthermore, 95% of pancreatic adenocarcinomas originate in epithelial cells [29, 30]. Other pancreatic malignancies include acinar cell carcinoma and mucinous cystadenocarcinoma [31]. Acinar cells that secrete digestive enzymes can differentiate into ductal cells during chronic pancreatitis and are precursors of pancreatic cancer [32, 33] (Figure 1.3). Although pancreatic cancer can result from genetic mutations in the ductal epithelial cells, these mutations, which include KRAS,

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Pancreas

Epithelial cells (Ducts )

Activates Mutations KRAS ,BRCA2,CDKN2

Inactivates Tumor suppressor genesTP53 Abnormal cell growth Proliferation Improper Immune responses Metastasis

Fig. 1.3 Alterations in ductal epithelial cells can alter the gene regulation resulting in uncontrolled cell growth and proliferation. In pancreas, alterations in epithelial cells activate mutations (KRAS, BRCA2, CDKN2) and inactivate the tumor suppressor genes Tp53. Thus, it causes the abnormal cell growth and proliferation followed by improper immune responses associated with the occurrence and development of metastasis of pancreatic cancer

CDKN2A, BRCA2, MLH1, SMAD4, and LKB1 mutations, can activate oncogenes and inactivate tumor suppressor genes such as INK4A, p53, and ARRF. These alterations lead to uncontrolled cell growth caused by the disruption of signaling cascades, such as NF-κB, mitogen-activated protein kinase (MAPK), and PI3K– AKT pathways, that regulate cell survival and growth [31].

1.8

KRAS Mutations

PC is correlated with mutations in key oncogenes and/or tumor suppressor genes. Of these, enhanced KRAS mutations are regularly linked with tumor progression. In fact, 90% of pancreatic patients have a mutation in the exon 12th codon of the KRAS protein [34]. KRAS is activated when it binds with GTP, initiating RAF family kinase activation including RAF1, ARAF, and BRAF. Phosphorylated RAFs trigger the MEK pathway, which regulates cell growth and inhibits apoptosis, leading to pancreatic cancer progression. Furthermore, altered levels of the upstream tyrosine kinase receptors EGFR and VEGFR activate RAS [35], thus stimulating multiple signaling pathways that actively participate in tumor development, including the NF-κB, MAPK, RAL, PLC–PKC, and PI3K–AKT pathways [36, 37]. Clinically, KRAS mutation assays are used as diagnostic tools to guide treatment options for pancreatic cancer, signifying that KRAS mutations could represent prognostic biomarkers for the disease [38]. Oncogenic KRAS is inhibited by 1)

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farnesyltransferase inhibitors (FTIs), which recognize the CAAX motif responsible for the transformational function of RAS downstream, the other signaling pathways [39], or 2) anti-RAS peptide vaccines at the RNA interference level in the posttranscriptional stage [38]. Recent studies have shown that TLN-4601 and salirasib, which constitute farnesyl isoprenoid small molecules, can act as PC antagonists by decreasing RAS–GTP levels and promoting cell death [40, 41].

1.9

P16/INK4/CDKN2A Mutations

Cyclin-dependent kinase (CDKN2A) (also well-known as MTS-1, P16/INK4, and CDK4I) gene is located on chromosome 9p21. Through the G1–S checkpoint, it suppresses the cell cycle development which is also necessary to regulate uncontrolled cell differentiation [42]. The CDKN2A gene is inactivated in pancreatic cancer by numerous factors such as loss of CDKN2A function, due to promoter slicing hypermethylation or homozygous deletion and loss of heterozygosity [43]. Inherited alterations in CDKN2A cause familial atypical different melanomas and greater risk of pancreatic cancer. It is one of the most commonly changed genes in cancer and the most prevalent of mutations in sporadic pancreatic cancer, with inactivation occurring in 98% of cases [43]. Some clinical studies have shown that the loss of INK4 provokes the activation of KRAS in pancreatic tumor development and promotes chemoresistance to pancreatic cancer. Hence, these mutations can act as predictive biomarkers for diagnosing the disease [38].

1.10

BRCA2 Mutations

BRCA2 is also known as FANCD1 [36]. The BRCA2 gene is positioned on chromosome no. 13Q12–13Q13 [44]. It is a multifaceted tumor suppressor and has a vital role in genetic integrity. BRCA2 exerts various functions in cell cyclespecific methods, including G2–M cell cycle checkpoint, chromatin remodeling, ubiquitylation, DNA repair, and transcriptional regulation [45]. Mitotic kinases control various functions of BRCA2 in cytokinesis, mitosis, and cell death. BRCA2 is involved in double-strand repair in the S-phase of the cell cycle, through the regulation of RAD51 recombinase [36, 46]. Inactivated and germline-mutated BRCA2 increases the risk for breast, stomach, ovarian, and pancreatic cancer [47, 48]. Furthermore, among the germline gene mutations that have been connected with greater risk of pancreatic cancer, BRCA2 mutations are the most common and identified in approximately 17% of familial pancreatic cancer (FPC) [49]. However, in the case of sporadic mutations, only a small number of cases habor somatic BRCA2 mutations [36]. Various numerical clinical studies were executed to evaluate the significance of BRCA2 as a prognostic biomarker for early diagnosis of pancreatic cancer. Huang, L. et al., 2013, carried out a study on two-stage association of pancreatic cancer, examining 981 cases and 1991 controls in the first stage followed by 2603 cases and 2877 controls. These studies revealed that the

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c.
532A > G mutagent situated in the 30 UTR is significantly correlated with sporadic pancreatic cancer (odd ratio, 1.30; CI, 95%; P < 0.0001) [50]. For instance, a pancreatic cancer patient carrying a BRCA2 617 del T mutation expresses the prolonged survival after treatment with a combination of docetaxel, gemcitabine, and capecitabine followed by single-agent irinotecan [51]. In another case, a patient with metastatic pancreatic adenocarcinoma responded to mitomycin C combination capecitabine [52], and this increased sensitivity to intercalating agents might be due to BRCA2 mutations [52]. These various cases suggest that BRCA2 mutations could be used as potential prognostic biomarkers of increased treatment sensitivity in pancreatic tumors.

1.11

SMAD4/DPC4 Mutations

The tumor suppressor gene SMAD4/DPC4 is located on chromosome no. 18q21. Fifty-five percent of pancreatic cancers harbor inactivating SMAD4/DPC4 gene mutations, which contribute to tumorigenesis by inhibiting growth suppression mechanisms and enhancing tumor progression through downregulation of the TGF-β signaling pathway [34, 53].

1.12

p53

p53 is a tumor suppressor gene, also termed as antigen NY-CO-13 [36]. It transcriptionally activates its target genes, which leads to cellular stress including DNA damage or oxidative stress and nutrient deficiency through restraining cellular functions increased by apoptosis or cell cycle arrest [54, 55]. Approximately 75% of human pancreatic cancers harbor mutations in the p53 tumor suppressor gene, thus underlining the significant role of p53 mutations or inactivation in pancreatic cancer development [56]. p53 plays key roles in stimulating cellular senescence in response to oxidative stress, hypoxia, ribonucleotide depletion, nutrient starvation, and DNA damage. Furthermore, p53 regulates several cellular functions such as metabolism, differentiation, stem cell function, motility, cell-to-cell communication, invasion, motility, and metastasis in the tumor microenvironment. These functions might be suppressed during tumor progression [57]. Recent studies show that p53 can stimulate classical DNA damage in response to cell senescence. Its target genes, associated with this response, namely, PmaiP1 (Noxa), P21 (CDKN1A), and Bbc3 (Puma), are not involved in the tumor suppressor functions [58]. Evidence from clinical studies suggests that p53 could serve as a potential prognostic biomarker for pancreatic cancer diagnosis and therapy prediction. A study performed in 57 patients with pancreatic cancer found an association between PC and p53 mutations as well as mRNA expression. Interestingly, the study results revealed that the patients with lowest p53 mRNA expression levels were associated with very deprived prognosis (p ¼ 0.032). In contrast, patients with high p53 mRNA expression were associated with significant progression (p ¼ 0.021) [59].

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BRAF Mutations

The Catalogue of Somatic Mutations in Cancer (COSMIC) lists more than 30 different BRAF mutations involved in the development of various tumors including pancreatic tumors [60]. Most of these mutations are point mutations located on exon 15 in (V600E/Val 600/c.1799 T > A) the BRAF gene. The BRAF protooncogene, one of the RAF family proteins, has three isoforms, ARAF, BRAF, and RAF1 (CRAF), which are direct mediators of MAP kinase. Hence, using allosteric mechanisms, BRAF can directly stimulate MEK and extracellular signal-regulated kinase (ERK), which regulate cell growth and proliferation and control apoptosis [61, 62]. Vemurafenib (reversible ATP-competitive inhibitor) is an active inhibitor of BRAF in BRAF-mutated cancers. It is used to treat patients with metastatic melanoma and aggressive growth of a KRAS-mutant pancreatic carcinoma [63]. Therefore, the BRAF V600/E/val assay is used as a prognostic biomarker to detect various cancers, including pancreatic cancer. Ninety percent of pancreatic tumors carrying mutations in KRAS stimulate several downstream signaling pathways, such as RAS–RAF–MEK–ERK (MAPK) and PI3K–AKT–mTOR pathways.

1.14

RAS–RAF–MEK–ERK (MAPK) Signaling Pathway

The RAS–MEK signaling pathways play a major role in cellular activities such as cell division, growth, differentiation, proliferation, progression, survival, and metabolism in response to upstream signals. The dysfunction of intermediates of the MAPK pathway results in diverse effects such as tumorigenesis and the ability to function without mitogenic signals, abnormal cell proliferation, and the inhibition the apoptosis. Hence, alterations and mutations in MAPK signaling promote tumor progression [64]. BRAF is the initiator of the MAPK pathway. The extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) cascade is characterized by sequential molecule activation, initiated by the small GTPase and different protein kinases that phosphorylate MAPKKK then MAPKK, which functions as a gate keeper and phosphorylates/stimulates the effector kinase–mitogen-activated protein kinase (MAPK). To date, the MAPK cascade has been divided in to six groups in mammals, namely, ERK1/2, ERK 3/4, ERK 5, ERK 7/8, JNK 1/2/3 (c-Jun amino N-terminal kinase), and p38 isoforms [65, 66]. The MAPK signaling pathway has various effects which can control cell cycle progression, differentiation, and cell senescence [67]. A review of literature documents the complexity of this pathway, revealing the various kinases, apoptotic regulators, caspase executioner families, and transcription factors, which can be either activated or inactivated through phosphorylation of proteins. Moreover, the MAPK pathway stimulates the transcription of multiple genes. For instance, RAF can stimulate the phosphorylation of protein involved in cell death either by downregulating MEK and ERK or self-regulating MEK and ERK.

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The irregular activation of MAPK signaling can lead to the development of cancers via various mechanisms, especially genetic mutations. These mutations can occur due to the upregulation of membrane receptors and BRAF and RAS genes as well as other genes involved in various pathways, viz., PTEN, PI3K, and Akt, which are essential for regulating RAF activity [68, 69]. RAS has been recognized as an oncogene that is mutationally activated in 25% of all the cancers. It is associated with either by downregulation of MEK and ERK or self-regulation of MEK and ERK and eminent incidence in colon (50%), lung (30%), pancreas (90%), melanoma (25%), and thyroid (50%) cancers [67, 70]. In view of these key functions, the RAS/RAF/MEK/ERK pathway is an essential pathway for remedial interventions. Accordingly, various inhibitors are currently being developed to target the MEK cascade and exert effective antiproliferative activity. For instance, CI-1040 represents the first MEK inhibitor and succeed in in vivo and in vitro clinical phases, although it did not demonstrate potent antitumor activity in the phase II clinical studies [71], indicating that further pharmacokinetic enhancements are required to improve the efficacy of these inhibitors.

1.15

PI3K–AKT–mTOR Signaling Pathway

1.15.1 PI3K Signaling Since PI3K innovation in the 1980s, the family of lipid kinases named phosphatidylinositol 3-kinases (PI3Ks) has gathered wide interest. It has been found to contribute to various cellular processes (cell growth, proliferation, migration, and survival) [72]. PI3K signaling is one of the most commonly dysregulated pathways in cancers [73]. PI3K is activated by G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). This leads to the PI3K autophosphorylation, which results in the activation of the serine/threonine kinase Akt pathway and other downstream effectors [72, 74]. Abnormal activation of this pathway affects various cellular processes, such as metabolism, cell cycle progression, survival, regulation of cell death, genomic instability, and protein synthesis [75]. Various agents have been developed to target the PI3K–Akt pathway, among which LY294002 and wortmannin act as potential inhibitors of the PI3K pathway [75]. The PI3K pathway inhibits cell death and regulates cell differentiation during tumor development. PI3K activates the phosphatidylinositol 3,4,5-triphosphate through the phosphorylation of phosphatidylinositol 4,5-bisphosphate leading to the stimulation of the AKT pathway, which targets various downstream effectors [76]. mTOR is a major target of this pathways. It is crucial for cellular growth and metabolism and is involved in the activation of the initiation factor 4E-binding protein (4EBP1) in eukaryotic translation [77].

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AKT signaling IGFIR PDGFR EGFR VEGFR

Growth Factors

PI3K

PIP2 PIP3 AKT

mTOR

FoXo

4EBP1

Cdk

eIF4E HIF1α

GERS/GAPS

Rac/Rho family Inhibits apoptosis Cell cycle arrest

Survival , Invasion, Proliferation , Metabolism

Cell cycle progression Cyto skeleton motility NADPH oxidation

Fig. 1.4 AKT signaling regulates several downstream substrates that are participated in the various cellular events. Growth factors (IGFIR, PDGFRS, EGFR, and VEGFR) activates the PI3K that phosphorylates and activates the AKT. Thus, activated AKT downregulates the multiple targets (FOXO, IKK, NF-κB) in order to inhibit the apoptosis and increase the cell proliferation and survival. Directly or indirectly, AKT activates the mTOR that promotes the inactivation of HIF-1α which results to cell proliferation and invasion. AKT activates the Rac/Rho family and promotes the cytoskeleton motility and NADPH oxidation

1.15.2 AKT Signaling AKT (protein kinase B) is an essential regulator of human physiology. It plays a significant role in promoting several cellular functions required for cell growth, proliferation, survival, and metabolism [78]. The AKT signaling cascade (Fig. 1.4) leads to the activation of various transcription factors including HIF-1α, NF-κB, and FOXO which play a vital role in cell cycle regulation and the suppression of cell death [79].AKT signaling alterations cause several genetic abnormalities in different types of tumors. These genetic abnormalities underline the key contribution of AKT in the development of pancreatic cancer.

1.15.3 mTOR Signaling Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and one of the downstream targets of the PI3K signaling pathway (Fig. 1.5). It exits as

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Energy

17 Nutrients Amino acids

Oxygen /stress

Growth factors

mTOR

Ribosome biogenesis

4EbP1

S6K1

Regulation of Transcription

Cell Growth Cell proliferation

Fig. 1.5 Functions of the mTOR signaling. Mammalian target of rapamycin (mTOR) is a serine/ threonine protein kinase. Oxygen levels, growth factors, nutrients, and intracellular energy activate the mTOR and thus regulate the anabolic and catabolic processes; downregulate the several targets which include ribosome biogenesis, 4EBP1 (eukaryotic initiation factor 4E-binding protein), and S6K1 (ribosomal S6 protein kinase 1); and result in the regulation of transcription followed by cell growth, proliferation, and cell metabolism

two functional catalytic subunits formed by mTORC1 and mTORC2. mTOR contributes to several growth-related functions including enhanced cell growth, proliferation and survival, rearrangement of actin cytoskeleton, and protein degradation [80]. mTORC1 is composed of mTOR, Gbl, and Raptor, which act as a scaffold protein interlinked to the mTOR kinase, which activates the mTORC1 signaling. Following PI3K/AKT signaling, mTORC1 stimulates anabolic processes and suppresses catabolic reactions in the cell. Several signaling molecules (growth factors, energy, nutrients, and amino acids) activate mTORC1, thus promoting diverse cellular processes, i.e., cell growth, cell differentiation, migration, and cell metabolism. On the other hand, inadequate levels of these molecules can alter mTORC1 function and promote macroautophagy. mTORC2, on the other hand, is composed of mTOR, Gbl, and rapamycin-insensitive companion of mTOR (Rictor) and participates in AKT signaling. It contributes to cell differentiation, and its main function is to regulate the actin cytoskeleton. The stimulation of the mTOR signaling cascade modulates various cellular processes including the translation of several proteins, such as HIF-1α, which activates VEGF expression in angiogenesis, and cyclin D1, which stimulates cell cycle development [81, 82]. mTORC1 is extremely susceptible to rapamycin, while mTORC2 is less sensitive to it. Driscoll et al. [82] revealed that the suppression of mTORC2/PI3K signaling is a possible therapeutic strategy for pancreatic cancer (Fig. 1.6.) The above survival signaling PI3K/AKT/mTOR cascade is fully involved in the regulation of cell proliferation and apoptosis, and more than 50% of cancers are

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Growth factors

PI3K

Gbl

mTORC2 Rictor AKT

TSC Nutrients Gbl

Cell survival Metabolism Proliferaon

mTORC1 Raptor

S6K

4EBP

HIF1α

Survival , Angiogenesis

Ribosome biogenesis

Fig. 1.6 Activities of mTORC1/2: mTOR is the catalytic components of the two subunits named as mTORC1 and mTORC2. mTORC1 is composed of mTOR, Gbl, and Raptor; mTORC2 is composed of mTOR, Gbl, and Rictor. Growth factors activate mTORC2 by activation of PI3K, and activated mTORC2 stimulates the AKT, thus regulating the various cellular functions such as cell growth, proliferation, survival, and metabolism. AKT activates mTORC1 by inhibiting the tuberous sclerosis complex (TSC) which leads to the activation of downstream targets S6K, 4EBP, and HIF-1α and promotes the ribosome biogenesis, cell survival, and angiogenesis

caused by alterations in this pathway, including pancreatic cancer [83]. The overexpression of AKT and its involvement in the cellular plasticity of the pancreas demonstrate how AKT signaling could play a crucial role in the development of pancreatic tumors [84]. Therefore, the PI3K–AKT pathway is crucial to the progression of pancreatic cancer. HIF-1α is a transcription factor and a key regulator of angiogenesis, necessary for the cellular response to low oxygen. The PI3K/AKT/mTOR pathway increases HIF-1α gene expression, thus allowing tumor cells to survive and develop under hypoxic conditions. Therefore, mTOR and HIF-1α could serve as prognostic targets for drug development [85]. Furthermore, Peng T and Dou QP showed in their recent study that the mTOR inhibitor everolimus can enhance the efficacy of gemcitabine chemotherapy in resistant pancreatic tumors. This indicates that mTOR inhibitors with improved efficacies are required to improve antitumor activity and increase the survival rate in pancreatic cancer patients [86]. In a phase II clinical study of patients treated with a combination of capecitabine and everolimus, Kordes S et al. observed moderate antitumor activity and minimal

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toxicity levels, thus signifying that combination therapies could be effective for treating pancreatic cancer [87]. One of the undergoing clinical phase I/II studies is investigating the sorafenib combination everolimus in patients with advanced pancreatic cancer (NCT00981162).

1.16

Endocrine Pancreas

Pancreatic endocrine tumors occur very rarely, with roughly 5 per in 1,000,000/year. Pancreatic endocrine tumors (PETs) are often termed as “islet cell tumors” or “pancreatic neuroendocrine tumors” (PNETs) [88]. PETs originate from the endocrine (hormonal) cells and nervous system within the pancreas [89] and are mostly associated with the lungs and gastrointestinal tract and also found in the pancreas [89, 90]. However, PETs have been shown to decelerate growing and are not as much aggressive as invasive ductal carcinomas of the pancreas. However, when they reach the metastatic stage, they become life-threatening and more difficult to cure [91]. The US Surveillance, Epidemiology, and End Results (SEER) survey from the year 1973–2014 indicates that PETs amount to 3.6% of all neuroendocrine tumors (NETs) [91]. PETs are a very rare subgroup of pancreatic tumors and represent 1–2% of all the pancreatic cancers [92]. The incidence of NETs has significantly improved, and the prevalence of PETs has also drastically increased in each primary tumor site [93]. Furthermore, about 20% of PETs are nonfunctional. Functional tumors might be benign or malignant in nature, with no environmental factors involved in tumor development [88]. Pancreatic NETs are classified as functional NETs and nonfunctional NETs. Functional NETs are associated with clinical symptoms and associated to the type of hormones released including gastrin, insulin, glucagon, somatostatin, and vasoactive intestinal peptide, whereas nonfunctional NETs are not associated with clinical symptoms either because no hormone is secreted or the other substances (subunits of human chorionic gonadotropin, ghrelin, neurotensin, neuron-specific enolase, chromogranins) that are secreted do not display clinical symptoms [91]. Common functional syndromes of this disease are gastrinomas, insulinoma, glucagonomas, VIPomas, and somatostatinoma, caused by the secretion of gastrin, insulin, vasoactive intestinal peptide, glucagon, and somatostatin [94]. PETs are non-inherited (sporadic) and approximately 10–30% of nonfunctional PETs display an endocrine tumor syndrome which includes tuberous fibromatosis (TSC), multiple endocrine neoplasia syndrome type 1(MEN 1), Von Recklinghausen’s syndrome (neurofibromatosis), and von Hippel–Lindau syndrome [95, 96]. The nonfunctional familial syndromes occur due to inherited germline loss and gain of functional mutations in tumor suppressor genes [96].

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Conclusion

The pancreas plays a critical role and various physiological functions and is associated with various deadly diseases (pancreatitis, diabetes, and pancreatic cancer), caused by improper immune responses associated with genetic mutations and lifestyle modifications. Recently, several studies elucidate that alcohol, smoking, and obesity are the main causative risk factors for development of pancreatitis to pancreatic cancer. Predominately, abnormal zymogen activation and genetic mutations particularly abnormal function of trypsinogen lead to acute pancreatitis and chronic pancreatitis. Furthermore, long-standing chronic pancreatitis is the development of diabetes mellitus. Moreover, activation of oncogenes and inactivated tumor suppressor genes contributes to the development and progression of chronic pancreatitis to pancreatic cancer. Among these disorders, pancreatic cancer has a very poor prognosis and survival. The lack of early detection methods reduces the chances of identifying the tumor at an early stage. Therefore, researchers are trying to develop enhanced diagnostic tools to aid in preventing and treating the disease to increase survival outcomes. Recently, the improved understanding of the molecular mechanisms mediating PC development has resulted in the design of enhanced targeted therapies including combination therapies and photochemical therapies that target numerous signaling pathways and prevent tumor reoccurrence.

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Pancreatitis: Clinical Aspects of Inflammatory Phenotypes Nyshadham S. N. Chaitanya and Aramati BM Reddy

Abstract

Pancreatitis is a condition in which the pancreas becomes inflamed and gets damaged when the digestive enzymes are activated before they are released into the small intestine and attacks the pancreas. When these activated digestive enzymes are activated still in the pancreas, irritating the cells of our pancreas causes inflammation. These cause complications such as the formation of the pseudocyst, breathing problem, failure in the kidney, diabetes and pancreatic cancer. Two forms of pancreatitis include acute and chronic. Acute pancreatitis is a condition in which inflammation lasts for short time, while chronic pancreatitis is a condition in which inflammation lasts for a longer time. Gallstones and the gallbladder in the pancreas can be removed by surgical treatment. The probability of developing pancreatitis can be reduced by alcohol cessation and preventing gallstone complication. Risk factors include hereditary, hyperlipidaemia, smok ing, cystic fibrosis and usage of certain medicines such as oestrogen and tetracy cline. The tests to detect pancreatitis include enzymes of the pancreas, liver and kidney. Signs of infection include fever/fatigue, anaemia and decreased electrolyte and calcium level. In acute pancreatitis, the patient’s diet consists of bowel rest, and for chronic pancreatitis, it includes low-fat diet and high carbohydrates. Keywords

Acinar cell injury · Acute pancreatitis · Chronic pancreatitis · Secretin-enhanced MR cholangiography · Fluid therapy

N. S. N. Chaitanya · A. BM Reddy (*) Laboratory of Cell Signalling, Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_2

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Abbreviations AP CP CRP ERCP FAIMS RDW VOC

2.1

Acute pancreatitis Chronic pancreatitis C-reactive protein Endoscopic retrograde cholangiopancreatography Field asymmetric waveform ion mobility spectrometry Red cell distribution width Volatile organic compounds

Introduction

Acute pancreatitis accounts for approximately 56 cases per one-tenth million persons per year in the United Kingdom, while in the United States over 220 thousand are hospitalised per year. Formation of gallstones is the common cause for acute pancreatitis, about 25 per hundred of acute pancreatitis cases were due to alcohol. Gallstones of patients smaller than 5 mm in size are thought to have higher risk of gallstone pancreatitis. Obstruction at sphincter of Oddi leads to injured pancreatic ducts. Acute pancreatitis includes improper release and activation of pancreatic enzymes, and trypsin plays a role in activating these proenzymes. Its inappropriate activation leads to pancreatic inflammation and triggers the release of IL-1, IL-6 and IL-8; TNF-α; and platelet-activating factor [24]. Advancement in magnetic resonance imaging had allowed the identification and characterisation of pancreatic disorders [25]. Chronic pancreatitis leads to pancreatogenic diabetes mellitus which also occurs secondary to pancreatic cancer [10]. The first category study of chronic pancreatitis (CP) in the United States is Prospective Evaluation of Chronic Pancreatitis for Epidemiologic and Translational Studies [27], and to better understand the pathophysiology and mechanisms, biorepositories are needed [8]. Research in pancreatic cancer, chronic pancreatitis (CP) and pancreatogenic diabetes mellitus were impeded due to inability in obtaining pancreatic tissue for study, genetic testing and biomarker development [23]. Genetic testing has been increasing clinical practice for the identification of susceptible genes for diseases [11]. The treatment of chronic pancreatitis includes pancreatectomy with autologous islet cell transplantation which is offered only in selected centres worldwide [22].

2.2

Causes

The factors responsible for chronic pancreatitis include smoking, autoimmune responses, genetics, alcohol, obstructive mechanisms, metabolic abnormalities and idiopathic mechanisms, whereas for acute pancreatitis, it includes autoimmune

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responses, use of alcohol, hypertriglyceridaemia, biliary hypercalcaemia and hereditary/genetic abnormalities.

2.3

Acute Pancreatitis

Amongst gastrointestinal system diseases, one of the major forms is acute pancreatitis (AP) [28]. It is a serious disease possessing a guarded prognosis and aetiology which involves either the bladder or alcohol; it leads to trauma which is extremely rare and is often associated with intra-abdominal lesions [12]. Paediatric pancreatitis is understudied and an emerging field with an increasing incidence of disease and therefore extrapolated from adults [1]. A patient suffering from shortness of breath and acute pulmonary oedema upon admission at third day of hospitalisation was diagnosed with takotsubo cardiomyopathy [15]. Acute myocardial infarction incidence in acute pancreatitis is rare, and recognition and diagnosis of such may be clinically challenging [20]. Autoimmune systemic disease of IgG4-related disease defined as infiltration of IgG4-positive plasma cells in the affected organs leads to tissue inflammation which is rare and involves every organ [5]. Type I autoimmune pancreatitis is an IgG4-related systemic disease which exhibits as a pancreatic disorder, whereas type II is characterised by absence of IgG4-positive cells [3]. Toll like receptors initiate the excessive inflammatory reaction which makes the immune function unbalanced and also damages the function of many organs. Studies show TLR-4 is closely related to the occurrence and development of acute pancreatitis [5]. Acute pancreatitis is a necro-inflammatory disease diagnosed by elevated lipase levels and distinct imaging findings. One of the strategies to prevent recurrent attacks involves providing alcohol cessation counselling to patient [14]. Recent reports on fluid therapy, antibiotics, analgesics and the management of AP have led to improvements in clinical care [7]. Pancreatitis and Wilson’s disease were taken as examples in the study of ER stress in liver and pancreatic diseases [16]. Acute pancreatitis is caused by acinar cell injury, defective intracellular transport and duct obstruction. In acinar cell injury, agents such as alcohol, drugs, trauma and ischaemia cause activation of digestive enzymes through release of intracellular proenzymes and lysosomal hydrolases. In defective intracellular transport and metabolic injury, alcohol intake leads to acinar cell injury through delivery of proenzymes to lysosomal compartment and intracellular activation of enzymes. In duct obstruction and ampullary obstruction, chronic alcoholism, ductal concentration and intestinal oedema lead to impaired blood flow and ischemia and finally to acinar cell injury. This acinar cell injury leads to lesions such as intestinal inflammation, proteolysis, necrosis and haemorrhage (Fig. 2.1).

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Fig. 2.1 Acute pancreatitis is mainly due to acinar cell injury which is caused by alcohol, smoking, metabolic injury and duct obstruction

2.4

Chronic Pancreatitis

Pancreatic inflammation with progressive loss of exocrine and endocrine gland compartments leading to atrophy or replacement with fibrous tissue in the affected area is known as chronic pancreatitis. Consequences of chronic pancreatitis include recurrent abdominal pain or constant abdominal pain, maldigestion and diabetes mellitus [13]. Characteristic features of chronic pancreatitis (CP) include pancreatic inflammation, scarring and fibrosis [17]. Clinical manifestations include pain in the abdominal region and insufficiency in exocrine and endocrine glands [18]. Even though acute pancreatitis and chronic pancreatitis were distinct [26], reports state that acute pancreatitis, recurrent acute pancreatitis and chronic pancreatitis represent a disease continuum [2, 21]. The risk factors include idiopathic, hereditary, autoimmune, severe, acute, pancreatitis-associated, chronic and obstructive pancreatitis [6]. This classification was based on individual’s risk of developing chronic pancreatitis [4] (Fig. 2.2).

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Fig. 2.2 Chronic pancreatitis is caused by factors such as idiopathic, hereditary, autoimmune, severe, acute and obstructive pancreatitis

2.5

Diagnosis

Primary onset of acute pancreatitis was not detected till symptoms appear, while later stages were detected by fibrosis, distortion of the pancreatic ducts, pseudocyst and portal vein thrombosis or splenic vein thrombosis [13]. Patients with a long-standing history of alcohol or tobacco use are at risk for chronic pancreatitis which helps in diagnosis, and management involves reduced usage of alcohol and tobacco, modifications in diet and treatment of pancreatic insufficiency. Pancreatic neoplasms of acute, chronic and autoimmune pancreatitis were detected using diffusionweighted imaging. Ductal anomalies and characterisation of pancreatic cystic lesions were evaluated in acute and chronic pancreatitis using secretin-enhanced MR cholangiography [25]. Volatile organic compounds (VOC) were detected using field asymmetric waveform ion mobility spectrometry (FAIMS) by comparing healthy controls with pancreatic cancer from a urine sample [19]. RDW was not specific, but the CRP/albumin ratio is an inexpensive and reliable marker in prognosis of AP [28]. Amongst coronary imaging techniques, optimal cohesion tomography helps us

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to observe coronary thrombus, inflammatory processes and presence of acute myocardial infarction. Intravascular imaging delineates details of intracoronary pathology which cannot be clearly predicted on standard fluoroscopy [20].

2.6

Treatment

Treatment includes medical, radiological, endoscopic and surgical [13]. Fluid therapy is used for acute pancreatitis. Endotherapy serves to achieve symptom remission and also reduces rates of recurrence, abdominal pain, hospitalisation and surgical arbitration [9].

2.7

Conclusion

Management of acute pancreatitis by fluid therapy yields better outcome. Acute pancreatitis complications can be prevented with limited usage of antibiotics as prophylaxis which shows beneficial effects. Severe acute pancreatitis benefits from ERCP, while mild patients are recommended to undergo single-stage laparoscopic cholecystectomy as they get benefit from single-stage and bile duct exploration which reduces length of hospitalisation and need for recurrent admissions. Acute pancreatitis is intent by alcohol, smoking and duct obstruction which proceeds to chronic pancreatitis which is caused by alcohol, obstruction of the pancreatic duct and hyperlipidemia. Progressive understanding of this disease had enhanced and changed the approach to management. Management of chronic pancreatitis involves counselling the patient regarding alcohol and tobacco and addressing the problems of malnutrition and osteoporosis. Acknowledgements NSNC acknowledges the ICMR for SRF fellowship and Laboratory of Cell Signalling supported partially by the University of Hyderabad through institutional funding from DST-FIST-II and PURSE, and ABMR acknowledges funding from DBT-RNAi, DAE-BRNS and DST-SERB, Government of India, through extramural research grants.

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6. Etemad B, Whitcomb DC (2001) Chronic pancreatitis: diagnosis, classification, and new genetic developments. Gastroenterology 120(3):682–707 7. Faghih M et al (2019) New advances in the treatment of acute pancreatitis. Curr Treat Options Gastroenterol 17(1):146–160 8. Fisher WE et al (2018) Standard operating procedures for biospecimen collection, processing, and storage: from the consortium for the study of chronic pancreatitis, diabetes, and pancreatic Cancer. Pancreas 47(10):1213–1221 9. Guo A, Poneros JM (2018) The role of Endotherapy in recurrent acute pancreatitis. Gastrointest Endosc Clin N Am 28(4):455–476 10. Hart PA et al (2018) Evaluation of a mixed meal test for diagnosis and characterization of PancrEaTogEniC DiabeTes secondary to pancreatic Cancer and chronic pancreatitis: rationale and methodology for the DETECT study from the consortium for the study of chronic pancreatitis, diabetes, and pancreatic Cancer. Pancreas 47(10):1239–1243 11. Hohmann M et al (2018) Practical genetic testing in gastroenterology. Dtsch Med Wochenschr 143(20):1477–1480 12. Khaoula Y et al (2018) Blunt abdominal trauma causing acute pancreatitis: presentation of the case study. Pan Afr Med J 30:126 13. Kleeff J et al (2017) Chronic pancreatitis. Nat Rev Dis Primers 3:17060 14. Klochkov A, Sun Y (2019) Alcoholic Pancreatitis. StatPearls. Treasure Island (FL) 15. Koop AH et al (2018) Acute pancreatitis-induced takotsubo cardiomyopathy and cardiogenic shock treated with a percutaneous left ventricular assist device. BMJ Case Rep 2018 16. Lukas J et al (2019) Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas. Adv Med Sci 64(2):315–323 17. Majumder S, Chari ST (2016) Chronic pancreatitis. Lancet 387(10031):1957–1966 18. Muniraj T et al (2014) Chronic pancreatitis, a comprehensive review and update. Part I: epidemiology, etiology, risk factors, genetics, pathophysiology, and clinical features. Dis Mon 60(12):530–550 19. Nissinen SI et al (2019) Detection of pancreatic Cancer by urine volatile organic compound analysis. Anticancer Res 39(1):73–79 20. Sanghvi S et al (2019) Coronary thrombosis in acute pancreatitis. J Thromb Thrombolysis 47 (1):157–161 21. Sankaran SJ et al (2015) Frequency of progression from acute to chronic pancreatitis and risk factors: a meta-analysis. Gastroenterology 149(6):1490–1500 e1491 22. Schrope B (2018) Total pancreatectomy with autologous islet cell transplantation. Gastrointest Endosc Clin N Am 28(4):605–618 23. Serrano J et al (2018) Consortium for the study of chronic pancreatitis, diabetes, and pancreatic Cancer: from concept to reality. Pancreas 47(10):1208–1212 24. Shah AP et al (2018) Acute pancreatitis: current perspectives on diagnosis and management. J Inflamm Res 11:77–85 25. Siddiqui N et al (2018) Advanced MR imaging techniques for pancreas imaging. Magn Reson Imaging Clin N Am 26(3):323–344 26. Singer MV et al (1985) 2d symposium on the classification of pancreatitis. Marseilles, 28–30 March 1984. Acta Gastroenterol Belg 48(6):579–582 27. Yadav D et al (2018) PROspective evaluation of chronic pancreatitis for EpidEmiologic and translational StuDies: rationale and study design for PROCEED from the consortium for the study of chronic pancreatitis, diabetes, and pancreatic Cancer. Pancreas 47(10):1229–1238 28. Yilmaz EM, Kandemir A (2018) Significance of red blood cell distribution width and C-reactive protein/albumin levels in predicting prognosis of acute pancreatitis. Ulus Travma Acil Cerrahi Derg 24(6):528–531

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Diabetes and Pancreatic Cancer: A Bidirectional Relationship Perspective Manoj Kumar Gupta, Vemula Sarojamma, and Ramakrishna Vadde

Abstract

An attempt was made to understand the bidirectional causal relationship between diabetes mellitus and pancreatic cancer. One theory claims that diabetes mellitus is a risk factor for developing pancreatic cancer. Supporting this, several studies have reported that four type 2 diabetes-associated polymorphisms, namely, rs8050136 of FTO, rs1387153 of MTNR1B, rs11039149 of MADD and rs780094 of GCKR, are associated risk factors for pancreatic cancer. Enhanced concentration of circulating C-peptide/insulin during hyperglycaemia is also reported to be the risk factors for developing both colorectal and pancreatic cancer. However, another theory claims that pancreatic cancer is risk factor for developing diabetes mellitus. Insulin sensitivity in pancreatic cancer patients is reported to increase after tumour resection supporting pancreatic cancer as a causal agent of DM. Incidence of type 2 diabetes (T2DM) leading to pancreatic cancer is more in comparison to type 1 diabetes. Successful treatment of obesity and T2DM decreases pancreatic cancer risk. Nevertheless, treatment of T2DM with insulin, insulin analogues, and insulin secretagogues enhances the risk for pancreatic cancer. Metformin, an antidiabetic and antineoplastic drug, is reported to reduce the risk of pancreatic cancer and should be considered as first-line therapy in all new-onset diabetes patients. Apart from metformin, antimalarial drug, namely, chloroquine, and other nutritional supplements, like limonene and melatonin, are also utilised in pancreatic cancer prevention and treatment. In near future, this information may be utilised in reversing the increasing incidence of pancreatic cancer. M. K. Gupta · R. Vadde (*) Department of Biotechnology & Bioinformatics, Yogi Vemana University, Kadapa, Andhra Pradesh, India e-mail: [email protected] V. Sarojamma Department of Microbiology, Sri Venkateswara Medical College, Tirupathi, Andhra Pradesh, India # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_3

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Keyword

Diabetes · Pancreatic cancer · Metformin · Insulin resistance · Hyperglycaemia

3.1

Introduction

Diabetes mellitus (DM) is a complex metabolic disorder clinically described by high blood glucose concentration because of reduced insulin production, insulin resistance, or both [89]. Though more than 50 forms of DM identified by the American Diabetes Association, DM is categorised mainly into two types, namely, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) [8]. T1DM is an autoimmune destruction of pancreatic β-cells. Unlike T1DM, T2DM has incapability of pancreatic β-cells to produce sufficient insulin required by the body. T2DM is a heterogeneous disorder caused by both genetic and environmental factors including oxidative stress [89, 107, 108]. T2DM constitutes about 90–95% of all forms of DM in human. In general population, incidence of T2DM increases with age [89]. Earlier studies have reported that chronic hyperglycaemia lead to various forms of cancer, for instance, urinary tract, liver and kidney cancer [2, 56, 145]. However, incidence of T2DM leading to cancer is more in comparison to T1DM [142]. Indeed, only few studies have reported association between T1DM and cancer [47]. Studies have also reported that women with DM are more prone to breast cancer in comparison to the women without DM [16, 76]. Relationship between DM and some cancer may be because they share same risk factor; for instance, obesity and aging are causing agent for both DM and pancreatic cancer [47]. Growing BMI increases the risk ratio for cancer. Obese patients having BMI greater than 30 kg/m2 are more prone to cancers in comparison to overweight (BMI between 25 kg/m2 and 30 kg/m2) patients [19]. Apart from insulin resistance, obesity also enhances oestrogen production, which in turn may increase the risk of oestrogen-dependent tumours. Weight gain is often associated with high risk for breast, cervix and endometrium cancer in women [37, 75, 131]. Recently, our team reported four T2D genes, namely, TCF7L2, CCL2, ELMO1 and VEGFA, along with FOS which plays an important role in causing T2D and its associated disorders, like rheumatoid arthritis, nephropathy, neuropathy and cancer via p53 or Wnt signalling pathways [54]. Thus, both DM and cancer are complex processes, and normal cell must undergo several transformations before turning into cancerous cell. For example, activation of KRAS and suppression of CDKN2A and SMAD4 initiate pancreatic cancer progression [34, 109]. Most plausible molecular functions by which DM influences neoplastic process are chronic inflammation, hyperglycaemia and hyperinsulinaemia [47]. A study reported that diabetes-associated diseases like steatosis, cirrhosis and nonalcoholic fatty liver disease are responsible for inducing liver cancer [47]. About 80% of pancreatic cancer is reported to occur because of glucose intolerance [79]. Incidence of DM amongst pancreatic cancer patients is high in comparison to other cancers [119]. About 50–80% of pancreatic cancer patients are reported

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to be diagnosed with DM at least 3 years prior to pancreatic cancer diagnosis [98, 100, 135]. A distinct segment on pancreatic cancer in the American Cancer Society’s ‘Cancer Facts & Figures 2013’ [123] suggests, ‘About 25% of patients with pancreatic cancer have diabetes mellitus at diagnosis, and roughly another 40% have pre-diabetes’, that ‘patients with long-term (five or more years) type II diabetes have a 50% increased risk of pancreatic cancer’ and ‘pancreatic cancer can cause diabetes, and sometimes diabetes is an early sign of the tumour’ proposing bidirectional relationship between DM and pancreatic cancer [111]. Overall 5 year survival rate of pancreatic cancer patients is 7–8% and is reported to be the fourth leading cause of cancer-related deaths [67, 106, 121]. As the causal mechanism of this bidirectional relationship between DM and pancreatic cancer is still a topic of debate, in this chapter, authors discussed about the bidirectional relationship between DM and pancreatic cancer. In the beginning, we have discussed about possible pathways, namely, insulin resistance, hyperglycaemia and chronic inflammation, by which both are interlinked. Later, we have discussed about various evidences supporting bidirectional relationship between them. Finally, we have discussed about various drugs or nutrient supplement that can be utilised in the treatment of pancreatic cancer. In near future, these information may be utilised in treatment of both DM and pancreatic cancer.

3.2

Plausible Link Between DM and Cancer

Insulin resistance, hyperglycaemia and chronic inflammation are three important mechanisms that interlink DM with cancer [138]. During insulin resistance, which is a common feature of T2DM, insulin level in circulating blood frequently increases, which in turn causes hyperinsulinaemia [138]. Hyperinsulinaemia may initiate development of cancer either by binding insulin to insulin receptor (IR) or increasing the concentration of circulating insulin growth factor (IGF)-1 [112]. Insulin signal transduction is regulated via isoforms of IR, namely, IR-A and IR-B. IR-A has affinity for both insulin and insulin growth factors (IGFs) and is expressed mainly in cancer cells and foetal tissues [138]. IR-B is insulin specific and is mainly associated with glucose homeostasis. When insulin ligates with IR-A, it initiates pro-growth mitogenic effect which in turn initiates carcinogenesis. Hyperinsulinaemia also enhances the expression of IGF-1 in the liver, further activating IGF-1 receptor, which in turn stimulates cell proliferation causing cancer [33, 94]. Hyperglycaemia may induce cancer either by ‘direct effect’ or ‘indirect effect’ [138]. During ‘direct effect’, hyperglycaemia directly affects tumour cells by inducing mutations, enhancing invasion as well as migration and further renewing cancerrelated signalling pathways. During ‘indirect effect’, initial action takes place at another organs/tissue, which will later influence tumour cells via stimulating production of circulating growth factors (insulin/IGF-1) as well as inflammatory cytokines [138]. Hyperglycaemia is reported to enhance Wnt/β-catenin signalling, which is a key cancer-associated pathway, by permitting nuclear retention as well as aggregation of transcriptionally active β-catenin [6, 26].

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Chronic inflammation and chronic oxidation are reported to work together. Hyperglycaemia produced reactive oxygen species (ROS) which damages protein, lipids and DNA, which in turn leads to cancer [138]. ROS-induced chronic inflammation enhances TNF-α concentration, which in turn stimulates nuclear factorkappa B (NF-κB), followed by development as well as progression of numerous tumours [31, 60, 69]. Thus, continuous exposure to oxidative stress as well as chronic inflammation transforms susceptible cells to cancerous cell [133].

3.3

Risk Factors for DM

As stated above, T2DM is a complex disorder that develops due to interaction between strong hereditary component and environmental factors. Though the environmental factors play significant role in the development of diabetes, their impact varies in each individual [5]. Even under the same environmental conditions, some people are more prone to T2DM, while some are not, suggesting hereditary factor plays important role in the development of T2DM. The advancement of genetic technologies had enabled researcher to identify various T2DM loci via linkage studies or candidate gene approach [5]. Linkage studies identified two important T2DM genes, namely, calpain-10 gene (CAPN10) [58, 61] and TCF7L2 [35, 51]. Few important T2DM genes identified via candidate gene approach are insulin receptor substrate 1 (IRS1) and IRS2, peroxisome proliferator-activated receptor gamma (PPARG), potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11), HNF1 homeobox B (HNF1B), HNF4A and Wolfram syndrome 1 (wolframin) [5]. Recent genome-wide association study (GWAS) in Asian population by Hu and team reported that SNPs in PPARG (rs1801282), IGF2BP2 (rs7651090), KCNJ11 (rs5219), CDKN2A-CDKN2B (rs564398 and rs10811161), CDKAL1 (rs10946398, rs7754840, rs9460546, rs7756992 and rs9465871), SLC30A8 (rs13266634) and IDE-KIF11-HHEX (rs10509645, rs1111875 and rs10748582) are risk factor for T2DM [146]. Another GWAS analysis reported rs5219 polymorphism of the KCNJ11 gene is a risk factor for developing T2DM in Caucasians [116], European and East Asia, but not in Mongolian [1, 24, 95], Indian [23, 117] or Ashkenasi Jewish [92]. The KCNJ11 gene, a member of the potassium channel gene family, is situated at 11p15.1. Potassium channels are found in most mammalian cells and are associated with many physiological activities including apoptosis, immunomodulation and insulin secretion. KCNJ11 encodes inward-rectifier potassium ion channel (Kir6.2). Kir6.2 binds with sulfonylurea receptor 1 (SUR1) to form KATP channel, which regulates production and secretion of insulin via glucose metabolism. Mutation in KCNJ11 (rs5219) gene disrupts the normal function of KATP channel and thus production and secretion of insulin which consequently causes T2DM in human [49, 129, 130]. Earlier studies have reported that pregnant women having T allele are at higher risk of developing gestational diabetes mellitus (GDM) ([87, 78]. Thus, the rs5219 (p.Lys23Glu) variation in the 11p15.1 region plays vital role in causing T2DM, thus making it a popular marker for controlling T2DM.

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GWAS analysis in Asian [25, 27, 115] and European [27] human populations reported that rs1535500 polymorphism of the KCNK16 gene is a risk factor for developing T2DM. KCNK16 encodes the potassium two-pore domain channel subfamily K member 16 (alias TALK-1) [91] and is situated at 6p21.2. These channels are mainly expressed in human and mouse pancreatic β-cells and generate outwardly rectifying, non-inactivating K+ flows, which increases with increase in extracellular pH [48]. This polymorphism increases TALK-1 channel activity, disturbing normal membrane potential of β-cell, Ca2+ influx and insulin secretion which ultimately leads to T2DM [132]. Recently, through computational approach, we have reported that polymorphism rs13266634 in ZnT8 transporter disrupts the normal movement and accumulation of zinc into intracellular vesicles from cytoplasm, which in turn have negative impact on the production, storage and secretion of insulin by β-cells of the pancreas and thus causes T2D in human [55]. Reactive oxygen species, like singlet oxygen, are also reported to increase the activity of TALK-1 channel [36, 48, 57, 68]. Vierra and team suggested that inhibition of β-cell TALK-1 channels may be a novel therapeutic approach for reducing hyperglycaemia in T2DM [132]. Recently, Someya and team reported that Japanese college students having BMI > 22.0 kg/m2 are more prone to diabetes later in life [124]. Adela and team via nonparametric-based machine learning approach identified numerous cellular network proteins, like IL1R2, PTPN1, IRS2, AKT1, INSR, LEPR, IRS1, IL6R, MYD88 and PCSK9 which are associated with insulin resistance, atherosclerosis and inflammation [3]. Epicardial fat volume in early-onset T2DM is signature of reduced renal function in future [110]. Zhang and team reported that obstructive sleep apnoea disorder has negative influence on glucose metabolism and causes T2DM [144].

3.4

Risk Factors for Pancreatic Cancer

Carcinogens are the most important factors for inducing pancreatic cancer. Smoking tobacco accounts for about 20 to 30% of pancreatic cancer [82]. Exposure to environmental tobacco smoke in childhood days enhances the risk of pancreatic cancer [134]. Analysis of Nurses’ Health Study data proposes that in utero exposure to smoking is also a plausible cause for pancreatic cancer [9]. Exposure to chlorinated hydrocarbon as well as organochloride compounds also enhances the risk of pancreatic cancer [97, 103]. Infection via Helicobacter pylori [105] and hepatitis B [59] and tropical pancreatitis, chronic pancreatitis and hereditary pancreatitis are also reported to be risk factors for pancreatic cancer [71, 83]. People belonging to older age and with Ashkenazi Jewish and African-American ancestry are more prone to pancreatic cancer [82]. Incidence is lowest amongst African, Southeast Asia and Indian populations [67]. Familial history also plays significant role in the development of 10–20% of pancreatic carcinomas [82]. Mutation in BRCA2 [17], PALB2 [17], p16 [50], STK11/ LKB [46], PRSS1 [83], SPINK1 [83], IGF-1 [127] and ATM [113] also reported to be associated with pancreatic cancer. Recently, one study observed decreased levels of

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HbA1c in benign pancreatic cancer patients in comparison with malignant patients, and recommending HbA1c may also serve as a biomarker for pancreatic cancer [38]. Chronic pancreatitis is also reported to increase the risk of pancreatic cancer [67]. DM patients are also reported to have 30% risk of developing pancreatic cancer [67]. Association between DM and pancreatic cancer has been known for almost 200 years. In 1833, Bright reported that DM patients died of pancreatic cancer within 6 month [18]. In late 1950s, Green and team reported that almost 50% of pancreatic cancer patients at the Mayo clinic have prior history of glycosuria or DM [52]. Positive relationship between pancreatic cancer and specifically high body mass index (BMI), obesity and centralised fat distribution has also been reported. Insulin secretagogues, for instance, sulfonylurea that stimulated β-cells to release insulin, are also reported to induce various cancers in T2DM patients [29, 86]. Compensatory hyperinsulinaemia, insulin resistance and high levels of circulating insulin-like growth factors (IGF) are the most important factors underlying in the association between T2DM and pancreatic cancer [79]. Animal studies have reported that initiation of pancreatic cancer is dependent on the turnover of islet cells. For instance, in hamsters, initiation of islet cell proliferation increases carcinogenesis of pancreatic duct [38]. Though earlier studies have reported that 50% of pancreatic cancer is reported to have prior DM history, it still remains unclear if early onset of pancreatic cancer initiates DM or DM is a risk factor for pancreatic cancer.

3.5

Evidences Proposing DM Is a Risk Factor for Pancreatic Cancer

Everhat and team reanalysed 20 epidemiology cases that have studied relation between DM and pancreatic cancer via meta-analysis and reported that pancreatic cancer may develop due to complication in DM [39]. Another meta-analysis of studies of breast, colon and rectum, endometrium and pancreatic cancers suggested that increased concentration of circulating C-peptide/insulin and other markers of hyperglycaemia is the risk factors for developing colorectal and pancreatic cancer [102]. One population-based study in the United States with 2153 controls and 526 pancreatic cancer cases reveals significant positive trend in risk (P ¼ 0.016) with increasing years before diagnosis of cancer [122]. In T2DM patients, as pancreas is exposed to substantial hyperinsulinaemia for several years, researchers believe that chronic hyperinsulinaemia may stimulate the growth of pancreatic cancer [45]. Binding studies have observed insulin receptors on pancreatic cancer cell [40, 42, 43]. In vitro studies have reported insulin stimulates growth of hamster [43], rat [88] and human [12, 32, 74, 128, 136] pancreatic cancer cell lines. Apart from hyperinsulinaemia, increased concentration of free fatty acids is also reported to initiate pancreatic cancer [41]. Numerous GWAS have reported various genetic variants that are responsible for causing both DM and pancreatic cancer. Pierce and team have studied T2DM 37 risk alleles and reported that three polymorphisms, namely, rs8050136 of FTO, rs1387153 of MTNR1B and rs11039149 of MADD, are

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associated with high risk of pancreatic cancer [101]. Prizment and team reported that rs780094 polymorphisms of glucokinase regulator (GCKR), which is responsible for causing T2DM, may also be a risk factor for pancreatic cancer [104]. Steven and team reported that risk for T1DM and ‘young-onset’ DM patients to develop pancreatic cancer are two-time higher than non-DM individual [125]. Another form of DM, namely, pancreatogenic, or type 3c diabetes (T3cDM), constitutes 5–10% of Western DM populations and is responsible for causing several numerous other diseases. T3cDM is characterised by hepatic insulin resistance and is caused by deficits of both pancreatic polypeptide and insulin. Chronic pancreatitis, which is one of the most important risk factors of pancreatic cancer, is responsible for causing 70% T3cDM [28]. T3cDM is also responsible for causing development of metabolic bone disease and nutritional deficiencies [28]. However, contrasting results proposing DM is not a risk factor for pancreatic cancer have also been proposed by several researchers [20, 44, 53]. Human pancreatic cancer cell line SOJ-6 [128] and PANC-1 [12] are reported not to be affected via insulin.

3.6

Evidences Proposing Pancreatic Cancer Is a Risk Factor for DM

Most of the pancreatic cancer-related DM is diagnosed either simultaneously with pancreatic cancer or 2 years prior to diagnosis of pancreatic cancer suggesting recent onset of DM may be an early sign of pancreatic cancer [53, 118]. Numerous studies have reported peripheral insulin resistance in pancreatic cancer patients with DM [100, 118]. Though few, insulin resistance is also reported in non-DM pancreatic cancer patients with lesser degree [99]. Permert and team reported that insulin sensitivity in pancreatic cancer patients increases after tumour resection supporting pancreatic cancer as a causal agent of DM [99]. Basso and team identified 2030 MW diabetogenic peptide in serum of pancreatic cancer patients [11]. A number of investigators have studied insulin resistance at the organ, tissue and cellular levels in pancreatic cancer [10, 63, 80, 81]. In human skeletal muscles, during initial steps of the insulin signalling cascade, no significant difference amongst healthy controls and pancreatic cancer patients were reported in insulin receptor binding, tyrosine kinase activity and insulin receptor substrate-1 content [81]. However, downstream steps of the insulin signalling cascade, namely, phosphatidylinositol 3-kinase (PI3-K) activity and glucose transport, were reported to be impaired in pancreatic cancer patients [63]. Moreover, glycogen synthase activity is also reported to be reduced in skeletal muscle of both pancreatic cancer human and rodent suggesting insulin signalling cascade gets impaired at various stages of pancreatic cancer [80, 81, 99]. Islet dysfunction is another interesting feature of DM related with pancreatic cancer [137]. As the amount of islet tissues depleted via tumour is very small, islet dysfunction is less likely to decrease islet volume. Indeed, endocrine pancreatic function can be modulated even with large depletion of pancreatic tissue

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[15]. Reduced insulin secretion in pancreatic cancer patient is due to reduce response to stimuli [21, 118]. Chemically induced pancreatic cancer in hamsters is also reported to impair release of normal glucose-stimulated insulin [4]. Ishikawa and team observed enhanced level of proinsulin, in comparison to insulin, in patients with pancreatic cancer patients, proposing that proinsulin maturation may also be affected by the tumour [64].

3.7

Drugs Utilized in the Treatment of Pancreatic Cancer

Thrombosis is a common problem in pancreatic cancer, and chronic pancreatic cancer causes venous thromboembolism [120, 139]. Anticoagulant treatment via aspirin is reported to improvise survival rate of pancreatic cancer patient via reducing thromboembolic complexity [84, 90, 114]. Two independent meta-analyses reported that metformin consumption could reduce the incident of pancreatic cancer by 46% [38, 143]. Metformin reduces blood glucose levels mainly via supressing hepatic glucose production, specifically hepatic gluconeogenesis, and increasing peripheral tissue insulin sensitivity [72]. Metformin is also reported to have a cardioprotective property, which in turn modulates fat metabolism as well as adipose tissue hormone production, mainly leptin [62]. Treatment of T2DM patients with metformin improvises endothelial functions, including soluble vascular cell adhesion molecule-1, tissue-type plasminogen activator and inhibitor, soluble E-selectin and vascular endothelial growth factor [30, 65, 85]. Majority of these molecules play significant role in angiogenesis, fibrosis and thrombosis. However, the important molecular mechanism of metformin’s effect on metabolism as well as growth inhibition is modulated via liver kinase B1 (LKB1)/50 AMPK (AMP-activated protein kinase) signalling pathway. In dormant cell, majority of the energy (in ATP form) is generated in mitochondria through oxidative phosphorylation. Metformin is reported to enhance glucose uptake as well as glycolysis and decreases mitochondrial ATP production, which in turn activates LKB1/AMPK signalling pathway further restricting the growth of pancreatic cancerous cell. Depletion of islet cells through streptozotocin, biguanide metformin and alloxan treatment restricts the induction of pancreatic cancer [13, 22, 96]. Chloroquine, antimalarial drug, is also reported to restrict the growth of pancreatic tumours via inhibiting ‘autophagy’ [140]. However, precaution must be taken with alcoholic pancreatic cancer patients. Nutritional supplements, like limonene [70]; melatonin [77]; vitamins A, C and E [14]; selenium [7]; vitamin K [93], vitamin B6 [126] and green tea [66] and some synthetic drugs, like polysaccharide K [73] and Ukrain (NSC-631570) [141], are also utilised in pancreatic cancer prevention and treatment. Hence, metformin, chloroquine and other nutritional supplements, like limonene and melatonin, may be utilised in pancreatic cancer prevention and treatment.

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43

Conclusion and Perspective

Earlier studies reported link between chronic DM and pancreatic cancer. However, exact mechanism linking DM and pancreatic cancer is a topic of debate. Some studies hypothesize that DM is a risk factor for pancreatic cancer, while other proposed pancreatic cancer is a risk factor for DM. Thus, in this review, an attempt was made to understand the bidirectional relationship between DM and pancreatic cancer. Insulin resistance, hyperglycaemia and chronic inflammation are three important factors that interlink DM with cancer. However, T2DM patients are more prone to pancreatic than T1DM patients. Successful treatment of obesity and T2DM and obesity decreases pancreatic cancer risk. Nevertheless, treatment of T2DM through with insulin analogues, and insulin secretagogues enhances the risk for pancreatic cancer. Metformin, an antidiabetic and antineoplastic drug, is reported to reduce the risk of pancreatic cancer and should be considered as first-line therapy in all new-onset DM patients over the age of 50. Apart from metformin, antimalarial drug, namely, chloroquine, and nutritional supplements, like limonene [70] and melatonin [77], are also utilised in pancreatic cancer prevention and treatment. In near future, these information may be utilised in treatment of both DM and pancreatic cancer as well as reversing the increasing incidence of pancreatic cancer. Besides, it seems that many aspects of molecular mechanism modulating bidirectional relationship between DM and pancreatic cancer are still unknown. Identification of significant genes, molecular pathways and environmental factors, through computational as well as in vivo/in vitro studies, may provide us with an opportunity to reduce cancer at higher speed. Conflicts of Interest Authors declare no conflicts of interest.

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Metabolic Adaptations in Diabetes Mellitus and Cancer Anil Kumar Pasupulati, Nageswara Rao Dunna, and Srikanth Talluri

Abstract

A web of interconnected and flexible metabolic pathways helps in maintaining cellular homeostasis. These flexible metabolic pathways are further deregulated to ensure cell survival during clinical conditions such as diabetes and cancer. In fact, complex metabolic programming is a hallmark of both diabetes and cancer. Normally, glucose absorbed by cells is catabolized through glycolysis to form pyruvate. Pyruvate fuels the citric acid cycle in the mitochondria of aerobic cells. Diabetic condition is presented with a battery of glycolytic abnormalities. Glucose-6-phosphate derived from glucose is converted back to glucose and pumped out of the cell. Similarly, pyruvate is also converted back to glucose. Import of glucose via GLUT4 is also impaired during diabetes. In spite of decelerated glycolysis, gluconeogenesis also occurs in diabetes. In diabetic settings, cells adapt to using acetyl-CoA derived from fatty acids to fuel citric acid cycle. Cancer cells also present with several metabolic abnormalities where glucose-6phosphate powers pentose phosphate pathway and contribute ribose required for the rapid proliferation of transformed cells. Cancer cells convert pyruvate to lactate, which is transported out of cells. Glutamine provides the majority of anaplerotic carbon for the citric acid cycle, and acetyl-CoA derived from citrate contributes to the synthesis of fatty acids. Both 3-phosphoglycerate and pyruvate are spared for the synthesis of amino acids. In the case of diabetes, cells uptake A. K. Pasupulati (*) Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India e-mail: [email protected] N. R. Dunna Cancer Genomics Lab, Department of Biotechnology, SASTRA Deemed University, Thanjavur, India S. Talluri Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_4

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very less glucose compared with normal cells, whereas in case of cancer, cells consume excess glucose due to aerobic glycolysis. In this chapter, we will discuss the major components of the metabolic deregulations in diabetes and cancer. Keywords

Diabetes · Cancer · Metabolism · Randle cycle · Warburg’s effect · Glutamine metabolism

Abbreviations ACC ACLY ACS ACTH AGEs AKT AMPK BRAF CCK CML c-Myc CREBP DAG ETC FAS G6P G6PDH GLP1 GLS1 GLUT4 GOLD GPAT GSH GSSG HIF1α IDH IDO IGFBP3 IKKb IRS LDHA MOLD mTORC NF-kB

Acetyl-CoA carboxylase ATP citrate lyase Acetyl-CoA synthetase Adrenocorticotropic hormone Advanced glycation end products Serine-threonine protein kinase AMP-dependent protein kinase B-Raf proto-oncogene Cholecystokinin Carboxymethyl-lysine Cellular myelocytomatosis cAMP-response element-binding protein Diacylglycerol Electron transport chain Fatty acid synthase Glucose-6-phosphate Glucose-6-phosphate dehydrogenase Glucagon-like peptide-1 Glutaminase-1/L-glutaminase Glucose transporter type 4 Glyoxal-lysine dimer Glycerol-3-phosphate acyltransferase Glutathione Glutathione disulfide Hypoxia-inducible factor 1α Isocitrate dehydrogenase Indoleamine 2,3-dioxygenase Insulin-like growth factor-binding protein Inhibitory kB kinase b Insulin receptor substrate Lactate dehydrogenase A Methylglyoxal-lysine dimer Mammalian target of rapamycin complex 1 Nuclear factor-kB

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Metabolic Adaptations in Diabetes Mellitus and Cancer

α-KG PC PDH PEPCK PFK PHGDH PTEN SCD SHMT1 SREBP1 T1D and T2D TDO TIGAR TPH1

4.1

55

α-ketoglutarate Pyruvate carboxylase Pyruvate dehydrogenase Phosphoenolpyruvate carboxykinase Phosphofructokinase Phosphoglycerate dehydrogenase Phosphatase and tensin homolog Stearoyl-CoA desaturase Serine hydroxymethyltransferase Sterol response element-binding protein Type 1 diabetes and type 2 diabetes Tryptophan 2,3-dioxygenase TP53-induced glycolysis and apoptosis regulator Tryptophan hydroxylase 1

Introduction

Metabolism, a central theme in biological chemistry, allows cells and organisms survive by providing metabolites and energy they require for maintenance, growth, and propagation. In the first stage of metabolism, all foods are fragmented into their monomers, mainly glucose, amino acids, and fatty acids, which are further subjected to complete breakdown into water and carbon dioxide via pyruvate and acetyl-CoA. Both pyruvate and acetyl-CoA serve as crucial hubs of intermediary metabolism. Transient storage of glucose in its polymeric form as glycogen helps in maintaining optimum glucose levels. Additional glucose can also be synthesized from pyruvate, which is derived from amino acids. Fatty acids that are either absorbed from diet or released from adipose tissue are converted to ketone bodies via acetyl-CoA. The central theme of intermediary metabolism is ensuring tight control of blood glucose levels that are about 100 mg/dl or 5.5 mM. The metabolic pathways diverge into two types: catabolic, those that degrade biomolecules to release free energy, and anabolic, those that polymerize monomers into macromolecules. A great portion of the biopolymers is broken down during catabolism to ensure ATP production. ATP helps meet the energy requirements of cells and also facilitate anabolic pathways. Anabolic pathways ensure the synthesis of macromolecules including fatty acids, proteins, and nucleic acids. Depending on the context, a few metabolic pathways can serve both as catabolic and anabolic. An example for such amphibolic pathway is citric acid cycle, which participates in the synthesis of amino acids in addition to break down of acetyl-CoA. Another example for amphibolic pathway is pentose phosphate pathway.

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Healthy Individuals Maintain Strict Normoglycemia

Tight regulation of blood glucose levels within a narrow range is referred to as glucose homeostasis. Glucose homeostasis (4–6 mM) is strictly regulated predominantly by the tug-of-war between insulin (that lowers blood glucose) and glucagon (that raises blood glucose) [1]. Classically, insulin and glucagon are considered to regulate blood glucose, but recent studies added several hormonal and neural glucoregulatory factors that influence blood glucose levels moderately (Table 4.1). Insulin and glucagon interact with tissues such as the liver, gut, brain, adipose, and muscle and act antagonistically to each other in regulating blood glucose levels [2–4]. Circulating glucose, through a series of events, stimulates the pancreatic islet (β) cells to secrete insulin. Insulin elicits its action via its receptors expressed in insulin-sensitive tissues such as the liver, muscle, and adipocytes. Insulin is the principal endocrine mediator that decreases the blood glucose levels by several means: (a) promoting glucose uptake by insulin-sensitive tissues, (b) suppressing hepatic and renal glucose production, and (c) reducing circulating free fatty acids (FFA). Metabolic events regulated by insulin are presented in Table 4.2. Low circulating glucose levels stimulate glucagon from pancreatic islet (α) cells, which elicits counterregulatory action against insulin. Glucagon enhances glucose production in the liver by activating glycogenolysis. The effect of glucose production by the glucagon is temporary. Epinephrine, which is secreted from the adrenal medulla, performs multiple actions in target organs: (a) stimulates glycogenolysis and gluconeogenesis in the liver, (b) induces gluconeogenesis in the kidney, and (c) lowers glucose uptake in the skeletal muscle. Pituitary growth hormone (GH) and cortisol

Table 4.1 List of predominant hormones that are associated with the regulation of glucose homeostasis Hormone Insulin

Effect on blood glucose Reduces

Glucagon Somatostatin Epinephrine

Increases Reduces Increases

Glucagon-like peptide-1 (GLP1) Thyroxin

Reduces

Amylin Cortisol ACTH

Reduces Increases Increases

GH

Increases

Increases

Key mode of action Increase glucose uptake by cells; increase glucose conversion to glycogen or fatty acids Increase glycogenolysis; increase gluconeogenesis Suppress glucagon and insulin secretion Increase glycogenolysis. Enhance fatty acids release from adipocytes Enhance insulin secretion; suppress glucagon secretion Increase glycogenolysis; increase intestinal absorption of sugars Suppress glucagon secretion Increases gluconeogenesis; antagonize insulin action Increase cortisol secretion; enhance fatty acids release from adipocytes Antagonize insulin action

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Table 4.2 Insulin-regulated metabolic events pertinent to carbohydrates, lipids, and proteins Metabolic events regulated by insulin Anabolic effects on carbohydrate metabolism Increase the transport of glucose into insulin-sensitive cells (muscle and adipocytes) Reduce the rate of glucose release by the liver via inhibiting glycogenolysis Reduce the blood glucose by (a) stimulating glucose uptake, (b) stimulating glycolysis, and (c) glycogenesis Inhibit gluconeogenesis Anabolic effects on lipid metabolism Stimulates lipogenesis Facilitates the conversion of fatty acids to triglycerides in the liver Reduces the rate of release of free fatty acids from adipose tissue Protein metabolism Increases the transport of free amino acids into hepatocytes and myocytes Stimulates protein synthesis in myocytes

act as glucose counterregulatory hormones, and both act synergistically. Both these hormones stimulate expression of gluconeogenic enzymes and lower glucose transport [4, 5]. Glucagon-like peptide-1 stimulates insulin secretion and suppresses glucagon secretion. Further, neuropeptides (brain-derived neurotrophic factor, gastrin-releasing peptide), enteroendocrine hormones (gastric inhibitory polypeptide, gastrin, and CCK), adipokines, and myokines partially contribute to glucose homeostasis [6].

4.3

Diabetes Mellitus

Diabetes mellitus (D) is a metabolic disorder with impaired secretion of insulin (type 1 diabetes mellitus (T1D)) and peripheral insulin resistance (type 2 diabetes mellitus (T2D)) resulting in elevated blood glucose levels (hyperglycemia) [7–9]. In T1D (previously known as insulin-dependent or juvenile), insulin secretion is absent due to autoimmune destruction of pancreatic β-cells, which occurs subclinically over months to years. In general, T1D develops in childhood or adolescence. However, it can also develop in adults, which often initially appears to be T2D. In T2D (previously known as non-insulin-dependent or adult-onset), insulin levels are relatively inadequate because target tissues developed insulin resistance. Insulin resistance is manifested in impaired suppression of gluconeogenesis in the liver. Peripheral insulin resistance impairs glucose uptake, and together, hepatic and peripheral insulin resistance results in hyperglycemia. In T2D, early in the disease, insulin levels are very high; however, insulin secretion depletes in the later course of the disease. Symptoms of both T1D and T2D include hyperglycemia in addition to polyuria, polyphagia, and polydipsia. Advanced complications include peripheral neuropathy, nephropathy, and predisposition to infection. In this chapter, the metabolic adaptations will be discussed in hyperglycemia and are relevant to both T1D and T2D.

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Metabolic Adaptations in Diabetes Mellitus

In this section, metabolic adaptation in diabetes will be discussed considering the conditions such as insulin deficiency and hyperglycemia. Diabetes is generally presented with reduced glucose uptake and glucose clearance by the tissues particularly the muscle and adipose. In addition to perturbations in glucose uptake and metabolism in extrahepatic tissues, deregulation in glucose cycling, suppression of hepatic glycolysis, and increase in hepatic gluconeogenesis contribute to hyperglycemia.

4.4.1

Altered Glucose Cycling in Diabetes

Diabetes is presented with decline in glycolytic flux, which is one of the main reasons for hyperglycemia [10]. Decline in glycolytic flux is explained by increase in glucose-6-phosphate conversion to glucose [11, 12]. Normally, glucose is converted to glucose-6-phosphate immediately after taken up by the liver. But, during diabetes, glucose-6-phosphate gets dephosphorylated and returned to the circulation. The possible reason for the observed effect of glucose flux into circulation is both stimulation of glucose-6-phosphatase activity and decreased glucokinase activity [13]. In decreased insulin, glucagon ratio in diabetes results in the repression of glucokinase system [14]. The rate of phosphorylation of glucose is 30% lower in diabetic liver when compared with healthy liver. Another issue with altered glucose cycling in diabetes is the reduced rate of hepatic glycolysis. Diabetic hepatocytes show a 60% reduction in the rate of glycolysis. The liver recycles a large portion of glycolytically derived pyruvate to glucose during diabetes [13]. This pyruvate to glucose cycling inhibits lactate accumulation. Unlike in normal cells where pyruvate is reduced to lactate in the cytoplasm, in the diabetic liver, glycolytically derived pyruvate is channeled to mitochondria. Mitochondrial import of pyruvate is facilitated by the counterion exchange of ketone bodies particularly acetoacetate. Pyruvate that enters into mitochondria is recycled to glucose via gluconeogenesis pathway, and only about 20% pyruvate is oxidized to CO2. Together, both glucose-6-phosphate and pyruvate cycling to glucose contribute to hyperglycemia. More than three-fourth of glucose taken up by the liver get recycled to glucose in diabetic condition. The increase in glucose cycling in diabetic liver is consequence of increased oxidation of free fatty acids.

4.4.2

The Randle Glucose-Fatty Acid Cycle

The Randle cycle (glucose-fatty acid cycle) explains the competition between glucose and fatty acids for their oxidation and uptake [15, 16]. The Randle cycle is posited to depict impaired glucose metabolism in T2D and insulin resistance. Depending on the availability of the fatty acids, metabolic adaptation in cardiac and skeletal muscle shifts back and forth between carbohydrate and fatty acids as

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oxidative energy source [16]. The Randle cycle explains the mechanisms that are involved in the switch from carbohydrate to fat oxidation and glucose-sparing effect in three possible ways as follows: 1. Increased fat oxidation inhibits glucose utilization; fatty acids are taken up from the plasma and are carried to the mitochondria by fatty acid-binding proteins. The fatty acids are transported into the mitochondria by the carnitine palmitoyltransferase system, where they undergo β-oxidation to yield acetylCoA. Citrate formed from acetyl-CoA inhibits pyruvate dehydrogenase (PDH) and phosphofructokinase, respectively. This leads to a buildup of glucose-6-P and inhibition of hexokinase, resulting in reduced glucose uptake and oxidation (Fig. 4.1).

Fig. 4.1 The Randle cycle explains that free fatty acid (FFA) oxidation yields acyl-CoA, which converts into acetyl-CoA in mitochondria. Acetyl-CoA and citrate inhibit pyruvate dehydrogenase (PDH) and phosphofructokinase (PFK) thus blocking glycolysis. FFA-derived acyl-CoA converts into diacylglycerol (DAG), which by a series of events inhibits insulin signaling and prevents GLUT4 translocation and glucose uptake. Acetyl-CoA formed from mitochondrial oxidation of FFA activates pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) and favors gluconeogenesis

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2. Free fatty acids prevent insulin-mediated glucose transporter 4 (GLUT4) translocation. Normally, insulin induces phosphorylation of insulin receptor substrates (IRS), which eventually results in translocation of GLUT4 to membrane for glucose intake. Free fatty acid metabolites such as ceramides, acyl-CoA, and diacylglycerol (DAG) activate several serine/threonine kinases (protein kinase C, nuclear factor-kB, inhibitory kB kinase b (IKKb)), which phosphorylate IRS, and protein kinase B/Akt thus inhibits insulin signaling. Therefore, free fatty acids impair GLUT4-dependent glucose uptake. 3. Free fatty acids induce gluconeogenesis; mitochondrial oxidation of free fatty acids generates acetyl-CoA, which activates pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK), thus promoting gluconeogenesis. Another concern is that peroxisomal oxidation of fatty acids generates acetate. Acetate ensures glucose synthesis via peroxisomal glyoxylate pathway.

4.4.3

Aldose Reductase Pathway

During hyperglycemia, hexokinase that has high affinity for glucose becomes saturated, and the excess glucose is sequestered through the aldose reductase (AR) pathway, also known as the polyol pathway [17]. This is a two-step pathway; in the first step, glucose is reduced to sorbitol by AR (EC 1:1:1:21), and this reaction oxidizes NADPH to NADP+ (Fig. 4.2). In the second step, sorbitol is oxidized to fructose, and this reaction is catalyzed by sorbitol dehydrogenase, which also produces NADH from NAD+. The rate of sorbitol conversion to fructose is slower than the rate at which glucose is converted to sorbitol. Therefore, AR pathway results in the accumulation of sorbitol and depletion of NADPH. Depletion of NADPH results in decreased levels of reduced glutathione, a cellular antioxidant. AR

Fig. 4.2 Aldose reductase reduces glucose to sorbitol. Sorbitol dehydrogenase catalyzes sorbitol conversion to fructose. The depletion of NADPH in this pathway results in the NADPH scarcity, which results in decreased synthesis of reduced glutathione, an antioxidant

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pathway is implicated in several complications of diabetes, especially in microvascular damage in the retina, kidney, and nerves [18, 19]. Sorbitol accumulates and produces osmotic stresses on cells by draining water into tissues. Alternatively, fructose formed during polyol pathway contributes to the formation of advanced glycation end products (AGEs). In normoglycemic conditions, polyol pathway is inactive as AR has high Km and low affinity for glucose. The biological significance of polyol pathway is not known; however, fructose has importance in production of spermatocytes.

4.4.4

Nonenzymatic Glycation

The events of nonenzymatic glycation (NEG) involve the irreversible attachment of reducing sugars with free amino groups (predominantly lysine or arginine) of proteins [20]. NEG reactions occur at a slow rate during normal aging; however, these reactions accelerate in diabetic subjects due to the abundance of glucose and fructose. NEG comprises sequel of events that trigger with formation of Schiff’s base between reducing sugar and amino group and intermediate Amadori product that further undergo Maillard reactions to yield advanced glycation end products (AGEs). These AGEs form adduct on afflicted proteins and include large number of heterogeneous chemical structures with cross-linking propensity (Table 4.3). AGEs are fluorescent and relatively insoluble and appear yellow-brown. Carboxymethyl-lysine (CML), pentosidine, argypyrimidine, pyrraline, carboxyethyl-lysine, fructoselysine, and methylglyoxal-derived hydroimidazolones are well-characterized AGEs [20]. Both biochemical and immunohistochemical evidence suggest that CML is predominant AGE in majority of tissues from diabetic patients [20]. NEG of client proteins adversely affects their structural and functional properties [20]. In addition to affecting the physicochemical properties of the afflicted proteins, NEG induces aberrant cellular signaling and alters gene expression profiles [20–23]. HbA1c, a predominant form of glycated hemoglobin, is an index of nonenzymatic glycation in diabetic patients. In addition to the secondary

Table 4.3 Commonly observed AGEs in the biological systems AGEs Carboxymethyl-lysine Pentosidine Argypyrimidine Imidazolone Carboxyethyl lysine GOLD MOLD

Carbohydrate source Glucose, threose Ribose Methylglyoxal 3-Deoxyglucosone Methylglyoxal Glyoxal Methylglyoxal

Table is modified from Ref. [20] GOLD glyoxal-lysine dimer, MOLD methylglyoxal-lysine dimer

Target amino acid Lysine Lysine + arginine Arginine Arginine Lysine Lysine Lysine

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complications of diabetes, AGEs are implicated in many age-related chronic diseases such as cardiovascular diseases, Alzheimer’s disease, and cancer.

4.5

Metabolic Adaptations in Cancer

The hallmark of cancer is an uncontrolled proliferation of aberrant cells. One aberrant cell typically proliferates up to 109 cancer cells, and to survive and proliferate, cancer cells activate certain metabolic pathways. Altered metabolic pathways in cancer cells are needed to generate enough precursors to meet elevated energy needs, cell maintenance, redox balance, and biosynthesis of macromolecules.

4.5.1

Glycolysis and Warburg Effect

Unlike normal cells, where most of the pyruvate from glycolysis enters the citric acid cycle, in malignant cells, pyruvate converts into lactic acid, even in the presence of oxygen [10]. This explicit conversion of most of the pyruvate to lactate by tumor cells was observed by Otto H. Warburg a decade ago. He showed malignant cells actively use glycolysis for ATP generation, even in the presence of oxygen, and the metabolic shift from oxidative phosphorylation to glycolysis in cancer cells might be due to a respiratory injury. Therefore, instead of 36 ATP from the complete oxidation of one glucose molecule, merely 2 ATP are formed in cancer cells by the conversion of pyruvate into lactate by aerobic glycolysis. Although Warburg proposed the mitochondrial defect to be the culprit for aerobic glycolysis in cancer cells, it is also debated that the observed metabolic derangements were as a result of oncogene activation and tumor suppressor inactivation. Nevertheless, majority of tumors display “glucose addiction” and are presented with increased dependence on glycolysis. Glycolysis provides both ATP and metabolic intermediates essential for cancer cell proliferation. The malignant cells demonstrate great demand for glucose and show a high rate of aerobic glycolysis that offers several advantages as follows: (1) Tumor microenvironment is presented with the hypoxia, and under such conditions of impaired oxidative phosphorylation, glycolysis provides sufficient ATP for cells. (2) The intermediates generated during glycolysis provide precursors for the synthesis of fatty acids and nucleic acids, and this seems to be particularly important for actively proliferating tumor cells (Fig. 4.3). Several intermediates of glycolysis including glucose-6-phosphate and fructose-6-phosphate are channeled via pentose phosphate pathway to generate ribose-5-phosphate and NADPH. Ribose-5-phosphate is building block for nucleic acid synthesis. NADPH is critical for maintaining reduced glutathione levels that in turn regulate cellular redox homeostasis. Furthermore, NADPH also supports lipid synthesis. Therefore, cancer cells adapt to glucose as their predominant source for both energy and key components necessary for nucleic acid and fatty acid synthesis. Metastatic cancer cells accumulate lactate. Lactate

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Fig. 4.3 The major metabolic alterations in cancer cells. 1. Aerobic glycolysis and glucose branching. Glycolytic intermediates transform to ribose-5-phosphate, a precursor for synthesis of nucleic acids. 2. Mitochondrial metabolism is propelled by glutamine. Glutamine converted to glutamate becomes the major carbon source for intermediates of citric acid cycle and particularly for anabolic precursors such as oxaloacetate and citrate. Oxaloacetate converted to aspartate participates in purine synthesis. 3. Citrate, which is derived from glucose or glutamine, converts into acetyl-CoA. Acetyl-CoA and its derivative malonyl-CoA undergo condensation to yield fatty acid palmitate

indirectly contributes to the invasion of cancer cells by activating extracellular matrix-remodeling enzymes.

4.5.2

Glutamine Metabolism in Cancer Cells

Glutamine is an indispensable metabolic substrate for cancer cells wherein it acts as a building block via anabolism and serves as an energy source through catabolism (Fig. 4.3). Glutaminase 1 (GLS1) converts glutamine to glutamate. Glutamate conversion to α-ketoglutarate (α-KG) and routing into the citric acid cycle appear to be crucial for the oncogene-induced tumor growth. α-KG is the main source of

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oxaloacetate and malate for fatty acid synthesis in malignant cells. Studies have shown that the expression of GLS1 correlates with growth of the tumor. It is noteworthy that inhibition of glutaminase suppresses growth of tumor. The precise role of glutamine in tumorigenesis remains to be characterized despite the fact that suppression of glutaminase inhibits oncogenic transformation. Glutamine provides the majority of anaplerotic carbon for the citric acid cycle. Further, glutamine contributes to the antioxidant pool in tumor cells. Malate derived from glutamine (via citric acid cycle) serves as a substrate for the malic enzyme. This enzymatic conversion yields NADPH, which is important for the conversion of glutathione (GSH), a cellular antioxidant from its oxidized form glutathione disulfide (GSSG). It was revealed that oncogene c-Myc contributes to glutamine metabolism directly and indirectly. c-Myc stimulates expression of glutamine transporters and helps glutamine import into cells. Alternatively, c-Myc also stimulates GLS1 expression by suppressing the miRNA (miR23A and miR23B), which otherwise represses GLS1 expression [24, 25].

4.5.3

Lipid Metabolism in Cancer Cells

Unlike normal cells that rely on lipid intake to produce molecules of membranes and signaling, cancer cells adapt fatty acid synthesis de novo from citrate, glutamine, and NADPH. Carbon units derived from glucose are utilized in the de novo fatty acid biosynthesis. Breakdown of glucose into citrate triggers this anabolic reaction. Citrate is exported from mitochondria to cytosol and is converted into acetyl-CoA by ATP citrate lyase (ACLY). Irreversible carboxylation of acetyl-CoA produces malonyl-CoA, and this reaction is catalyzed by acetyl-CoA carboxylase (ACC). These two precursors (acetyl-CoA and malonyl-CoA) are repeatedly assembled to yield palmitate (Fig. 4.3). Palmitate is a key source for mono- and polyunsaturated fatty acids, and stearoyl-CoA desaturase (SCD) converts palmitate into palmitoleic acid, which is a monounsaturated fatty acid. During hypoxic conditions, citrate is synthesized from glutamine, thus contributing to lipid synthesis. It is worth mentioning that in rapidly growing tumors, hypoxia prevails, and during hypoxia, SCD activity is significantly reduced. This could explain the reason for decreased amount of glutamine-derived unsaturated fatty acids. To overcome SCD deregulation in hypoxia, cancer cells rely on extracellular unsaturated lysophospholipids [26]. It is interesting to note that aggressive tumors depend on lipids obtained from exogenous sources for their survival, and particularly, metastatic cancer cells skew toward adipocytes [27]. NADPH is another indispensable source required for fatty acid biosynthesis. Cancer cell couples fatty acid oxidation with fatty acid biosynthesis for maintaining NADPH levels. In conditions of nutrient depletion and stress, malignant cells maintain NADPH levels both by reducing fatty acid biosynthesis, where NADPH is consumed, and increasing fatty acid oxidation, where NADPH is generated. It is suggested that these metabolic rearrangements in cancer cells are orchestrated by the AMP-dependent protein kinase (AMPK), which allows survival of cancer cells

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during anchorage-independent growth and nutrient depletion [28]. It is interesting to note that either inhibition of fatty acid oxidation or de novo lipid biosynthesis similarly sensitizes leukemia cells to apoptosis suggesting that these two metabolic events might share common substrates and these two events occur simultaneously [29]. Several enzymes that facilitate lipid metabolism such as ACL, ACC, FAS, and SCD are under transcriptional control by sterol response element-binding protein (SREBP1). This lipogenic transcription factor SREBP1 is a key downstream target of oncogenic BRAF signaling. Sustained lipogenesis through the maintenance of active SREBP1 contributes to therapeutic resistance in BRAF-mutant melanoma [30].

4.5.4

Protein Metabolism in Cancer Cells

The synthesis of proteins is a highly complex and synchronized process and requires involvement of all the amino acids. The rate of protein synthesis is high in cancer cells that increase the overall need for amino acids. Malignant cells have a high requirement for particularly serine and glycine owing to their involvement in nucleotide biosynthesis. mTORC1 is a major signaling pathway involved in protein translation and amino acid metabolism and is activated by multiple oncogenic insults, particularly oncogene c-Myc. Cancer cells are influenced by growth factor signaling to express the amino acid transporters on the surface that facilitate their uptake from surrounding media. Amino acid availability stimulates mTORC1, which in turn stimulates the synthesis of proteins by effecting translation and biogenesis of ribosomes. As discussed above, glutamine uptake is another important trait of cancer cells. Glutamine uptake and the activity of glutaminase enzyme are both triggered by mTORC1, thus providing glutamate for transamination reactions and for the upkeep of the citric acid cycle. Citrate acid cycle intermediates further contribute for the synthesis of various amino acids. When there is a glutamine surplus, it is often exchanged for essential amino acids to trigger mTORC1 and protein biosynthesis. Thus, an abundance of glutamine and essential amino acids empowers mTORC1-mediated activation of protein biosynthesis.

4.6

Factors Responsible for Metabolic Adaptations in Cancer Cells

4.6.1

Hypoxia

It is very well known that cancer cells are often presented with hypoxic conditions, and hypoxia is one of the main culprits contributing to alterations in tumor metabolism. Hypoxia is an important contributor to intra- and inter-tumor cell diversity and stimulates pronounced but nonuniform expression of hypoxia-driven genes in solid tumors [31]. Majority of glycolytic enzymes are upregulated during hypoxia and provide the rationale for increased rate of glycolysis in cancer cells. In contrast,

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expression of lactate dehydrogenase, which converts pyruvate to lactate, increases during hypoxia. Hypoxia-inducible factor 1α (HIF1α) and HIF2α are the two major transcription factors that transduce hypoxic effects on cells. Of the two, HIF1α is considered as the master sensor that coordinates cellular responses in relation with oxygen homeostasis. HIF1α stimulates 9 of the 10 genes involved in glucose uptake and glycolysis including GLUT1, hexokinase, and lactate dehydrogenase [32]. HIF1α induces pyruvate dehydrogenase kinase, which in turn blocks pyruvate dehydrogenase, thereby inhibiting pyruvate conversion to acetyl-CoA. HIF2α elicits cell multiplication during hypoxic conditions via surging c-Myc function. The targets of c-Myc include enzymes of the glycolytic pathway, glutaminolysis, and fatty acid synthesis. It also mediates the overexpression of the glutamine transporters in the membrane and mitochondrial glutaminase enzyme.

4.6.2

Inactivation of Tumor Suppressors and Activation of Oncogenes

The predominant event in cancer is the occurrence of inactivating mutations in tumor suppressor genes and activating mutations in oncogenes. Most cancers are presented with mutations in Tp53 tumor suppressor gene. Anabolic pathways branching off from glycolysis are regulated by p53. PARK2 (Parkinson’s disease-associated gene) is a p53 target and acts as a tumor suppressor gene. PARK2 deficiency ensures cancer cells to adopt the Warburg effect. TP53-induced glycolysis and apoptosis regulator (TIGAR) is an allosteric activator of phosphofructokinase and inhibits glycolysis. Moreover, p53 also suppresses GLUT1 and GLUT4 transcription. Furthermore, p53 reduces cytochrome c oxidase, which helps in assembly of complex IV of electron transport chain (ETC). Therefore, p53 loss in cancer cells aborts ATP production from ETC and shifts the ATP production to glycolysis [33]. Further, p53 regulates mTOR, PTEN, IGFBP3, and AMPK, thus playing a bigger role in affecting tumor metabolism [34]. In most cancer cells, AKT (serine/threonine kinase) is hyperactive. It is considered to be responsible for cancer cells’ addiction to glucose metabolism for survival and proliferation [35]. AKT contributes to glucose metabolism by (1) enhancing GLUT1 transcription, (2) aiding in phosphorylation of glucose by hexokinase, and (3) eliciting expression of glycolytic enzymes through HIF1 and mTOR signaling [32]. Cancer cells are often presented with hyperactive mTOR pathway [36]. mTOR serves as an energy sensor and fosters nutrient uptake, and activation of mTOR increases glycolysis by stimulating HIF1α expression. c-Myc is another oncogene, and its elevated expression is associated with 40% of all cancers [37]. c-Myc affects pathways associated with energy metabolism, particularly glycolysis. c-Myc induces GLUT1, hexokinase, phosphofructokinase, enolase, and lactate dehydrogenase (Table 4.4). As described earlier, c-Myc plays a key role in glutamine metabolism in cancer cells by inducing the expression of glutamine transporters and GLS1 enzyme that converts glutamine to glutamate.

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Table 4.4 List of the oncogenes and transcription factors that regulate metaboic events in cancer cells Oncogenes/ transcription factor HIF1α HIF1α SP1 MYC/HIF1α Lipid metabolism SREBP1

CHO metabolism/aerobic glycolysis PDK1 IDH1/2 G6PDH GLUT1/3, HK2, PFK1/2, LDHA ACLY, ACC, FASN, GPAT, ACS, SCD1

Amino acid metabolism MYC GLUD1/2, GLS2, SHMT1, PHGDH MYC/HIF1α ASS1 NF-kB IDO, TDO CREBP TPH1

4.6.3

Stages of cancer progression Primary tumor cells

Primary tumor cells and proliferating tumor cells Primary tumor cells and proliferating tumor cells

Mitochondrial Dysfunction

Cancer cells are presented with mitochondrial dysfunction resulting in increased levels of succinate, fumarate, and NADPH oxidase. Accumulation of citric acid cycle intermediates shifts energy production to glycolysis. Studies suggest that suppression of the NADPH oxidase gene results in decreased tumor cell proliferation. Mutations in mitochondrial proteins affect events of the citric acid cycle and oxidative phosphorylation. Additionally, p53 upregulates the expression of cytochrome c oxidase 2 and thus surges the mitochondrial respiration rate. In transformed fibroblasts, the morphology and function of the mitochondria are affected, and oxidative phosphorylation is attenuated due to reduced expression of respiratory complex I proteins [38, 39]. Cancer cells possess low mitochondrial respiration and compensate with an increased dependency on glycolysis. It is interesting to note that mutations in mitochondrial DNA occur at higher frequency when compared with normal cells. Predominantly, mutations occur in genes encoding mitochondrial ETC complex I, III, IV, and V, tRNAs, and rRNAs [32]. Possible reason that explains, at least in part, for higher frequency of mutations in mitochondrial DNA is increased production of free radicals in mitochondria of cancer cells [40].

4.7

Summary

It is now accepted that reprograming of metabolic pathways is a hallmark of both diabetes and cancer. In diabetes, it is more clear that either insulin resistance or absolute deficiency of insulin is major culprit in eliciting metabolic reprograming. In

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case of cancer, it is not yet clear what comes first between metabolic reprograming and uncontrolled proliferation. It is intriguing because mutations in genes whose products encode metabolic enzymes are associated with certain cancers. On the other hand, mutations in tumor suppresser genes and oncogenes are directly attributed with some cancers. Studies are being focused on metabolic alterations in cancer cells with an aim to better understand the biology and improve therapeutic responses. Glycolysis (lactate dehydrogenase/hexokinase), mitochondrial metabolism (ETC), and glutamine metabolism (glutaminase) have emerged as therapeutic targets in cancer cells. It is interesting to note that metformin originally considered as antidiabetic drug also exhibits anticancer properties by inhibiting ETC complex and thus mitochondrial ATP production. Cells demonstrate contrasting phenotype pertinent to glucose uptake in case of diabetes and cancer. Cells uptake very less glucose in diabetes, whereas cancer cells consume excess glucose. Nevertheless, it appears that controlling strict hyperglycemia appears to be a common strategy to combat both complications of both diabetes and cancer. Acknowledgments Authors thank Rajkishor Nishad, Lakshmi Prasanna, and Deepti Nabariya for their assistance during the preparation of this chapter.

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Role of Mitochondria in Pancreatic Metabolism, Diabetes, and Cancer Noble Kumar Talari, Ushodaya Mattam, and Naresh Babu V. Sepuri

Abstract

Ever since from the discovery of mitochondria, it attracted the field of medicine due to its involvement in numerous aspects of cellular metabolism. Mitochondria, the powerhouse of the cell, are extremely important for maintenance of several vital processes such as TCA cycle, generation of cellular energy source adenosine triphosphate (ATP), cell growth, cell death, and signal transduction. Therefore, it is evident that any type of mitochondrial perturbations would result in myriad of diseases. Mitochondrial diseases are also results from nuclear DNA mutations because most proteins involved in mitochondrial metabolism and mitochondrial DNA maintenance are nuclear encoded. Mitochondrial DNA variations are also observed in aging, diabetes, cancer, and neurological diseases such as Parkinson’s and Alzheimer’s diseases. So far, the research accentuated the relationship between mitochondrial dysfunction and multitude of diseases. Pancreas is one such organ that is frequently affected by mitochondrial perturbations. Over the past decade, significant amount of research has been done on mitochondria in pancreatic dysfunction. Heretofore, accumulating evidences suggest that pancreatitis, diabetes, and pancreatic cancer have highlighted the relationship with mitochondrial dysfunction. In this book chapter, we explore the advances that have been made toward identifying the mitochondria as therapeutic target in pancreatic malignancies including pancreatic metabolism, diabetes, and cancer. Keywords

Pancreatic ductal adenocarcinoma · Mitochondria · Type 2 diabetes mellitus · Pancreatic β cells · Insulin and metformin

Co-correspond author: Noble Kumar Talari. N. K. Talari (*) · U. Mattam · N. B. V. Sepuri (*) Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_5

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Abbreviations αKG β-Lap Ala AM ARE ASK1 Asp BPTES Cit Cys DON ER Fum GCL GEM Gln Glu GLUD1 Gly GOT GS HA HBP HIF IGF-1 Iso Lac LDHA Mal MDH Met MMP NOX NQO1 NR5A2 NRF2 OAA OAA PDAC PPP PTEN Pyr

Alpha-ketoglutarate β-Lapachone Alanine Adrenomedullin Antioxidant response element Akt-stimulated phosphorylation of apoptosis signal-regulating kinase 1 Aspartate Bis-2-(5-phenylacetamido-1,3,4-thaidazole-2-yl) ethyl sulfide Citrate Cysteine 6-Diazo-5-oxo-L-norleucine Endoplasmic reticulum Fumarate Glutamate cysteine ligase Gemcitabine Glutamine Glutamate Glutamate dehydrogenase Glycine Aspartate transaminase Glutathione synthase Hyaluronan or hyaluronic acid Hexosamine biosynthetic pathway Hypoxia inducible factors Insulin-responsive growth factor Isocitrate Lactate Lactate dehydrogenase A Malate Malate dehydrogenase Metformin Matrix metallo proteinase NADH oxidase NADPH-quinone oxido-reductase 1 Nuclear receptor 5 A2 Nuclear factor erythroid 2-related factor 2 Oxaloacetate Oxaloacetic acid Pancreatic ductal adenocarcinoma Pentose phosphate pathway Phosphatase and tensin homolog Pyruvate

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Role of Mitochondria in Pancreatic Metabolism, Diabetes, and Cancer

R5P ROS Suc T2DM TCA UCP2 UDP-GlcNAC

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Ribose 5-phosphate Reactive oxygen species Succinate Type2 diabetes mellitus Tricarboxylic acid cycle Uncoupling protein 2 Uridine diphosphate N-acetylglucosamine

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the aggressive pancreatic malignancies with increased incidence over the years and becomes the second leading cause of cancer-related death. The 5-year survival rate of PDAC is 8% which pinpoints the high mortality rate of this cancer. The high mortality rate results from several factors such as challenges associated with surgical excision, lack of early diagnostic and biomarkers, and delay in occurrence of clinical symptoms. Hence, 80% of pancreatic cancers are at advanced stage during diagnosis. In addition, limited efficacy of therapeutic treatment options also hampers the outcome of the disease. Therefore, new therapeutic treatment options are required to manage the disease. In recent years, a new phenomenon has been observed that certain cancer cells depend on metabolism for survival and proliferation, which is defined as metabolic addiction. This non-oncogenic addiction is tissue specific and influenced by both genetic and environmental factors. Evidences showed that pancreatic cancer cells depend more on metabolic addiction, and therefore, it is a new area for therapeutic intervention. Pancreatic cancers consist of a dense fibroblastic stroma rich in fibroblasts and immune cells. The fibroblasts deposit huge amount of extracellular matrix proteins during activation, mainly hyaluronan (HA) (non-sulfated glycosaminoglycan), which results in elevated interstitial pressure of PDAC [50, 87]. Eventually, this creates lack of oxygen and lack of nutrient availability condition. In this condition, cancer cells are challenged to maintain redox, metabolic homeostasis, and macromolecular biosynthesis. Under these circumstances, cancer-stromal cell interactions provide metabolically supportive niche for cancer cell survival. This metabolic adaption can occur by numerous mechanisms such as immune suppression and cross talk mechanisms (by swapping cytokines, metabolites, and growth factors) and further by the physical change in the tumor microenvironment that promotes the tumor progression process [40]. Growing evidences suggest that pancreatic cancer is consequence of diabetes, or diabetes is the consequence of the pancreatic cancer. Therefore, the risk for developing other condition is more as they show bidirectional association. The characteristic features of diabetes such as hyperglycemia and insulin resistance are known to contribute for tumor formation. The induction of pancreatic cancer among diabetes mellitus population is indefinable, and it could be due to metabolic, hormonal, and immunological alterations. Several hypothesized mechanism defines the association between these two conditions such as involvement of insulin, insulin-responsive growth factor (IGF-1), leptin, and adiponectin [22, 69, 85, 86, 113].

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Growing evidences also suggest that pancreatic cancer stimulates the occurrence of diabetes. It has been suggested that diabetes is the early symptom of PDAC as it has been observed with 30% of the PDAC patients [14, 71]. Unlike in type 2 diabetes (T2DM), the glucose intolerance is much worse in pancreatic cancer-induced T2DM. Researchers have found that the manifestation of T2DM among pancreatic cancer patients is likely to be a paraneoplastic observation and is potently induced by several factors such as adrenomedullin (AM); pancreatic cancer-derived S-100A8, a N-terminal peptide; and islet amyloid polypeptides (IAPP) [83] that impair glucose tolerance, thus acting as potential mediators of diabetes mellitus in pancreatic cancer. Additionally, chronic pancreatitis and infectious diseases such as Helicobacter pylori, hepatitis B, and HIV virus are also associated with this PDAC [120]. Therefore, identifying a common target which is important for all these pancreatic malignancies would be beneficial. One such target is mitochondria as it is crucial for the maintenance of several vital processes in cells.

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Mitochondria in Pancreatic Malignancies

5.2.1

Mitochondria in Cancer

Since from years, it is known that cancer cells utilize glucose and other building blocks high rate than its counterparts. The discovery of Warburg effect initially leads to the concept that even under oxygen-rich conditions tumor cells tend to shift their metabolism toward glycolysis instead of oxidative phosphorylation. This evidences that mitochondria are either damaged or trivial in cancers. Briefly, it is demonstrated that cancers associate with mutations in mitochondrial DNA, declined mitochondrial copy number, and reduced transcriptional and translation of nuclear genes that encode for mitochondrial proteins [10, 109]. Nonetheless, this perception has been ruled out by the discoveries that oxidative phosphorylation is the ultimate energy source in several malignancies [76]. Furthermore, mitochondria play a pivotal role in cellular metabolism, calcium signaling, and redox signaling. Importantly, in all wellknown hallmarks of cancers such as angiogenesis, resistance to cell death, sustained cellular proliferation, escape from immune response, and deregulated cellular bioenergetics [42, 43], mitochondria play an important role. Hence, mitochondria could be a promising therapeutic target in the treatment of cancers.

5.3

Mitochondrial ROS in Cancer

Mitochondria consist of a double-layered membrane, namely, outer and inner membranes. Mitochondria are the sites of oxidative phosphorylation and respiratory chain complexes present on the inner membrane which facilitates this process. During this process, some of the electrons might escape from electron carriers. So, mitochondria are liable for the production of reactive oxygen species (ROS). This mainly occurred at the sites of mitochondrial electron transport chain (ETC) complexes such as complex I and complex III. The ROS can cause deterioration

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effect to the mitochondrial DNA. The number of mitochondria varies from cell to cell depending on the bioenergetics of the cell. Each cell has multiple copies of mitochondria, and each mitochondrion contains several copies of mitochondrial DNA. Mitochondria have their own DNA and encode 37 genes among which 13 genes encode polypeptides required for oxidative phosphorylation, 2 genes encode rRNAs, and the remaining 22 genes encode tRNAs. Mitochondrial DNA is more prone for mutations compared to nuclear DNA as it is exposed to ROS and furthermore the absence of histones in mitochondrial genome also enhances the probability of mutation in mitochondrial DNA [57]. Therefore, mitochondrial DNA variations are responsible for dysregulated oxidative phosphorylation and eventually increase the ROS production [35]. In cells, other sources are also responsible for the production of ROS [45]. To combat the harmful effects associated with ROS production, cells have antioxidant defense mechanisms. The function of each antioxidant enzyme is different, for instance, superoxide dismutases act on superoxide, whereas catalases and peroxiredoxin act on hydrogen peroxide. The enzymatic action of superoxide dismutase converts the superoxide produced in the mitochondria to H2O2, while its conversion to O2 and water is mediated by catalases.

5.4

ROS in Progression of Pancreatic Cancer

The most frequent genetic abnormality associated with PDAC is mutation in KRAS gene. However, targeting KRAS is proven yet challenging, and therefore, a combined strategy of targeting tumor-driven signaling pathways could be a promising target to manage PDAC [54]. The tumor dormant cells which survived with KRAS oncogenic ablation are responsible for tumor relapse or recurrence. These cells have the characteristic feature of stem cells and depend more on OXPHOS. The dense stromal network of PDAC also hampers the delivery of nutrients and oxygen, creating a hypoxic stress condition. In such condition, increased glycolysis and hexosamine biosynthesis pathways facilitate the aggressiveness of PDAC [38]. In addition, oncogenic ablation and metabolic changes also create the circumstances that foster the production of ROS in PDAC. Oncogenic KRAS has shown to induce ROS production in numerous mechanisms. Oncogenic KRAS alters the mitochondrial function by suppressing electron transport chain (ETC) complexes such as complex I and complex III [114] and regulates hypoxia-inducible factors (HIFs) via transferrin receptor (Tfr1). Tfr1 regulates Fe homeostasis and is responsible for the production of ROS. As it is evident that mutant KRAS cells depend more on oxidative phosphorylation, Tfr1 levels have been elevated in several malignancies including pancreatic cancer [51]. The altered efficiency of mitochondrial function results in increased ROS production as well as ROS-driven occurrence of 4-hydroxy-2-nonenal (4-HNE) adduct formation with macromolecules. This inhibits mitochondrial proteins and damages mitochondrial DNA to induce pancreatic cancer initiation [64].

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The mitochondrial respiratory chain complexes as well as NADH oxidase (NOX) enzymes promote the production of ROS via KRAS. NADH oxidases are flavoenzymes and catalyze the O2 to generate ROS, including superoxide radicle (O2 ) and hydrogen peroxide (H2O2). NADH oxidases are activated by small GTPase Rac1, and Rac1 expression is seen high in PDAC cell line [28]. Further KRAS-induced ROS formation is regulated by NOX activation via its cytosolic regulatory subunit, i.e., p47phox [81]. Other several groups have also reported the KRAS-NOX-ROS production in human PDAC which again supports the mechanistic regulation of ROS production is mediated by NOX family members [60, 66, 116]. As mitochondria are important for the production of ROS and most of the NOX enzymes act in mitochondria, various studies have demonstrated the altered mitochondrial function in PDAC. Others reported that 70 mitochondrial proteins were differentially altered by KRAS induction, showing the impact and importance of KRAS on mitochondria and cell metabolism. Among these, NADH dehydrogenase 1 alpha sub-complex assembly factor 1 acts as an important molecule for KRAS-induced mitochondrial dysfunction [111]. Another study has shown that KRAS transformation leads to metabolic adaptation to high glycolytic activity, suppresses mitochondrial respiratory chain complex I activity, and creates ROS stress in cancer cells [47]. Similarly, mitochondrial ROS also drives the formation of pancreatic cancer via growth factor signaling in acinar cells [64]. Others have reported that KRAS-induced tumorigenicity also mediated via ROS generated at the complex III maintains the anchorage-independent growth and cell proliferation. Together, all these evidences describe the importance of mitochondrial metabolism in tumor invasiveness [114].

5.5

ROS-Apoptosis-Pancreatic Cancer

In pancreatic cancer, ROS acts like pro-survival mechanism and inhibits apoptosis. Because, NAD(P)H oxidase stimulates the ROS production, its inhibition via siRNA treatment induces the apoptosis via diminishing the phosphorylation of Akt and ASK1 pathway proteins [75, 108]. Additionally, ROS generated by NADPH oxidase also confers antiapoptotic effect by inhibiting the protein tyrosine phosphatases (PTP). Inhibition of PTP in pancreatic cancer results in enhanced phosphorylation of JAK2 and hampers the apoptosis [60]. It is evident that activation of NF-Kb in cancers confers resistance to apoptosis. Therefore, the role of apoptogenic agent Brucein D (BD) has been studied in pancreatic cancer. This study showed BD triggers ROS production via NADPH oxidase, reduces the activation of NF-Kb signaling pathway, and promotes the activation of p38 mitogen-activated protein kinases (MAPK) signaling pathway [59]. The combined treatment of GEM/cannabinoid in pancreatic cancer is more effective to inhibit the pancreatic tumor growth and triggers autophagy process through ROS-dependent mechanism [26]. As most solid tumors exhibit low levels of antioxidant enzymes, the research work from several groups demonstrated that overexpression of adenovirus-MnSOD construct of mitochondrial superoxide dismutase (MnSOD) suppresses the pancreatic tumor growth. Thus, the inhibition of ROS formation

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Oncogenic KRAS

HIF NADH Oxidase

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Fig. 5.1 Role of KRAS-induced ROS in pancreatic cancer. The mutant form of KRAS induces ROS by modulating mitochondrial metabolism, hypoxia-inducible factors (HIFs), and NADH oxidase. The altered efficiency of mitochondrial function leads to increased ROS production as well ROS mitochondrial proteins and DNA damage to induce pancreatic cancer initiation. Elevated ROS results in activation of p38 MAPK which initiates epithelial mesenchymal transition. In addition, ROS activates antiapoptosis pathways such as NF-κB and JAK/STAT leading to cancer progression

by superoxide dismutases is beneficial for suppression of pancreatic tumor growth [21, 107, 115] (Fig. 5.1).

5.6

KRAS Alters Glucose and Glutamine Metabolism in Pancreatic Cancer

Although the majority of the PDAC harbors oncogenic KRAS, it drives the tumor development by diverse mechanisms; until now, no effective treatment that targets oncogenic KRAS has reached to the clinic. As RAS inhibitors are unsuccessful, it leads to the understanding that RAS is not an endurable target; however, recent

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studies demonstrate that still this approach may abide to treat KRAS-induced malignancies [73, 98, 105]. Early in the 1900s, a lot of efforts have been put forwarded to develop anti-RAS strategies. One such strategy is inhibiting posttranslational modifications of the RAS by using farnesyltransferase inhibitors (FTIs). Farnesyltransferase transfers the farnesyl group to the RAS protein after translation process, and this step is required for the attachment of RAS to the cell membrane and to transmit signals to initiate cell proliferation. However, the results are disappointing because RAS is alternatively activated by geranylgeranyl transferases (GT). Therefore, researchers have tried with Geranylgeranyl transferase inhibitors (GTIs) and combined therapy of FTIs and GTIs. Conversely, this strategy also failed due to high toxicity effect. As all these results are disappointing, this enforces the discovery of small-molecule inhibitor of phosphodiesterase delta (PDEδ). PDEδ is known as prenyl-binding protein, and it interacts with farnesylated KRAS protein and is important for its localization to plasma membrane (PM). Because KRAS signal transduction chiefly depends on its recruitment toward PM, targeting this may suppress the growth and proliferation of cancer cells. Accumulating evidences also showed that PDE inhibitors are effective therapeutic targets [79, 94]; however, further studies are also required to evaluate its efficacy. Another anti-RAS strategy that targets STK33 kinase is also proven unsuccessful by few research groups [3, 67]. Notwithstanding, with these results, the search for RAS as therapeutic target still continued by researchers. Understanding the fact that cancer cells display altered cellular metabolism, the interest have been shifted toward identifying KRAS-driven potential cellular pathway for therapeutic intervention. Therefore, therapeutic targeting of Warburg effect in pancreatic cancer has gained considerable amount of interest in recent years [91]. Encouraging this, a novel CPI-613, a lipoate analog agent known to inhibit cancer-specific mitochondrial metabolism, has shown to be effective as anticancer drug [68, 74, 80]. Therefore, mitochondria gained attraction as a therapeutic target.

5.7

Metabolic Addiction in Pancreatic Cancer

Cancer cells require adequate energy and building blocks for cellular proliferation. Hence, metabolic pathways are rewired in cancer cells so that the nutrients and energy resources are shifted toward anabolic pathways for their survival. Accumulating evidences suggest that reprogramming of cancer cell metabolism is chiefly controlled by various oncogenic signals, particularly KRAS [17, 18, 61, 90]. Integrated genomic, biochemical, and metabolomics approaches have revealed that KRAS controls multiple metabolic pathways for tumor maintenance in pancreatic cancer. It is evident that glucose transporters and rate-limiting enzymes of glucose metabolism are transcriptionally controlled by KRAS. In pancreatic cancer, in consistent with Warburg effect, the metabolic pathways are reprogrammed, consuming glucose and glutamine more and utilizing them toward biosynthetic pathways. In acceptance with this, in pancreatic cancer, it has been shown that the nitrogen donor for nucleotide biosynthetic pathways chiefly comes from glutamine [121]. The altered glucose metabolism contributes high rates of glycolytic flux,

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activates hexosamine biosynthetic pathway, and promotes ribose biosynthesis. The pentose phosphate pathway (PPP) generates ribose moiety of DNA and is involved in tumor maintenance of pancreatic cancer. Ribose 5-phosphate is generated from non-oxidative arm of PPP utilized in the biosynthesis of DNA/RNA. Supporting this, KRAS activation has shown to upregulate ribose 5-phosphate isomerase A and ribulose-5-phosphate 3-epimerase, the enzymes of non-oxidative arm of PPP [121]. Importantly, KRAS activation regulates the expression of key enzymes of glycolysis such as HK1, HK2, and PFK [33, 90, 121]. In pancreatic cancer, oncogenic KRAS also increases the transcription of LDHA expression so that the pyruvate is more converted toward lactate [19, 121]. Indeed, the posttranslational modification of LDHA also plays an essential role in pancreatic cancer. LDHA is acetylated at K5 position and inhibits its activity there by negatively regulating pancreatic cancer [123]. Another study also suggested that lactate acts as an alternative fuel to support the proliferation of PDAC [38]. Given the importance of lactate as an alternative fuel, researchers have shown that pancreatic cancer growth is reduced by inhibiting the LDHA expression by RNA interference experiments [123]. Furthermore, Raf/MEK signaling pathway is the major effector pathway of KRAS-induced altered glucose metabolism in pancreatic cancer [121]. Thus, pharmacological inhibition of MEK signaling pathway decreases the KRAS-induced enzymes and their downstream metabolites. Additionally, the promoter region of oncogenic KRAS consists of c-Myc-binding elements, thus suppressing c-Myc expression by RNA interference which results in the suppression of same enzymes which were regulated by KRAS [121]. Together, these results recommend that activation of KRAS effectively alters pancreatic glucose metabolism, and therefore, it is an attractive area to treat pancreatic malignancies. The differences in metabolic dependencies distinguish cancer cells different from its normal counterparts. In addition to glucose, several cancer cells have evolved to rely on glutamine because it is a major source of carbon, precursor for glutathione (a major antioxidant), and source of amino group for amino acids such as glycine, serine aspartate, and alanine. The other remarkable feature of altered metabolism in cancer cells is to accentuate the importance of biosynthetic pathways. Cancer cells utilize glutamine for the synthesis of nucleic acids, proteins, and lipids. Therefore, targeting glutamine metabolism is an alluring area of investigation in cancers. Strikingly, in pancreatic cancer, glucose deprivation has shown minimal effect on growth, and cellular redox state of PDAC suggests that pancreatic cancer cells also depend on alternative mechanisms to maintain redox status. Indeed, withdrawal of glutamine from cells results in altered redox status in PDAC [101]. The mitochondria consume glutamine to generate α-ketoglutarate (α-KG) via glutaminase (GLS1) and glutamate dehydrogenase (GLUD1). This α-KG is used as a fuel in the tricarboxylic acid (TCA) cycle. However, in contrast, PDAC cells use transaminases such as aspartate transaminase (GOT2) for the production of α-KG in mitochondria. Simultaneously, this reaction leads to the production of aspartate from oxaloacetic acid (OAA) in cytoplasm. Further, the cytosolic aspartate transaminase (GOT1) acts on aspartate and generates aspartate back into OAA. The OAA produces malate and pyruvate via by malate dehydrogenase (MDH1) and malic enzyme, respectively. Ultimately, this pathway is essential for the production of

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cytosolic NADPH and maintains redox status in PDAC. Hence, targeting of glutamine metabolism (GOT, MDH, and malic enzyme) suppresses the in vitro and in vivo growth of PDAC [101]. In addition to this pathway, KRAS induces Nrf2dependent ROS detoxification to promote tumorigenesis in pancreatic cancer. In general, ROS are mutagenic and known to promote cancer through oxidation of DNA and subsequent mutations in genes that promote cancer cell proliferation. However, in contrast, ROS detoxification program is also known to promote carcinogenesis in pancreatic cancer [25]. During cellular stress, ROS levels were tightly RSP Pentose phosphate pathway

NAD+

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Fig. 5.2 Rewiring of glucose and glutamine metabolism in pancreatic cancer. Metabolic pathways are reprogrammed and shifted toward anabolic pathways for pancreatic cancer. Oncogenic KRAS upregulates the glycolysis process via regulation of glucose transporters GLUD1. The altered glucose and glutamine metabolism activates hexosamine biosynthetic pathway, and pentose phosphate pathway generates ribose moiety of DNA. NAD+ for glycolysis maintenance will be generated by conversion of pyruvate to lactate through LDHA. Cancer cells utilize aspartate transaminase (GOT2) instead of GLUD1 to generate α-ketoglutarate in the mitochondria and to fuel TCA cycle. This reaction simultaneously creates aspartate from oxaloacetate (OAA) in cytoplasm. The cytosolic aspartate transaminase (GOT1) acts on aspartate and generates aspartate back into OAA. This OAA is then converted into malate and to pyruvate via malate dehydrogenase (MDH1) and by malic enzyme, respectively. Eventually, this pathway is essential for the production of cytosolic NADPH and thereby GSH levels. On the other hand, increased ROS levels also regulate GSH levels through ROS/NRF2/ARE pathway. NRF2 enhances GSH levels by inducing the expression of GS, GCL, and enzymes involved in GSH synthesis. α-KG alpha-ketoglutarate, Ala alanine, Cit citrate, Cys cysteine, Fum fumarate, Gly glycine, GSH reduced glutathione, GSSH oxidized glutathione, NRF2 nuclear factor erythroid 2-related factor 2, ARE antioxidant response element, GS glutathione synthase, GCL glutamate cysteine ligase, Iso isocitrate, Lac lactate, Mal malate, R5P ribose 5-phosphate, Pyr pyruvate, OAA oxaloacetic acid, Suc succinate, UDP-GlcNAC uridine diphosphate N-acetylglucosamine

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controlled by detoxification program by antioxidant system that is regulated by transcription factor Nrf2. However, mutant KRAS confers a reduced intracellular environment by constitutively activating this antioxidant program. Nrf2 also generates GSH through a series of reactions. The glutamine (Gln)-derived glutamate (Glu) converts to aspartate (Asp) in the mitochondria, shuttles to the cytosol, and generates NADPH in the cytosol. This NADPH alters the redox homeostasis which enables the cell proliferation via reducing glutathione (GSH) levels. The ability to cope up with oxidative stress and nutrient-poor and hypoxic microenvironment in PDAC utilizes the glutamine to maintain the redox homeostasis, thereby supporting proliferation of PDAC (Fig. 5.2).

5.8

Glutamine Metabolism as Therapeutic Target in Pancreatic Cancer

Several metabolic phenotypes have been observed in PDAC among which is Warburg phenotype which heavily depends on glycolysis producing lactate as final product. Reverse Warburg phenotype utilizes the lactate to fuel the TCA cycle intermediates, eventually less dependent on glucose for proliferation. Glutaminolysis phenotype maintains redox balance by providing TCA cycle intermediates via glutamine. In addition, lipid-dependent phenotype promotes tumor growth by synthesizing lipid from TCA cycle intermediates and further utilizes the energy by lipolysis process [62]. Considering the role of glutamine metabolism in pancreatic cancer proliferation, considerable amount of research has been done to demonstrate its efficacy in therapeutics. 13C-labeling experiments show that the majority of the Asp (aspartate, 50–75%) in pancreatic cancers is derived from glutamine. To test this, initially, researchers have inhibited the glutaminase (GLSi) which hampers the production of glutamate (Glu) in the mitochondria. As GLS is activated by Rho GTPases, researches have inhibited the Rho GTPases by small-molecule inhibitor 968 and found that it inhibits the oncogenic transformation in cancers [110]. Other GLS inhibitors CB-839, bis-2-(5-phenylacetamido1,3,4-thaidazole-2-yl)ethyl sulfide (BPTES), had shown antiproliferative activity in many cancers including pancreatic cancer [7, 37, 118]. Based on the efficacy of CB-839 in pancreatic cell culture system, its efficacy has also been tested in vivo in a treatment-resistant autochthonous mouse model of pancreatic cancer and showed a paradoxical effect. Inhibition of glutaminase by CB-839 has shown no antitumor effect in autochthonous mouse model. Treatment of CD-839 on different orthotopic mouse models such as implanting CD-839-sensitive MPDAC cell line into the organ of tumor origin, i.e., pancreata of nude mice, subcutaneous transplantation of MPDAC cell line into mice, and mice bearing flank tumors has failed to show the decrease in in vivo tumor growth [7]. The ineffectiveness of GLSi by CD-839 is further explained as an adaptive reaction to chronic exposure of GLSi, and multiple compensatory pathways have been activated to sustain the proliferation in pancreatic cancer. This shows the evidence that targeting these pathways in combination with

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GLSi is more effective to enhance the therapeutic strategy [7]. The metabolic plasticity observed in pancreatic cancer utilizes both the glutamine-dependent and glutamine-independent pathways to replenish the nutrients. These results relevantly support previous findings that pancreatic cancer cells obtain energy from multiple fuel sources including autophagy, macropinocytosis, and uptake of free amino acids [20, 102, 119]. Pancreatic cells treated with glutamine and without glutamine affect the expression of several differentially regulated genes essential for JAK/STAT and MAPK signaling pathway. The PPI network analysis has shown that MYC, HSPA5, IL18, and CXCR4 have highest connectivity degree and could play major role in pancreatic cell proliferation [52]. Others have reported that pancreatic cells overexpress MUC1 and replenish the TCA cycle intermediates via glutamine but not glucose. Glucose limitation arrests the cells at G1 phase so that the cells can’t enter into S phase as they can’t synthesize DNA due to the disruption in pyrimidine biosynthesis. MUC1 reprograms glutamine metabolism and provides the important metabolite of pyrimidine nucleotide biosynthesis, i.e., aspartate (Asp), via glutamine [36]. Intracellular pH also regulates cancer cell proliferation. A pH value above 7.2 allows cells to enter into S phase of the cell cycle rapidly and eventually into G2 and M phases. It is also evident that high intracellular pH suppresses the mitotic arrest occurred due to DNA damage. Therefore, in cancers, cell cycle checkpoints are bypassed at high intracellular pH which facilitates the cellular proliferation [63, 88, 112], Putney et al. 2003. Despite, intracellular acidic pH promotes apoptosis by allowing a conformational change in Bax protein (proapoptotic cytosolic protein) and permitting its translocation to mitochondria [55]. Increase in intracellular pH results in low pH in extracellular milieu which further promotes malignancies [32, 82]. At low pH condition, pancreatic cancers rely more on mitochondria in terms of increased glutamine uptake and oxidative phosphorylation. The chronic acidosis stress condition observed due to low pH can be overcome by glutamine metabolism as it serves bioenergetic needs and maintains ROS balance. Aspartate transaminase (GOT1) which converts Asp to OAA is further converted to pyruvate via malic enzyme (ME1). ME1 catalysis produces the NADPH utilized to quench ROS. Thus, anaplerotic glutamine metabolism maintains ROS balance during chronic acidosis condition for the survival of pancreatic cancer [1]. Markedly, even after the improvements in surgical resection, most pancreatic cancer patients will experience recurrence even after complete resection. Therefore, adjuvant systemic therapy helps to overcome the recurrence rate and improves the outcome. Earlier, gemcitabine (GEM) treatment therapy has been preferred as a therapeutic target; however, it fails due to development of innate or adapted drug resistance, eventually leading to poor patient outcomes. Hence, identifying drug or drug combinations which improves the current treatment options greatly benefits the patients. In view of pancreatic cancers’ dependency on glutamine metabolism, in multiple aspects, targeting this pathway may represent an effective therapeutic approach for pancreatic cancers. Accordingly, researchers have used glutamine analogs such as 6-diazo-5-oxo-L-norleucine (DON, interfere with nucleotide and protein biosynthetic pathways). Interestingly, it has been shown that DON reduces

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the tumor growth and inhibits the metastasis in a mouse model of systemic metastasis [97]. Others have reported that disrupting glutamine metabolism in pancreatic cancers sensitizes the chemoresistant PDAC to GEM therapy. DON treatment to GEM-R pancreatic cell lines effectively reduces the EGFR and AKT signaling pathways, reduces the proliferation, and sensitizes the chemoresistance in PDAC. Indeed, disruption of hexosamine biosynthetic pathway (HBP) via glutamine analogs (DON) also affects glycan biosynthesis and protein glycosylation [15]. In search for glutamine metabolism as an effective therapeutic treatment regime in pancreatic cancers, GLS inhibitors in combination with β-lapachone (β-Lap) were also tested and showed high tumor selectivity. β-Lap is a known targeted cancer therapeutic and forms ROS in tumor cells in an NADPH-quinone oxido-reductase 1 (NQO1)-specific manner. Further, in PDAC, NQO1 is highly elevated, and therefore, it is an attractive target. NQO1 consumes β-Lap as a substrate and produces hydroquinone form of β-Lap. It reacts with oxygen and is converted into its original form by consuming NADPH, eventually generating superoxide molecules. Furthermore, NADPH pools are reduced by treating pancreatic cells with bis-2-(5-phenylacetamido-1,3,4-thaidazole-2-yl) ethyl sulfide BPTES or CB-839 and inhibit the glutamine metabolism (GLSi) along with the tumor-selected ROS formation. Therefore, GLS inhibitors (GLSi) in combination with β-lap are expected to enhance the efficacy for the treatment of PDAC [13].

5.9

Pancreatic Cancer and Diabetes

The association between pancreatic cancer and diabetes has been first published in the year 1995. This study showed that the risk for developing pancreatic cancer is twofold high in diabetes mellitus patients [30]. The second meta-analysis (includes 17 case controls, 19 cohort and nested cohort studies), which has been published in the year 2005, demonstrates that individuals with diabetes have 50% higher chances for developing pancreatic cancer [49]. Few other cohort studies have also determined that manifestation of pancreatic cancer is high in people with diabetes [5, 34, 100]. Another large cohort study (29, 133) revealed that pancreatic cancer malignancy could be predicted by serum insulin levels [103]. All these findings confirm that people who have long-term diabetes are having higher chances for the manifestation of pancreatic cancer. Growing evidences suggest that insulin-like growth factor (IGF) have been associated with increased risk for several cancers. IGF-1 is more potent mitogen than insulin, known to inhibit tumor suppressor phosphatase and tensin homolog (PTEN), and eventually promotes pancreatic cancer proliferation by activating IGF-1/PI3K/Akt signaling pathway [69, 85]. Altered hormonal regulation among adiponectin and leptin (adipose tissue secreted hormones) is known to be associated with pancreatic cancers. Adiponectin hormone circulating levels are inversely proportional to plasma insulin. Thus, low levels of adiponectin increase the risk for developing type 2 diabetes mellitus obesity-related malignancies [22, 113]. Indeed, in vivo studies in pancreatic cancer showed that tumor growth is inversely correlated

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with serum adiponectin levels [124]. A genome-wide analysis also demonstrates that pancreatic-susceptible loci on chromosomes 10p 14 is nuclear receptor 5 A2 (NR5A2), which is essential for transactivation of adiponectin gene. Together, all these findings suggest the possible role of adiponectin in the devolvement of pancreatic cancer. Another hormone leptin which usually associates with obesity and type 2 DM also increases the risk for developing pancreatic cancer [104]. Leptin promotes the pancreatic cancer via JAK/STAT3 signaling pathway by upregulating the expression of matrix metalloproteinase (MMP-13) [31]. Another peptide hormone adrenomedullin (AM) also shown to impair the glucose tolerance by obstructing the insulin secretion in β cells. Hence, AM also acts as a mediator of inducing diabetes [2]. On the other hand, pancreatic cancer-derived S-100A8, an N-terminal peptide [4], and islet amyloid polypeptides (IAPP) [83] and adipose tissue inflammation also impair glucose tolerance, thus acting as potential mediators of diabetes mellitus in pancreatic cancer.

5.10

Metformin as Therapeutic Target in Pancreatic Cancer and Diabetes

Due to the unique relationship of diabetes and pancreatic cancer, antidiabetic drugs that lower insulin resistance and hyperinsulinemia are considered as cancer prevention strategies. One such drug is metformin (Met), a synthetic analog of naturally occurring biguanides. Met is used as standard therapy for diabetes since from 1960s [29] and is recognized as a safe drug. At molecular level, metformin exhibits antiproliferative characteristics in several ways such as it elevates AMP/ATP ratio and activates AMPK/mTOR pathway [8, 84], decreases circulating insulin and blood glucose levels [27], and represses gluconeogenesis by inhibiting the glycerophosphate dehydrogenase of the mitochondria [70], reducing the mitochondrial ETC complex I, thereby tumor respiration [11]. Metformin exits with hydrophobic cation that is responsible for Met uptake by tumor cells. However, metformin lipophilic analogs such as phenformin are taken up by cells via alternative mechanisms [96]. Except from its role in increasing acidosis during diabetic therapy, it has more potential than metformin in inhibiting pancreatic cancer proliferation [58]. Met decreases the ATP production by inhibiting the mitochondrial ETC complex I. This leads to the AMPK activation which in turn disrupts the insulin IGF-1 signaling via mTOR inhibition. Increase in mTOR signaling has been associated with many human pathological conditions such as obesity, type 2 diabetes, and cancer [23, 72, 117]. Therefore, targeting mTOR could be an attractive therapeutic target. Inhibition of mTOR by metformin results in decrease in protein synthesis and cell growth. Interestingly, in the absence of AMPK, metformin and phenformin also inhibit mTOR via Rag GTPases and REDD (mTOR negative regulator) which deciphers the AMPK-independent inhibition of mTOR by metformin and phenformin [53]. Rag GTPases are known to interact with mTORC1 and promotes its recruitment toward its activator Rheb (activator of protein kinase activity of

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mTORC1) [93]. Metformin increases the REDD expression in p53-dependent manner by inhibiting mTOR and arrests the cell growth [6]. In mice xenografts, Met has shown to reduce the pancreatic cancer growth, when administered orally and intraperitoneally. It also reduces the phosphorylation S6 ribosomal protein and ERK and affects cell growth [56]. Interestingly, at physiological glucose concentration (5 mM), metformin-induced mTOR inhibition in pancreatic cancer is elevated, while it is low at supraphysiological glucose concentrations (25 mM). This highlights the concept that at low glucose levels pancreatic cancer cells rely more on mitochondrial metabolism, and in such condition, they are more sensitive to mitochondrial respiratory inhibitors [92, 99]. Additionally, metformin in combination with 2-deoxyglucose (2-DG, glycolysis inhibitor) enhances the ATP depletion along with antiproliferative effects that have highlighted targeting mitochondrial bioenergetics and glycolysis pathway are productive in the treatment of pancreatic cancer [16].

5.11

Targeting Mitophagy in Pancreatic Cancer and Diabetes: Does the Shoe Fits?

Mitophagy is the selective mode of autophagy that targets the damaged mitochondria to autophagosome and their subsequent catabolism in lysosomes, maintains the mitochondrial integrity and function, and ensures the cellular homeostasis. Mitophagy is induced by several factors such as mitochondrial membrane depolarization, hypoxia, nutrient deprivation, inflammation, and DNA damage. Since mitochondrial homeostasis is an important phenomenon which maintains mitochondrial quality control, the defects in mitophagy have been seen in several pathological situations including cancer and diabetes. Many mitophagy adapter proteins have shown to be altered in such conditions including p62/SQSTM1, BNIP3, BNIPL, PARK2, and FANCC. During mitophagy, the formation of pre-initiation and initiation complex are comparable with general autophagy. Therefore, the defects in mitophagy are also attributed to general autophagy machinery. Strikingly, the nascent phagophore membrane is formed by the contribution from the endoplasmic reticulum (ER) and mitochondria. Hence, mitochondrial integrity is essential to modulate the production of phagophore membranes [9, 41]. Mitochondrial perturbations are vital in the progression of type 2 diabetes. Defects in mitochondria impair insulin signaling through decrease in respiration capacity, ATP production, and high ROS levels. High cytoplasmic ATP levels from glucose oxidation are sensed by pancreatic β cells to secrete insulin. Briefly, in such condition, ATP-sensitive KATP channel is closed causing depolarization of the plasma membrane, calcium influx, and finally insulin secretion. During respiration, uncoupling protein (UCP2) is known to mediate proton leak and decreases the ATP production. Therefore, UCP2 negatively regulates insulin secretion and acts as a mediator of pancreatic β cell dysfunction, obesity, and type 2 diabetes [122]. In addition, damaged mitochondria also increase the apoptosis of pancreatic β cell reducing the mass of β cell in type 2 diabetes [106]. Moreover, the ultrastructural

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analysis of diabetic β cell reveals that mitochondria are compromised in its shape such as round and distorted cristae, and incorporation of mitochondria into autophagic vacuoles suggests the importance of mitochondria in pancreatic β cell function [46, 65]. PINK1 is a serine/threonine protein kinase, and it detects and accumulates in damaged mitochondria. The accumulation of PINK1 recruits the translocation of Parkin (E3 ubiquitin ligase) from cytosol to mitochondria. Further, mitochondrial outer membrane proteins are ubiquitinated by Parkin, and this facilitates the recruitment of mitophagy cargo proteins such as SQSTM1, calcium-binding coiled-coil domain 2 CALCOCO2/NDP52, and optineurin to trigger the mitophagy [77]. PARK6 gene which encodes PINK1 has shown to reduce in obesity and type 2 diabetes implicating its importance in glucose metabolism [89, 95]. Loss of PINK also results in mitochondrial dysfunction in pancreatic β cells and impairs glucose uptake [24]. Additionally, protein folding, aggregation, and accumulation are also crucial for the manifestation of type 2 diabetes. This confers T2DM as protein misfolding disorder (PMD). Pancreatic β cells also secrete amylin protein along with insulin, and its aggregation mediates pancreatic β cell dysfunction and type 2 diabetes [39, 48]. Amylin overexpression in pancreatic β cell has shown to activate mTOR and hampers mitophagy [44]. The levels of chaperone proteins (Bip, protein disulfide isomerase, chemical chaperone such as tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA)) also alter the pancreatic β cell viability and function and improve the glucose homeostasis in T2DM [12, 78]. To sum up, as the defects in mitophagy have been associated with all pancreatic malignancies, targeting mitophagy could be a promising therapeutic target.

5.12

Concluding Remarks

Mitochondria gained a substantial amount of interest in pancreatic malignancies due to its involvement in several vital cellular processes. Predominantly, mitochondrial ROS, mutations in mitochondrial DNA, altered mitochondrial proteins, mitochondrial antioxidant system, respiration, oxidative phosphorylation, defects in mitophagy, metabolic addiction such as rewiring of glucose, and glutamine metabolism have been reported in pancreatic malignancies. Because pancreatic malignancies are bidirectionally association with diabetes, antidiabetic drugs are popular and known to be effective for the treatment options. Therefore, mitochondria as a personalized treatment for pancreatic malignancies are an interesting approach and need to be validated more in the near future. Acknowledgements Authors would like to acknowledge funding from DST-SERB (File number CRG/2018/001028) and DBT (File number BT/PR10319/BRB/10/1267/2013) to Prof. Naresh Babu V Sepuri. Noble Kumar Talari would like to acknowledge CSIR (09/414(1192)/2019EMR-1) for the Research Associate fellowship. Ushodaya Mattam would like to acknowledge DBT (BT/PR19439/BIC/101/431/2016) for the fellowship. The authors also thank their lab members for suggestions.

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Targeting Mitochondrial Enzymes in Pancreatic Cancer Gowru Srivani, Begum Dariya, Afroz Alam, and Ganji Purnachandra Nagaraju

Abstract

Mitochondria is a well-known dynamic intracellular organelle. It performs emerging functions in the cell, such as production of energy (ATP generators), apoptosis, and cell cycle regulation. Over the past years, researchers have concentrated on the complexity of pancreatic cancer by mapping genetic mutations associated with it. In these efforts, the role of mitochondrion to the pathogenesis of pancreatic cancer has been neglected. However, recently a growing body of research showed the evidence that mitochondrial enzymes play a crucial role in development of pancreatic cancer. In fact, deregulated mitochondrial enzymes not only participate in the metabolic reprogramming of tumor cells but also modulate cellular processes involved in tumor proliferation and progression. In this review, we described the association between the deregulated mitochondrial enzymes and development of pancreatic cancer. Keywords

Mitochondria · ATP · Pancreatic cancer and mitochondrial enzymes

Abbreviations 2-HG 2SC AcCoA

2-hydroxyglutarate S-2-(succino)cysteine Acetyl coenzyme A

G. Srivani · B. Dariya · A. Alam Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India G. P. Nagaraju (*) Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_6

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Aco1 AMPK ATP Ca CAFs CS EMT ETC FADH FH FHD FMN HIF HLRCC ICDH IMM IRE IRP1 JHDMS KDMS KEAP MDH MTATP6 NADPH NRF2 OAA OG2 OMM OXPHOS PC PDAC PGC/PCC PHD PRMT4 R-2-HG ROS SDH Succ CoA TCA TET α-KG

G. Srivani et al.

Aconitase1 AMP-activated protein kinase Adenosine triphosphate Calcium Cancer-associated fibroblasts Citrate synthase Epithelial to mesenchymal transition Electron transport chain Flavin adenine dinucleotide Fumarate dehydrogenase Fumarate hydratase Flavin mononucleotides Hypoxia-inducible factor Hereditary leiomyomatosis and renal cell cancer Isocitrate dehydrogenase Inner membrane of mitochondrion Iron-responsive elements Iron regulatory protein-1 Jumonji-C histone demethylases Histone lysine demethylases Kelch-like ECH-associated protein-1 Malate dehydrogenase mt DNA gene encoding for the CV subunit 6 Nicotinamide adenine dinucleotide phosphate Nuclear factor erythroid 2-related factor-2 Oxaloacetate Oxoglutarate Outer membrane of mitochondrion Oxidative phosphorylation Pancreatic cancer Pancreatic ductal adenocarcinoma Paraganglioma and pheochromocytoma Prolyl hydroxylases Protein arginine methyltransferase 4 2-Hydroxyl glutamate Reactive oxygen species Succinate dehydrogenase Succinal coenzyme A Tricarboxylic acid Ten-eleven translocation family α-Ketoglutarate

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Targeting Mitochondrial Enzymes in Pancreatic Cancer

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Introduction

Pancreatic cancer (PC) is one of the fourth most aggressive cancers, diagnosed at a very late stage, with poor survival rate [1, 2]. It has greater metastatic potential and resistance to chemoradiotherapy [3]. Its development and resistance to treatment depend on the inhibition of the apoptosis. Although mitochondrion and their enzymes are identified as crucial regulators for cell death, their significance as target for cancer therapy has not been clinically explored [4]. Mainly, its initiation is associated with multiple interactions between host and causative factors. These interactions induce the genetic and metabolic alterations. The deregulated cellular energetics are correlated with mitochondrial dysfunction caused by defects in mitochondrial enzymes, altered tumor suppressor genes, as well as mutations (point mutation and copy number change) in the mitochondrial DNA. The mitochondrial enzymes including citrate synthase, succinate dehydrogenase (SDH), fumarate hydratase (FHD), and isocitrate dehydrogenase (ICDH) [5] are defected and are thus connected with sporadic or familial form of cancer. This contributes to the deregulating cellular energy, upregulation of enzymes, increasing proliferative signaling, activating metastasis, invasion, endorsing inflammation, angiogenesis, and resistance to apoptosis [6]. Mitochondria are an important master regulator of intracellular organelles present in the cell, which are involved in biosynthetic and bioenergetic functions to reach metabolic need of the cell [5, 7]. In addition, participated in the cellular homeostasis, like the formation of ATP via electron transport chain (ETC), and oxidative phosphorylation (OXPHOS) in combination with the oxidation of metabolic intermediates through TCA (tricarboxylic/citric acid) cycle. Moreover, it also participates in fatty acid catabolism by β-oxidation; production of heme, steroids, and pyrimidine; and production of excess reactive oxygen species (ROS) via regulation of calcium concentrations (Ca2+) and through trafficking of small metabolic substances. It is associated with the initiation and implementation of the cell senescence [8, 9]. Therefore, the abnormal communication between the mitochondrion and other cells may cause the changes in cellular homeostasis and organismal dysfunction. Certainly, dysfunction of mitochondrion has been associated with various pathological conditions such as neurodegenerative disorders, muscular degeneration, cardiovascular disorders, and cancer [10, 11]. Historically, the association between cancer cells and dysfunction of mitochondrion aimed at the metabolism [12]. In 1956, Warburg discovered the association between cancer cell, mitochondrial dysfunction [13], and the altered mitochondrial function that modulates the gene function, cell cycle, cell viability, and metabolism [14]. In this review, we discussed about the key role of altered mitochondrial enzymes and mitochondrial DNA mutations in initiating a complex cellular reprogramming that supports the development of PC growth.

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Mitochondria and Their Enzymes

Mitochondria are an important organelle for cellular activities. It is the powerhouse of the cell. It is a double membrane organelle [15] and differs from organism to organism. The number of mitochondrion varies per tissue, organelle, and cell type. There are no mitochondrion present in red blood cells, and more than 2000 mitochondrion are present in the liver cells [16]. Mitochondria has a double-stranded circular DNA with 16.6 kb molecular weight, 22 transfer RNAs, 13 respiratory enzyme complex polypeptides, and 2 ribosomal RNAs that are involved in posttranslation of mitochondrial protein synthesis and respiration [17]. Mitochondria are differentiated from the cytoplasm through its outer and inner membranes. The outer membrane of mitochondrion (OMM) is characterized porous. The small uncharged molecules and ions can be freely crossed through the porins (pore-forming membrane proteins) like voltage-dependent anion channels (VDAC). Additionally, OMM also have particular translocases for importing large molecules such as protein molecules. Inner membrane of mitochondrion (IMM) is rigid and has a diffusion barrier for small molecules and ions. All these molecules or ions pass through the specific membrane transport proteins that are specific for particular molecule or ions. Thus, formed ion selectivity develops an electrochemical membrane potential ~ 180 mV in inner membrane. Further, the oxidative phosphorylation (OXPHOS) takes place in group of membrane protein complexes that generates the electrochemical gradient potential throughout the inner membrane and is utilized for the synthesis of ATP [18]. The OMM and IMM of mitochondrion are classified into three compartments, each having its significant role in protein synthesis. The innermost compartment is mitochondrial matrix, which is present surrounding the inner membrane. Mitochondrial matrix contains ribosomes, organic molecules, mitochondrial DNA, inorganic ions, nucleotide factors, and soluble enzymes. It is the place for DNA replication, transcription, synthesis of proteins, and various enzymatic reactions. The biosynthetic reactions of citric acid cycle, electron transport chain, respiration, and urea cycle occurred within the mitochondrial matrix. These cycles play a vital role to produce substrates for the formation of various macromolecules such as amino acids, nucleotides, heme, iron sulfur clusters, lipids, and nicotinamide adenine dinucleotide phosphate (NADPH) in regulated concentration for antioxidant protection [10]. The matrix contains various enzymes including citrate synthase, isocitrate dehydrogenase (IDH), alpha-ketoglutarate dehydrogenase, fumarate, succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) that play a crucial role in citric acid cycle/ Krebs/TCA cycle and electron transport chain intermediates [10, 19]. These enzymes are frequently deregulated or mutated leading to tumorigenesis [20]. TCA cycle generates NADH and FADH2 through the electron transport chain that form a proton gradient throughout cristae (mitochondrial inner membrane) and produce ATP with the help of enzyme proton-ATP synthase. This permits protons to pass through the membrane in a unidirectional way, similar to chemiosmosis process [21, 22]. This metabolic process is called OXPHOS (oxidative phosphorylation), which oxidizes the nutrients to generate ATPs. In addition to

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this, redox reactions also happen during this process that generates ROS (reactive oxygen species) such as peroxide or superoxide via oxygen reduction; ROS are harmful intermediates of OXPHOS. This process is of high potentiality as it reproduces 36 ATP molecules, produced during glycolysis. This process mainly takes place at complex-I, cytochrome-C oxidase, and complex-IV [23, 24]. The deregulated mitochondrial functions are mainly caused by dysfunction of mitochondrial enzymes or mutations in the mitochondrial DNA. These disturb the cellular bioenergetic process and support the metabolic reprogramming for tumor cell proliferation and survival as well as activating the tumor-promoting changes through the harmful intermediates like ROS, calcium, and small metabolites like glutamine produced by the mitochondrion. Moreover, various oncogenes or deregulated tumor suppressor proteins including P53, HIF (hypoxia-inducible factor), and RAS regulate cellular metabolism and mitochondrial respiration. These play a vital role in determining how the mitochondrial enzymes are utilized in the tumor cells. Thus, the altered tumor suppressors or oncogenes provide a direct association between the dysfunction of mitochondrion and tumorigenesis [19].

6.3

Dysfunction of Mitochondrial Enzymes and Pancreatic Cancer

Deregulated mitochondrial enzymes including citric acid cycle enzymes can alter the cellular energetics, resulting in the characteristic metabolic changes that are associated with the tumor progression and development. A series of biochemical reactions in the citric acid cycle and electron transport chain (ETC) catalyzed by various enzymes may play a vital role in pancreatic cancer initiation, proliferation, and progression (Fig. 6.1).

6.4

Citrate Synthase

Citrate synthase is the first and rate-limiting step in citric acid cycle. This is an irreversible condensation reaction of AcCoA (acetyl coenzyme A) and OAA (oxaloacetate) into citrate [25]. Continuously citrate from the matrix to the TCA cycle, this step is indeed crucial for transport of AcCoA in the form of citrate from mitochondrion to cytosol, which is then involved in synthesis of fatty acids or acetylation of proteins [26]. Overexpression and increased enzymatic activity of citrate synthase cause tumors, including pancreatic cancer, ovarian cancer, and renal oncocytoma [27]. Unfortunately, the changes made by citrate synthase like reprogramming the mitochondrial function have not been determined. Furthermore, there is no clarity how dysfunction of citrate synthase can promote the tumor proliferation and progression. Moreover, there are two conditions assumed – in one condition, the elevated levels of citrate synthase can produce excess amount of citrate, promoting tumor cells for the biosynthesis of fatty acids. This is mostly seen in the pancreatic cancer [28]. In the other condition, loss of citrate synthase

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Fig. 6.1 Role of mitochondrial enzymes in pancreatic cancer development [85] Oncogenic metabolites R-2-HG, succinate, and fumarate are the substrates of the gain of function of cytosolic mitochondrial isoforms of IDH, SDH, and FH. These are intermediates of the citric acid cycle. Mutant tumor suppressor genes IDH, SDH, and FH generate the intracellular accumulation of succinate, fumarate, and 2-oxoglutarate (2-OG) dioxygenases, and also inhibit the PDH, KDMS, and TET, resulting in the HIF-1α gene modifications via epigenetic alterations that may be involved in the tumor development. Fumarate irreversibly alters the cysteine molecules in proteins through succination that leads to the activation of Keap1 and the integral activation of NRF2, resulting in the transcriptional genes associated with antioxidant response. Succination of the citric acid cycle enzyme aconitase 2 bound with the IRP1 proteins upregulated the HIF-1α activity and led to the unpaired aconitase function in FH-deficient cells IDH isocitrate dehydrogenase, SDH succinate dehydrogenase, FH fumarate dehydrogenase, MDH malate dehydrogenase, CS citrate synthase, PHD prolyl hydroxylases HIF hypoxia-inducible factor, TET ten-eleven translocation family, KDMS histone lysine demethylases, KEAP Kelch-like-ECH-associated protein-1, NRF2 nuclear factor erythroid 2-related factor-2, OAA oxalo acetate, AcCoA acetyl coenzyme A, RH R denotes HIF, DNA, histone, OG2 oxologltarate, R-2-HG 2-hydroxyl glutamate, Succ CoA succinal coenzyme A

could alter the mitochondrial dysfunction that activates glycolytic switch to promote tumor progression. In addition, loss of citrate synthase is also associated with the initiation of (EMT) epithelial to mesenchymal transition. This suggests that the loss of citrate synthase activity not only induces the metabolic reprogramming but also supports the tumor cell proliferation, invasion, and metastasis [29].

6.5

Aconitase

Aconitase is the second mitochondrial enzyme in the citric acid cycle. It converts citrate into isocitrate through the cis aconitase. It is also known as IRP1 (iron regulatory protein-1) and Fe-S cluster enzyme. It plays a major role in maintaining homeostasis of intracellular iron. Based upon the intracellular iron concentrations, it

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may either act as an aconitase or exist attached to IRE (iron-responsive elements) in the untranslated regions of mRNA elements [30, 31]. Modified translated proteins control the uptake, excess utilization, and storage of iron [31]. In addition, these IRP1 play a vital role in iron absorption and erythropoiesis by regulating HIF-2α (hypoxia-inducible factor 2 α). Thus, these IRP1 or iron-regulating functions of aconitase are also associated with the tumor initiation and progression. The alteration in iron metabolism is indeed a key element for tumor cell survival [31]. In 2016, Yuan et al. [32] revealed that increased circulating levels of TCA cycle metabolites like aconitase and isocitrate and mutations in ACO1 (aconitase 1) gene are associated with tumor cell progression and metastasis. Moreover, this approximately increases twofold high risk for mortality of pancreatic cancer. Furthermore, their study data identified that ACO1 gene can be taken as the novel prognostic marker for pancreatic cancer.

6.6

Isocitrate Dehydrogenase

Isocitrate dehydrogenase (IDH) plays a vital role in cellular metabolism, tumor initiation, and metastasis. IDH catalyzes the conversion of isocitrate into 2-OG (2-oxoglutarate) with NADP as a cofactor. IDH exists in three isoforms: IDH1, IDH2, and IDH3. Among them, IDH3 is NADH (nicotinamide adenine dinucleotide) dependent, and the rest, IDH1 and IDH2, are NADPH (nicotinamide adenine dinucleotide phosphate) dependent [33, 34]. In the cytosol, IDH1 is encoded by the IDH1 gene which is located on 2q33.3. It is involved in oxidative decarboxylation of isocitrate to α-ketoglutarate to form NADPH from NADP+. The mitochondrion IDH2 is encoded by the IDH2 gene located on 15q26.1. It is associated with the reversible conversion of α-ketoglutarate to isocitrate and reduces NADPH to NAD+. Moreover, NADPH provides enough energy for reductive carboxylation [35]. Both IDH1 and IDH2 are homodimers that are functionally and structurally similar. They play a vital role in the oxidative reductase reactions and cellular activities against the oxidative destructions, where NADPH acts as a co factor. These reversible reductive decarboxylation also play a key role in various cellular processes including glycolysis and lipogenesis regulated through isocitrate transferase (ICT) that also produces citrate through the aconitase enzyme [36–38]. The NAD-dependent IDH3 plays a well-established function in citric acid cycle and, in addition, also catalyzes the irreversible conversion of isocitrate into α-ketoglutarate. NAD+ reduces NADH [34]. These reactions and isoform of IDH3 are mainly regulated by the substrate concentrations, negative and positive allosteric effectors like ATP/ADP ratio, calcium levels, citrate, and NAD+/NADH levels. Calcium, citrate, and ADP activate IDH3 function, whereas NADH, NADPH, and ATP suppress its function. Further, the α-ketoglutarate is metabolized into succinate which is a four-carbon molecule, and NADH is utilized in the ETC (electron transport chain) to produce ATP [39]. IDH3 is a heterotetramer having two α-subunits, one β-subunit, and one γ-subunit. IDH3 A gene is located on 15q25.1–

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q25.2, IDH3 B on 20p13, and IDH3 G on Xq28 sequentially. Here, α-subunit acts as a catalytic unit, and the rest of the two β- and γ-subunits function as regulatory units [34, 40, 41]. Inherited IDH isoforms-cytoplasmic IDH1 and mitochondrial IDH2 have been identified in several cancers, including pancreatic cancer [42], colon cancer [43], glioma [44], acute myeloid leukemia [45], osteosarcoma [46], prostate cancer [47], glioblastoma [48], B-acute lymphoblastic leukemia [47], and intrahepatic cholangiocarcinoma [49]. The inherited IDH1 and IDH 2 convert α-KG (α-ketoglutarate) to reductive oncometabolite, i.e., 2-HG (2-hydroxyglutarate). The accumulation of high concentration of 2-HG in tumor cells enhances the cytokine independency, DNA, and histone methylation, which eventually results in the tumor cell dedifferentiation [50, 51]. The frequently occurring IDH mutants, IDH1-R132 and IDH2-R172, have the ability to produce 10–100-fold increased concentrations of R 2-HG. In 2012, Koivunen et al. showed in their study that mutant R-2-HGinduced PHD1 and PHD2 expression (also known as EgIN and EgIN2), thereby inhibiting HIF-1α activity, thus increases the tumor cell growth, proliferation, and cloning in soft-agar growth in astrocytes, indicating that HIF-1α in this regard may function as a tumor suppressor gene [52]. Recently in another study, Brody et al. revealed that IDH1 mutations were involved in the pancreatic cancer development and metastasis. This, for the first time, reveals a proof for intragenic mutations of IDH1 R132H in pancreatic cancer. As per the literature, IDH1 mutation-associated pancreatic cancer is very rare incident. Mutations in IDH1 causes the hypermethylation due to the increased level of 2-HG, which leads to neomorphic proliferation and dedifferentiation [53]. In support of this study, the identified mutant gain and the function of IDH1 have been found less efficient in catalyzing the reductive carboxylation reaction that converts α-KG (α-ketoglutarate) into isocitrate, leading to enhanced production of 2-HG. In addition, it was also observed that mutant IDH1 is responsible for the morphological changes and driver of epithelial to mesenchymal transition (EMT) in the later stages of pancreatic cancer, where it is extreamly susceptible to the harsh effects of hypoxia and chemotherapy [42, 54].

6.7

Succinate Dehydrogenase

Succinate dehydrogenase (SDH) is the only membrane-bound enzyme in the citric acid cycle. It is associated with the inner mitochondrial membrane that converts succinate (four-carbon molecule) into four-carbon molecule fumarate. It catalyzes the oxidation reaction without decarboxylation (reduction of FAD to FADH2) and produces FADH2. This also shows a unique connection between citric acid cycle and electron transport chain, where it is also notified as respiratory chain complex-II [54] and succinate-ubiquinone oxidoreductase. It is a highly conserved heterotetrameric enzyme complex that includes SDHA, SDHB, SDHC, and SDHD. SDHA and SDHB are mitochondrial matrix catalytic subunits; SDHA have FAD as a cofactor that binds to fumarate and succinate substrates; SDHB

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contains the electron carriers of Fe-S center (iron-sulfur); SDHC and SDHD comprise the cytochrome b which is inner membrane protein and forms the complex-III (coenzyme Q-CoQ) binding site. The complex of SDH needs two factors: SDHAF1 (SDH assembly factor 1) and SDHAF2 [55, 56]. It is the only enzyme encoded by the nuclear DNA (nDNA) and lacks proton-pumping activity. Mutations in the SDH subunits are responsible for succinate accumulation. Thus, resulted metabolic reprogramming of the tumor microenvironment, despite normoxic conditions, provides a strategic atmosphere for tumor growth, proliferation, and survival. However, succinate is well known as a classical housekeeping gene [57]. Deactivating mutations of SDH subunits have been associated with various cancers like sporadic and hereditary cancers that include paraganglioma/pheochromocytoma (PGC/PCC) [58], breast cancer, pancreatic neuroendocrine tumor [59], renal cancer, and gastrointestinal stromal cancer [58]. Suppression of SDH enhances cytosolic, mitochondrial succinate levels and behaves as an oncogenic metabolite. The suppression of α-KG-PHDS (α-ketoglutarate-dependent prolyl hydroxylase) leads to stabilization of HIF-1α. Subsequently, it is translocated into the nucleus and induces the neovascularization and changes energy metabolism from oxidation to glycolysis [55, 60–62]. Stabilization of HIF-1α also resulted from the deactivation of PHDs via ROS which are produced from the respiratory complex-III; in addition, the lack of SDH cells enhanced the oxidative stress and ROS generation [63, 64]. Activation of HIF-1α under hypoxic conditions, together with succinate accumulation, enhances malignancy progression of pancreatic cancer [65], though SDH act as a tumor suppressor gene. Oncometabolites such as succinate and fumarate also suppress other α-KG-dependent dioxygenases, including TET family of 5-methylcytosine hydroxylases, histone, DNA demethylases, and JHDMs (Jumonji-C histone demethylases), resulting in epigenetic alterations and genome-wide changes of histone methylations [66].

6.8

Fumarate Hydratase

Another citric acid cycle enzyme is fumarate hydratase (FH), which catalyzes the oxidation reaction that converts fumarate into malate. Similar to SDH, FH also behaves as a tumor suppressor. However, germline-mutated FH is identified in HLRCC (hereditary leiomyomatosis and renal cell cancer) [67]. Recently, in another research, germline-mutated FH is also identified in pheochromocytoma and paraganglioma (PGC/PCC) [68] and is found downregulated in sporadic clear cell carcinoma and glioblastoma [69]. Fumarate possesses some common characteristics like 2-HG and succinate and has a unique feature associated with its chemical structure; it can suppress various OG-dependent enzymes, including histone, DNA demethylases, and PHDs [68]. Loss of FH function leads to the accumulation of intracellular fumarate, stabilization of HIF-1α, glycolysis, and activation of HIF-1-dependent pathways. Under physiological conditions, fumarate is indeed quietly reactive with α,

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β-unsaturated electrophilic metabolites; it can covalently bind with cysteine residues in proteins to generate 2SC (S-(2-succino)-cysteine), and this process is called succination. This process stimulates loss of function in various proteins, including mitochondrial citric acid cycle enzyme aconitase, Keap1 (Kelch-like ECH-associated protein1), and activation of NRF2 (nuclear factor (erythroidderived2)-like protein) which is an antioxidant response sensor protein promoting the ROS signaling. Thus, the activation of various genes plays a vital role in tumor initiation and progression [70–73]. In one research study, it was observed that metabolic enzymes SDH and FH alterations of CAFs (cancer-associated fibroblasts) were associated with the development of pancreatic cancer with the upregulation of MiR-21. This study shows that FH also plays a key role in the progression of pancreatic cancer [74]. Similarly, another study suggested that FH mutations in kidney cancer are linked with decreased function of metabolic sensor and AMPK (AMP-activated protein kinase), resulting in the enhanced synthesis of proteins and fatty acid to support the continuing cellular anabolic process [75]. It is clear that FH is indirectly or directly linked with the development of various cancers, including pancreatic cancer.

6.9

Malate Dehydrogenase

Malate dehydrogenase is another key enzyme that reversibly converts malate into oxaloacetate using NAD/NADH coenzyme system [76]. There could be a special metabolic pathway in tumor mitochondrion that favors tumor progression, as several studies suggested that mitochondrial malate dehydrogenase can form binary complexes with citrate synthase, glutamate dehydrogenase, aspartate aminotransferase, and fumarase [77]. The upregulation of malic enzyme is observed in various types of human cancers. Furthermore, malic enzyme mRNA upregulation in bladder cancer tissue is observed and was suppressed due to overexpressed microRNA-612. It is clear that malate dehydrogenase is linked with cancer development [78]. Later, it was suggested that malate dehydrogenase 1(MDH1) is associated with pancreatic ductal adenocarcinoma (PDAC). Importantly, MDH1 R248 methylation was downregulated in human PDAC clinical samples and was observed along with low-level expression of protein arginine methyltransferase 4 (PRMT4/CARM1) [79]. In this regard, it is confirmed that MDH1 is a key metabolic enzyme in pancreatic and other cancers and could be a promising therapeutic target for drug development. Mutations in citric acid cycle enzymes CS, IDH, SDH, and FH have therefore been revealed as a significant metabolic reprogramming that also includes mitochondrial complex network, and thus, these modifications directly or indirectly support the development of tumorigenesis.

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NADH-Ubiquinone Oxidoreductase

NADH-ubiquinone oxidoreductase is a well-known complex-I (CI) and is the largest electron transport complex, composed of NADH dehydrogenase, flavin mononucleotides (FMN), Fe-S center, and ubiquinone. It catalyzes the transfer of two electrons from the NADH to ubiquinone FMN and generates four protons and NAD+, which are further pumped from the mitochondrial matrix into intermembrane space for the generation of ATP [25]. It is the first place for receiving the electrons in the electron transport chain and is the active site for the generation of ROS. Hence, mutations in CI can expressively modify the redox homeostasis and bioenergetics [80]. Mutant mitochondrial genes encoding CI have been associated with the development of several malignancies including pancreas, breast, renal, thyroid, colon, prostate, and bladder as well medulloblastoma, head, and neck cancers [81].

6.11

Adenosine Triphosphate Synthase

Adenosine triphosphate (ATP) synthase is known as the complex V (CV), which is the last enzyme in the oxidative phosphorylation. CV generates ATP molecules from ADP and inorganic phosphate through the utilization of the electrochemical potential gradient across the inner mitochondrial space. It is recently found that ATP synthase is a part of PTP (permeability transition port) [82], which is a membrane-embedded mitochondrial complex. It is also involved in various mitochondrion-dependent events such as apoptosis and calcium buffering. Mutations occurred in ATP synthase encoded by the mtDNA have been involved in tumor development including pancreatic, thyroid, and prostate cancers [83]. Recently, Shidara et al. showed oncogenic functions of CV, and they observed two different point mutations in MTATP6 (mt DNA gene encoding for the CV subunit 6). Interestingly, mutant ATP 6 enhances the cell growth in 2D culture and showed increased oncogenic activity. Further, reintroducing the wild-type ATP6, which is nuclear encoded, results in inhibiting tumor growth. This study thus suggested mutant cells showing decreased activity of apoptosis enhanced ROS production and also clearly showed the linkage between CV mutations and tumor development [84] (Fig. 6.2).

6.12

Conclusion

In this review, we explained the link between mitochondrial enzymes and pancreatic cancer that are caused by mutations in the mitochondrial enzymes. We also discussed the dysfunction of mitochondrion and its importance in cancer development, genetic changes, and activities of oncogenic metabolites SDH, IDH, and FH involvement in progression of pancreatic cancer. Thus, these mitochondrial enzymes in pancreatic and other cancers could be a promising therapeutic target for drug development.

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Fig. 6.2 Role of mutant IDH, SDH and, FH effect on ETC I and V resulting in the metabolic deregulation, modifying the amount of oxidative phosphorylation in tumor development. mt DNA mutations identified in NADH-dehydrogenase-UQ oxidoreductase (complex-I) and ATP synthase (complex-V) Complex-II succinate-UQ oxidoreductase, complex-III reduced UQ-Cytc reductase, Complex-IV reduced Cytc-Cyt oxidase, ATPIF ATP synthase inhibitory factor, UQ ubiquinone

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Diabetes with Pancreatic Ductal Adenocarcinoma Gowru Srivani, Begum Dariya, Afroz Alam, and Ganji Purnachandra Nagaraju

Abstract

Diabetes and pancreatic ductal adenocarcinoma (PDAC) are common diseases and affect the same organ, pancreas. PDAC has a poor prognosis and response to conservative therapy. Diabetes is recently been correlated with mortality and morbidity from PDAC. The association between diabetes and PDAC stems from the structural association between the endocrine and exocrine pancreas and aberrant expression of hormones from islets. It can also result from other etiological factors including stress, inflammation, smoking, alcohol consumption, change in the diet, as well as inherited syndromes that affect PDAC tissue. Epidemiological evidence suggests that diabetes increases the risk for PDAC development. Insulin resistance, hyperinsulimenia, hyperglycemia, chronic inflammation, and their elementary mechanisms can contribute to the development of diabetes-associated PDAC. Signal transduction pathways that regulate metabolic functions also play a crucial role in the development of PDAC, promoting tumor proliferation, cell growth, differentiation, angiogenesis, and metastasis. In another way, PDAC is also a causative factor for diabetes, although the mechanisms are not well understood. Effective biomarkers might thus help detect the increased risk of PDAC. Furthermore, greater understanding of the pathological mechanisms linking diabetes to PDAC could guide the development of new therapeutic agents to prevent diabetes associated with PDAC.

G. Srivani · B. Dariya · A. Alam Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India G. P. Nagaraju (*) Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_7

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Keywords

PDAC · Type 2 diabetes · Exocrine · Endocrine · Hyperglycemia · Hyperinsulinemia

Abbreviations 4-HNE Akt AMP AMPK ATP ASK-1 CI COX2 DNA ERK ETC FGD-PET FTZ-F1 GI GLUT GWAS HNF-3β IER IGFBP-1 IGF IGFR IKK IL-6 IL-8 IR IRS JNK LKB1 LOOH LRH1 MDA MEK MMP-7 mTOR NADPH NF-κB

4-hydroxyl-2-nonenal Protein kinase B Adenosine monophosphate AMP-activated protein kinase Adenosine triphosphate Apoptosis signaling kinase-1 Confidence interval Cyclooxygenase Deoxyribonucleic acid Extracellular signal-regulated kinases electron transport chain F-18-Fluoro-deoxyglucose (FDG)-positron emission tomography (PET) Fushi-tarazu factor-1 Glycemic index Glucose transporter Genome-wide association studies Hepatocyte nucleoside factor-3β Intermittent energy restriction Insulin growth factor-binding protein-1 Insulin growth factor Insulin growth factor receptors Inhibitor of kB kinase Interleukin-6 Interleukin-8 Insulin receptor Insulin receptor substrate 1 c-Jun N-terminal kinases Liver kinase B1 Lipid hydroperoxides Liver receptor homolog-1 Malondialdehyde Mitogen-activated protein kinase Matrix metalloproteinase-7 Mammalian target of rapamycin Nicotinamide adenine dinucleotide Nuclear factor kappa B

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NO NR5A2 PDAC PDX-1 PI3K RIP-1 RO ROS RR SO4 SODD STAT TNF-α TRADD VAT VEGF

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Nitric oxide Nuclear receptor superfamily member Pancreatic ductal adenocarcinoma Pancreatic duodenal homeobox Phosphatidylinositol 3 kinase Receptor interacting protein Alkoxyl radical Reactive oxygen species Relative risk Sulfate radical Silencer of death domain Signal transducer and activator of transcription 3 Tumor necrosis factor-α TNF receptor-associated death domain Vascular adipose tissue Vascular endothelial growth factor

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer and fourth cause for cancer-related mortalities in the world. Its prevalence and mortality rates of PDAC are almost equal (~ 38,000 deaths and 45,000 new cases in the USA, 2013) [1]; it has a poor survival rate of 6 months, and survival for 5 year is less than 8%. Ninety-five percent of pancreatic malignancies arise in the pancreatic exocrine glands [2, 3] leading to PDAC and are usually diagnosed at the head and neck region of the pancreas [2].The life span risk of PDAC is 1% in 65-year-old patients and prevalence increases with age [4]. PDAC is predicted to become the second leading cause for cancer mortality in the USA by 2030 [5]. It is mainly caused due to the lifestyle modifications including exposure to tobacco, irregular diet habits, high alcohol consumption, chronic stipulation (chronic pancreatitis and chronic inflammation), obesity, and diabetes (Fig. 7.1) [1]. Among these factors, long-standing diabetes can enhance the risk of PDAC development. Although the connection between PDAC and diabetes is biologically complex, it has been thoroughly investigated to elucidate its mechanisms of action. There seems to be a bidirectional association between diabetes and PDAC [6]. For instance, recent epidemiological studies have proven that the risk of developing different cancers (PDAC, breast, colorectal, liver, urinary bladder, endometrial) is increased in patients with elevated levels of diabetes [7]. This is due to the fact that both cancer and diabetes have related risk factors (RR, 1.5–2.0) including obesity, diet, heredity, and being physically inactive, which elevate the incidence of PDAC in patients [8]. Moreover, diabetes associated with PDAC is detected 3 years prior to the development of PDAC and is thus considered as a risk factor (1.73–1.94) for PDAC [9].

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Evidence for Diabetes as a Risk Factor for PDAC

Everhart et al. have observed a close relation between diabetes and PDAC in their 30 epidemiological studies. Using 20 studies for meta-analysis [10], they found that the pooled RR is 2.1 for diabetes within the time period of 1 year prior to PDAC diagnosis, while the death of pooled RR was 2.1 for diabetes for a time period of 5 years [10]. Finally, authors emphasized that PDAC can be considered as one of the complications of diabetes [10]. Furthermore, another study analyzing 35 cohort studies described an association between diabetes and PDAC [11], on the metaanalysis basis of 27 studies to evaluate the relative risk on prevalence and mortality. These results confirmed that high risk of PDAC was identified in less than 1 year of diagnosing diabetes (i.e., relative risk 5.4; 95% confidence interval (CI); 3.49–8.30) [11]. This data shows that a shorter duration is equal to a higher risk at 1.5-fold after 5–9 years [11]. The authors strongly concluded that diabetes is an early indicator for PDAC and that newly diagnosed diabetes patients should be extremely attentive to PDAC development [11]. In the USA, a case-control study based on the population conducted in 2153 controls with 526 prevalence cases significantly revealed that diabetes shows an optimistic trend (P ¼ 0.016) in high-risk patients with increasing years before cancer diagnosis. Risk (OR 1.3; 95% CI, 0.4–4.0) of onset of diabetes slightly increased within 1 year of cancer diagnosis [12]. A larger study on meta-analysis was conducted on pooled data from three casecontrol studies including 2192 incidence cases and 5113 controls of diabetes Diabetes

Inflammation

Insulin resistance Mutagens

Hyperinsulimenia Hyperglycemia Loss of physical activity Metabolic syndrome Fructuose

Susceptivity Family history Inheritated syndrome

Metabolites of alcohol Environmental carcinogens Processed food Ex.Red meat Saturated animal fat

Excess alcohol consumption Exposure to tobacco Obesity Pancreatitis Helicobacter pylori

PDAC

Fig. 7.1 The main causative factors for PDAC development and progression from diabetes. The main risk factors are hyperinsulimenia, metabolic syndrome, obesity, excess alcohol abuse, and helicobacter pylori

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associated with PDAC [13]. The findings revealed that PDAC risk was reduced as the duration of diabetes increased. PDAC risk estimated as per pooled data was as follows: individuals diagnosed with diabetes less than 2 years versus individuals with diabetes diagnosed greater than 2 years prior to diagnosis had ORs with 95% CIs of 8 years showed long duration of low plasma IGFBP-1 and higher risk for PDAC (RR 3.30, 95% CI, 1.48–7.35). Hence, these results indicate that low plasma IGFBP-1 levels significantly promote PDAC risk. Another study investigated the IGF-1 gene polymorphism to illuminate the underlying processes that mediate the correlation existing between diabetes and PDAC development. Results from this study revealed an association between the IGF-1 gene polymorphic variant IGF-1 3’UTR Ex4 (G > C) C allele, diabetes, and increased risk of PDAC development [31]. Finally, evidence from epidemiological

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studies indicates that the associations between long-standing diabetes, insulin resistance, and IGF-1 are important targets for conventional treatment of PDAC.

7.6

Inflammation

Evidences from various studies suggest that inflammation plays a crucial role in insulin effects, and type 2 diabetes upregulates the signaling pathways leading to PDAC development. A recent review suggests that adipose tissue in endocrine organs can regulate the release of fatty acids, adiponectin, leptin, PAL-1, VEGF, and proinflammatory cytokines including TNF-α, IL-6, and resistin [32]. Dysfunctions in the adipose tissue may thus play a vital role in the progression of diabetes and various cancers. Increased levels of proinflammatory cytokines released by dysregulated adipose tissue can promote tumorigenesis [33]. TNF-α acts as a key regulatory molecule in innate immunity and regulates apoptosis under physiological conditions. Increased levels of TNF-α bind to TNFRS, resulting in the production of inhibitory proteins such as SODD (silencer of death domain) and TRADD (TNF receptor-associated death domain) that bind to the extra adapter protein RIP-1 (receptor-interacting protein). This leads to the formation of the TRADD-RIP1-TRAF complex, which initiates various signal transduction pathways including the MAP3K cascade (ASK-1: apoptosis signaling kinase-1). This cascade is linked to the TRADD-RPI1-TRAF complex that activates MAP2Ks, MEK-4, and MEK-6, enhancing the expression of JNK (c-Jun N-terminal kinases) and TPL 2-MEK-ERK pathways and TNF-α-PAI-1/VEGF. RIP1 engages with MEKK-3 and TGF-β–TAK1 to activate the IKK (Inhibitor of kB kinase) complex, which undergoes phosphorylation leading to the ubiquitination of IkBα. This results in the release of free NF-kB subunits, subsequently translocated to the nucleus, and induces the gene transcription involved in cell proliferation and survival [34–36]. IL-6 plays a critical role in the inflammation reaction and B-cell maturation under physiological conditions. Elevated IL-6 levels resulted from the JAK–STAT3 signaling cascade activation induced cell cycle progression and increased the cell survival and invasion [36, 37]. These pathways contribute to PDAC development by diminishing immune function. Recently, Hertzer et al. [38] emphasized in their in vivo study on high-fat, high-calorie diets that induced Kras G12 mice displayed elevated levels of VAT (vascular adipose tissue), and obesity and inflammation were correlated with the highest risk for PDAC development. These results strongly propose that adipose tissue may serve as a therapeutic target to treat and prevent PDAC. Furthermore, studies investigating energy homeostasis support epidemiological data indicating a correlation between obesity, diabetes, and cancer death. For instance, few studies have shown that tumor cells can become more aggressive in overfed animals when compared to animals fed with controlled caloric diets [39, 40]. These studies show that caloric restriction can mediate intermittent energy restriction (IER) and inhibit the inflammation mediated-insulin effect of the diet on

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tumor development. They also specify the variation between tumors with respect to the signaling cascade involved and the type of diet that influences tumor progression. Moreover, these results suggest that IGF and IGF signaling can constitute therapeutic targets for the development of novel therapeutic strategies against various diseases including PDAC [40, 41].

7.7

Oxidative Stress

The evidences also revealed that insulin, IGF-1, and type 2 diabetes may promote increased risk for PDAC by enhanced oxidative stress [42]. This is mainly due to the increased release of free radicals in the mitochondria, including ROS (reactive oxygen species), that disrupt the functions of DNA, proteins, and lipids, resulting in imbalance of metabolism [43]. High oxidative stress can release large amounts of fatty acid peroxidases that are expressed in high toxicity mutagenic functions. These compounds are (MDA) malondialdehyde and 4-HNE (4-hydroxyl-2-nonenal), both released in the adipose tissue leading to dysfunctions of the metabolic system [44]. This oxidative stress is mainly caused by imbalances between antioxidants and free radicals, especially ROS. ROS are key regulatory molecules that can disrupt gene transduction and act as intermediate molecules in signaling pathways. They can affect mitogenic functions, viz., cell growth and survival, through growth factor receptors that stimulate cell migration and can alter the tumor microenvironment by inducing angiogenesis and inflammation. In fact, tumor cells adapt and maintain their ROS levels to escape from apoptosis [45]. In PDAC, NADPH (nicotinamide adenine dinucleotide) is the major factory for intracellular ROS generation. Most of the enzymes in the mitochondria are engrossed in oxidative stress. Numerous varieties of free radicals can contribute to oxidative stress, including peroxynitrite, nitric oxide (NO), alkoxyl radical (RO), lipid hydroperoxides (LOOH), sulfate radical (SO4), and nitrogen-centered radical and metal-oxygen complexes. In addition, other cell components are also engaged in the internal oxidative stress, viz., xanthine oxidase, peroxisome, and their enzymes, especially P450 complex-detoxifying enzymes and NADPH oxidase complex, which contains NOX family [46]. Along with these free radicals, ROS generates various redox agent groups that play a vital role in many intra- and extracellular processes [47]. ROS can also enhance various signaling cascades essential for triggering tumor progression via cell growth, proliferation, angiogenesis, invasion, and apoptosis. This includes cell differentiation mediated through MAPK, via NF-kB and ERK1/2 [48]; evasion of cell death by stimulation of JAK/STAT, c-SRC, and PIK3/Akt signal cascade [49]; angiogenesis mediated by the generation of VEGF and angiopoietin [50]; and metastasis and tissue incursion by matrix metalloproteinases (MMP) inflection into the extracellular matrix [50]. Indeed, various epidemiological studies have unveiled a connection between oxidative stress and insulin resistance-mediated diabetes with PDAC. Studies additionally indicate that oxidative stress activates the initiation of signaling events that promote

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inflammation and insulin resistance, leading to tumor proliferation, growth survival, and adaptation of metabolic activities to apoptosis evasion [42, 51, 52]. In PDAC, hypoxia is a distinguishing factor. In fact, the release of HIF by ROS induces PIK3/Akt pathways [53]. Furthermore, ROS can hinder autophagy through the upregulation of the signaling Akt/mTOR pathway. This data suggests the ROS can induce malignancy and is capable of promoting sensitivity to chemo drugs by inhibiting the mTOR pathway [54]. Some experimental studies have shown that antioxidant supplementation (α-lipoic acid or vitamin E) may reduce the oxidative stress caused by insulin resistance [55]. Inducing insulin resistance in the experimental models by feeding them high sucrose and fructose diet has revealed a direct relation between insulin resistance and oxidative stress. In this study, the postprandial time period showed that hyperglycemia might cause oxidative stress through different signaling mechanisms that increase ROS and superoxides by the ETC (electron transport chain) [56]. This leads to the cellular redox state, downregulates tyrosine phosphorylation, and upregulates the insulin receptor substrate 1, thus suppressing the insulin signaling pathway [56].

7.8

Genomic Associations of Diabetes and PDAC

In diabetes and PDAC, heterogeneity is a common feature. The epidemiological and clinical studies indicate that diabetes increases PDAC risk. Although the susceptibility of genetic variants is extensively different, there is a modest overlap. Interestingly, both diabetes and PDAC share common features: i) Etiological risk factors, i.e., obesity, exposure to smoke, and alcohol consumption, may influence the genetic factors to increase risk. ii) Segregation of Mendelian analysis shows that a number of families display hereditary patterns. iii) Diagnosing at different age periods shows the related risk in some families [57]. GWAS (genome-wide association studies) have revealed that certain unpredicted genetic variants might change the risk for causing diabetes, and few diabetes susceptibility gene loci are often involved in tumor differentiation and progression [58]. In diabetes, there are 50 gene variants identified as genetic risk factors with minimal effect, including PPPARG, TCF7L2, KDNJ11, HDF1B, FTO, SLC20A8, and WFS1, and are detected via GWAS analysis [59]. Interestingly, GWAS analysis has identified one of the PC susceptibility genes, namely NR5A2, also called as LRH1 (liver receptor homolog-1). NR5A2 is one of the orphan nuclear receptor superfamily members that encodes the nuclear receptor subfamily gene FTZ-F1 (fushi-tarazu factor-1). It is highly expressed in the liver, intestine, exocrine pancreas, and ovaries [60, 61]. Functionally, it plays a vital role in developing digestive organs, steroidogenesis, cholesterol, bile acid synthesis, inflammation regulation in gut and liver, and cell proliferation in certain diseases including PDAC [62–64]. Studies have revealed that NR5A2 overexpression can promote cell growth, proliferation, and metastasis in PDAC [65, 66]. Thus, it might be considered as a potential target for PDAC. NR5A2 regulates the expression of several genes, which

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play a crucial role in the development of pancreas, insulin secretion, and β-cell function. These include hepatocyte nucleoside factor-3β (HNF-3β), HNF-1α, HNF-4α, and PDX-1. Conversely, NR5A2 expression is controlled by HNF-3β and HNF-1 [67]. Among these, PDX-1 (pancreatic duodenal homeobox) is an important regulator of endocrine pancreas development and is also involved in maintaining the endocrine pancreas functions through triggering gene transcription of islet amyloid polypeptide, insulin, glucokinase, GLUT-2, and somatostatin [67, 68]. Mutations in the HNF-3β and PDX-1 genes can cause type 2 diabetes and maturity-onset diabetes [69]. PDX-1 overproduction in pancreas upregulates the expression of MMP-7 in PDAC leading to tumor cell proliferation and invasiveness [70]. Moreover, the mutation in K-ras gene in PDAC may promote the overexpression of PDX-1, resulting in tumor cell proliferation, invasiveness, and survival [71]. Furthermore, cells undergoing embryogenesis and organogenesis require PDX-1 for survival and development of endocrine pancreas, but the overexpression of PDX-1 leads to dysregulation of islet cells through enhanced PI3K/AKT- and RAS-mediated signaling, leading to PDAC development, including proliferation, invasiveness, angiogenesis, and survival [71]. A better understanding of the NR5A2 gene significance and underlying mechanisms of HNF and PDX-1 in the development of PDAC may serve as potential molecular biomarker to diagnose and treat diabetes-associated PDAC.

7.9

Antidiabetic Therapy as a Risk Factor for PDAC

Controlling glucose levels is the major objective of effective diabetic maintenance, which leads to diminish mortality and morbidity by decreasing the risk of diabetesassociated PDAC. Clinicians consider numerous factors when choosing diabetic medications such as the type of diabetes, chronic adverse effects (including hyperglycemia, weight gain, fluid retention, gastrointestinal intolerance), patient characteristics, comorbidities, and cost of the treatment. Type 1 diabetic patients account for ~5–10% of the diabetic population globally. The deficiency of insulin affected due to autoimmune damage of the pancreatic β-cells requires life-long insulin therapy. In contrast, type 2 diabetes is frequent and is the reason for 95% of the diabetic population worldwide. However, 80% of type 2 diabetes is connected with decreased physical activity and obesity and usually develops from the prediabetic stage, distinguished by hyperinsulinemia (insulin resistance). Furthermore, it is always accompanied by reduced insulin release. Hence, loss of insulin increases both during fasting and post meal hyperglycemia [72]. Continued loss of insulin production and decreased incretin effects as well as various pathophysiological deficiencies lead to hyperglycemia of type 2 diabetes, which requires increased insulin therapy. The selection of the pharmacological agent prescribed to the patient is based on clinical symptoms and ongoing risk factors. Singh et al. [73] conducted a meta-analysis on 11 studies (6 cohort, 3 casecontrol, and 2 randomized control trails (RCTs)) and reported 1770 PDAC cases in

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730,664 patients suffering with diabetes on several antidiabetic medications. This analysis shows a significant relationship between insulin, metformin, and sulfonylureas use and the risk of developing PDAC in diabetic patients. Metformin users did not show PDAC risk in diabetes patients (P ¼ 0.073) as compared with non-users of metformin. In contrast, the use of sulfonylureas showed a significant associated risk of PDAC in diabetic patients [73].

7.10

Metformin

Numerous diabetic studies have investigated treatment with biguanide metformin, an antidiabetic drug frequently prescribed alone or in combination therapy. The risk of cancer-related mortality is decreased by metformin therapy when compared to insulin and insulin secretagogues treatment [74]. An epidemiologic meta-analysis of two studies was carried out to evaluate the outcome of metformin on cancer prevalence and death rate in patients with diabetes. The results showed a 32% decrease with overall summary of RR for cancer as 0.69 and 95% for CI (confidence interval) as 0.61–0.79 following metformin treatment as compared to other antidiabetic drugs [75]. Furthermore, a meta-analysis of a retrospective cohort of 62,809 patients with diabetes [76] and another case-control study on 973 patients with PDAC versus 863 case controls [77] were conducted separately with same time period. These studies reveled that metformin users display reduced risk of PDAC compared to sulfonylurea and insulin users. The glucose levels are reduced by metformin by decreasing the production of hepatic glucose and enhancing the insulin sensitivity. It slightly elevates plasma insulin concentration and suppresses the growth-regulating effect of insulin and IGFs [78]. In diabetic patients, metformin treatment significantly induces endothelial function, such as reduced plasma levels of soluble vascular cell adhesion molecule 1, vascular endothelial growth factor, von Willebrand factor, and soluble E-selectin tissue-type plasminogen inhibitor and activator [78]. Metformin suppresses the proinflammatory cytokines (IL-6 and IL-8) through the inhibition of NF-kB activation by the PI3k-Akt pathway [79]. These molecules play a crucial role in fibrosis, thrombosis, and angiogenesis which are essential for the development and progression of PDAC [78].

7.11

Molecular Mechanism of Metformin

Metformin acts mainly by inhibiting hepatic gluconeogenesis, thus reducing circulating glucose levels, increasing insulin sensitivity, and reducing the intestinal absorption of glucose [80]. Various mechanisms have been suggested to elucidate the inhibitory function of metformin on hepatic gluconeogenesis and glucose uptake such as suppression of the mitochondrial respiratory chain complex and AMPK activation (AMP-activated protein kinase) signaling pathway [81].

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AMPK is a master controller of several metabolic pathways and can inhibit glucose production in hepatocytes through liver kinase B1 (LKB1). AMPK activation by LKB1 is necessary for mediating the blood glucose effect of metformin [82]. A study performed by Shaw et al. (2005) observed that loss of LKB1 leads to phosphorylation and increased activation of AMPK, suggesting that treatment with metformin can enhance the activity of AMPK and decrease blood glucose levels in a LKB1-dependent process [83]. ATP production is mainly carried out in the mitochondria by oxidative phosphorylation, such as catabolic mechanism, namely, amino acids and fatty acids oxidation. Metformin treatment inhibits mitochondrial electron transport chain complex-I to enhance the activation of AMPK [81, 84]. This decreases cellular ATP production and elevates AMP levels, leading to increased glucose uptake and glycolysis [84]. AMPK contributes to cellular energy (ATP) levels and is activated by low intracellular ATP concentrations. Metformin suppresses the mTORC1 pathway through AMPK [85], which responds to energy stress by inhibiting cell proliferation and protein synthesis, suppressing the mTOR1 pathway [86]. AMPK plays a crucial role as a metabolic checkpoint for harmonizing energy levels and cell growth to ensure the initiation and maintenance of normal cell division and polarity [87]. In another study, He et al. [88] revealed that metformin suppresses hepatic gluconeogenesis by phosphorylating CBP (CREB-binding protein) at serine 436 through AMPK-PKCi/l, resulting in the separation of the CREB-CBP-CRTC2 transcription complex and downregulating gluconeogenic genes. Pearce et al. [89] showed in their study that long-term therapy of metformin increases CD8 T-cell half-life and improves the efficacy of anticancer vaccination. Metformin promotes apoptosis in cancer cells through the LKB1/AMPK pathway. This pathway regulates the phosphorylation of p27 at Thr 198 to stabilize p27, which permits cells to survive growth factor withdrawal and metabolic stress by autophagy [90] (Fig. 7.2). The antitumor activity of metformin not only regulates hormonal and metabolic functions but also regulates cell division and immune function.

7.12

Metformin and Its PDAC Preventive Effects

Epidemiologically, antidiabetic medications have been associated with decreased incidence of PDAC recurrence and death in diabetes-related PDAC patients. Recently, Li et al. [91] performed meta-analysis studies on nine retrospective cohort studies and two randomized controlled trials. These studies showed a considerable development in survival (HR, 0.86%; 95% CI, 0.76–0.97, P < 0.05) in metformin users as compared with controls. This study suggests that the metformin effect depends on the stage of tumor, with noticeable improvement in the survival rate of patients with locally advanced diseases, however, not with metastatic PDAC patients [91]. Another study by Kisfalvi et al. [92] suggested that metformin significantly suppressed PDAC growth via disturbing the signaling crosstalk between GPCR (G protein-coupled receptor) and IGF (insulin-like growth factor) receptor. Furthermore, oral metformin administration showed that metformin can significantly reduce

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PANC-1 and MIA PaCa-2 cell growth, xenografted on the flank nude cells. Hence, these results indicate that metformin could be a potential preventive agent for human PDAC. Additionally, Li et al. [77] revealed that the risk of progressing PDAC in metformin users decreased by 62% (OR, 0.38; CI, 95%, 0.22–0.69; P ¼ 0.001), as compared with the non-users of metformin. Another retrospective study performed by Sadeghi et al. [93] revealed that PDAC patients with diabetes show improved survival when treated with metformin [93]. Metformin has also been shown to check the endorsement effect of high fat diet on N-nitrosobis (2-oxopropyl) amine-induced pancreatic carcinogenesis in Syrian hamsters [94] and to suppress PANC-1 and MIA PaCa-2 tumor growth in xenograft models using athymic nude mice [95]. Furthermore, metformin inhibited PDAC growth by various inflammatory and tumorigenesis signaling pathways. For instance, Tan et al. [96] showed in their study that metformin treatment significantly reduced tumor growth, volume, burden, and weight by downregulating the NF-κB-STAT3 signaling pathways and activating AMPK. In another study, Metformin suppressed angiogenesis by inhibiting VEGF release in the tumor microenvironment and reducing tumor neovascularization. Additionally, it increased the chemosensitivity of gemcitabine by inactivating pancreatic stellate cells in PDAC [97]. These findings illustrate the antitumor activity of metformin in preventing diabetes-associated PDAC and suggest a novel approach for PDAC therapy and prevention. Insulin secretagogues, such as sulfonylureas, are rapid acting glinides [8]. Sulfonylureas are crucial for raising the plasma insulin levels but effectively act when pancreatic β-cells are present [98]. Moreover, they induce β-cells to release plasma insulin leading to β-cell depolarization [8]. For the past 50 years, sulfonylureas, for example, glipizide, glyburide, and glimepiride, have been used to treat type 2 diabetes patients. While sulfonylurea drugs can effectively reduce blood glucose concentrations, they cause weight gain and hyperglycemia [8].

7.13

Molecular Mechanism of Sulfonylureas

Sulfonylureas increase the plasma insulin concentrations by blocking the ATP-sensitive K-channels in the pancreatic plasma membrane. The release of insulin is initiated by a series of events. In pancreatic β-cells, the (potassium) K+-channel plays a vital role in regulating insulin secretion in order to respond to sulfonylureas and nutrient secretagogues. The K+-channel is mainly located in smooth muscles, skeletal muscle, and β-cells of the pancreas, kidney epithelia, and neurons. Plasma insulin levels can be increased through two different mechanisms: first, physiological stimulation through pancreatic β-cells and, second, reduction in hepatic clearance of insulin. Given the secretary function of sulfonylureas, it acts by binding to specific receptors on pancreatic β-cells. Stimulated insulin secretion raises blood glucose levels. Glucose enters into the pancreatic β-cells where it is metabolized by glycoly-

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sis and mitochondria, producing ATP and leading to increased intracellular ATP levels. This provokes the closure of ATP-dependent K+-channels in the plasma membrane of β-cells, thus decreasing the membrane’s K-permeability and causing the depolarization of the cellular membrane. This in turn leads to the opening of voltage-dependent Ca 2+ channels that enhance Ca 2+ inflow to the cytosol, thus triggering the exocytosis of insulin. This process is mainly caused by the actomyosin contraction and mediates the release of large amounts of insulin (Fig. 7.2) [98, 99]. A number of studies have shown increased risk of cancer and cancer-related mortality among patients with diabetes treated with sulfonylurea as compared with patients treated with metformin and other drugs [77, 100, 101]. It is uncertain whether this greater risk is associated with the effects of sulfonylurea or the more protective effect of metformin or if it is due to the effects associated with therapy selection and cancer risk [100]. Therefore, studies evaluating the duration, age, dose, and persistence of use are required to examine the association with particular cancer sites. The tumor-inducing effects of increased insulin levels and resulting IGF-1, insulin use in diabetic patients, and its association with greater risk of cancer have harnessed a wide attention [102, 103]. The diabetic patients using insulin for longer periods have been associated with increased risk of various cancers, including PDAC; however, the patients who depend for short period of insulin dependency say for less than 3 years are also at higher risk [104, 105]. In a study evaluating pooled data from three large case-control studies, long-term (> 10 years) insulin users had reduced risk as compared with short-time insulin users. Therefore, shorttime (3–10 years of insulin) users showed a 20% increased risk compared to patients that never used insulin [13]. In another case study of PDAC patients, more than 80% of patients using insulin only started taking it at least 2 years prior to their cancer diagnosis, which resulted from diabetes causing PDAC or long-standing diabetes provoked by the PDAC [77]. These above studies shown that PDAC risk in diabetic patients is correlated with a reduced number of patients using insulin. Therefore, a larger study sample is required to review the feasible effect of insulin use on PDAC risk.

7.14

Conclusion

The association between diabetes and PDAC is biologically complex. The risk of PDAC progression is higher in diabetic patients, with significant associated factors that modulate metabolic functions. Greater understanding of these mechanisms may lead to prognostic biomarkers for the early detection of PDAC. The protective effect of the antidiabetic drugs against PDAC provides an opportunity for intervention and prevention of this cancer (Fig. 7.3).

126 Fig. 7.3 Molecular mechanism of sulfonylureas: insulin secretion from pancreatic β-cells. Sulfonylureas increases the plasma insulin levels by inhibiting the ATP-dependent K+-channels through Ca 2+ channels-mediated exocytosis process

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Sulfonylureas Binds to high affinity of sulfhonylaurea receptor on pancreatic β – cell Closures the influx of ATP dependent K+ - channels β – cell depolarization opens the voltage –dependent Ca 2+ channels

Increased Ca 2+ inflow in to the cytosol exocytosis Secreted large amount of insulin

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92. Kisfalvi K, Eibl G, Sinnett-Smith J, Rozengurt E (2009) Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth. Cancer Res 69(16):6539–6545 93. Sadeghi N, Abbruzzese JL, Yeung S-CJ, Hassan M, Li D (2012) Metformin use is associated with better survival of diabetic patients with pancreatic cancer. Clin Cancer Res 18 (10):2905–2912 94. Schneider MB, Matsuzaki H, Haorah J, Ulrich A, Standop J, Ding XZ, Adrian TE, Pour PM (2001) Prevention of pancreatic cancer induction in hamsters by metformin. Gastroenterology 120(5):1263–1270 95. Krisztina K, Aune M, James S-S, Guido E, Enrique R (2013) Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas 42(5):781 96. Tan X-L, Bhattacharyya KK, Dutta SK, Bamlet WR, Rabe KG, Wang E, Smyrk TC, Oberg AL, Petersen GM, Mukhopadhyay D (2015) Metformin suppresses pancreatic tumor growth with inhibition of NFκB/STAT3 inflammatory signaling. Pancreas 44(4):636–647 97. Qian W, Li J, Chen K, Jiang Z, Cheng L, Zhou C, Yan B, Cao J, Ma Q, Duan W (2018) Metformin suppresses tumor angiogenesis and enhances the chemosensitivity of gemcitabine in a genetically engineered mouse model of pancreatic cancer. Life Sci 208:253–261 98. Sola D, Rossi L, Schianca GPC, Maffioli P, Bigliocca M, Mella R, Corlianò F, Fra GP, Bartoli E, Derosa G (2015) Sulfonylureas and their use in clinical practice. Arch Med Sci 11 (4):840 99. Ashcroft FM (1996) Mechanisms of the glycaemic effects of sulfonylureas. Horm Metab Res 28(09):456–463 100. Bowker SL, Majumdar SR, Veugelers P, Johnson JA (2006) Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin. Diabetes Care 29 (2):254–258 101. Monami M, Lamanna C, Balzi D, Marchionni N, Mannucci E (2009) Sulphonylureas and cancer: a case–control study. Acta Diabetol 46(4):279 102. Smith U, Gale EAM (2009) Does diabetes therapy influence the risk of cancer? Diabetologia 52(9):1699–1708 103. Gerstein HC (2010) Does insulin therapy promote, reduce, or have a neutral effect on cancers? JAMA 303(5):446–447 104. Wang F, Gupta S, Holly EA (2006) Diabetes mellitus and pancreatic cancer in a populationbased case-control study in the San Francisco Bay Area, California. Cancer Epidemiol Biomark Prev 15(8):1458–1463 105. Andersen DK, Korc M, Petersen GM, Eibl G, Li D, Rickels MR, Chari ST, Abbruzzese JL (2017) Diabetes, pancreatogenic diabetes, and pancreatic cancer. Diabetes 66(5):1103–1110

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Role of Inflammatory Cytokines in the Initiation and Progression of Pancreatic Cancer Madanraj Appiya Santharam and Vignesh Dhandapani

Abstract

It is essential to identify different targets for pancreatic cancer (PC) as it is asymptomatic until it has metastasized to other organs. After which time, the convention methods to control cancer such as surgery and chemotherapy is no longer an option. Over the years, it has been noted that the inflammation is a major cause of initiation of pancreatitis, leading to cancer. Various inflammatory cytokines initiate and play a role in progression of this cancer. In this chapter, we will be discussing about major inflammatory cytokines that have been identified over the years. For each cytokine, we will be looking at the source of the cytokines, the signaling mechanism of that cytokine, its role in other cancers followed by pancreatic cancer. Since the survival of many diagnosed PC patients are limited to months, understanding the origins and basic cascades of these different cytokines could better help in identifying the targets and developing different therapeutic approaches in screening of the patients with this disease. Keywords

Cytokines · Pancreatic Cancer · Inflammation · Extra-cellular Matrix · Interleukins

Abbreviations AP-1 APC

Activating Protein 1 Antigen Presenting Cells

M. A. Santharam (*) University of Leicester, Leicester, UK e-mail: [email protected] V. Dhandapani Technische Universität Dresden, Dresden, Germany # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_8

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ASK BAD BMP CAF CD CLC CNTF DC ECM EMT ERK FADD GDF GDNF GM-CSF IFN IKK IL JAK JNK LAP LIF LLC LTBP MAPK/MEK MDSC MHC MLK MMP NF-κB NK OSM PanIN PDAC PI3K PSC PTEN RAGE RIP ROS RTK SBE SCID SMA SMAD

M. A. Santharam and V. Dhandapani

Apoptosis Signal-regulating Kinase BCL2 Associated Death Promoter Bone Morphogenetic Protein Cancer Associated Fibroblasts Cluster of Differentiation Cardiotrophin-Like Cytokine Ciliary Neurotrophic Factor Dendritic Cells Extra Cellular Matrix Epithelial-Mesenchymal Transition Extracellular Signal-Regulated Kinases Fas-Associated protein with Death Domain Growth and Differentiation Factors Glial cell line-Derived Neurotrophic Factor Granulocyte Macrophage Colony Stimulating Factor Interferon IκB Kinase Interleukin Janus Kinase c-Jun N-terminal Kinase Latency-Associated Peptide Leukemia Inhibitory Factory Large Latent Complex Latent TGF-β Binding Proteins Mitogen Activated Protein Kinases Myeloid Derived Suppressor Cells Major Histocompatibility Complex Mixed-Lineage Protein Kinases Matrix Metalloproteinase Nuclear Factor-κB Natural Killer Oncostatin M Pancreatic Intraepithelial Neoplasia Pancreatic Ductal Adenocarcinoma Phosphoinositide 3-Kinase Pancreatic Stellate Cells Phosphatase and Tensin Homolog Receptor for Advanced Glycation End products Receptor Interacting Protein Reactive Oxygen Species Receptor Tyrosine Kinase STAT Binding Elements Severe Combined Immunodeficiency Smooth Muscle Actin Small Mothers Against Decapentaplegic

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SOCS SODD SRF STAT TAK TGF TME TNF TRADD TRAF Tregs Tyk VEGF

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Suppressor of Cytokine Signaling Silencer of Death Domain protein Serum Response Factor Signal Transducer and Activator of Transcription TGF-β-Activated Kinase Transforming Growth Factor Tumor Microenvironment Tumor Necrosis Factor TNF Receptor-Associated Death Domain protein TNF Receptor-Associated Factor Regulatory T-cells Tyrosine Kinase Vascular Endothelial Growth Factor

Introduction

Pancreatic Ductal Adenocarcinoma (PDAC) being asymptomatic during its progression makes it very lethal with the 5-year survival rate as low as 6% [147], as conventional methods such as surgery or chemotherapy would not be beneficial due to the advanced stages of the disease. PDAC is second, just behind colon cancer, in estimated new cases and mortalities in the US for cancers originating in the digestive system [147]. Familial history increases the risk of getting mutation in oncogenes, contributing to the increase in number of new patients [15, 87]. Recent developments in the immunotherapy has made people develop multiple clinical trials involving the PD-1/CTLA-4 combination [34, 48, 78]. The limitations of these trials could stem from the ability of the cancer to maintain an immunosuppressive tumor microenvironment [41]. The tumor microenvironment (TME) of PDAC has an extensive layer of stroma (known as desmoplasia) due to the presence of cancer associated fibroblasts (CAFs) [159] and a large number of immunosuppressive cells that is consisted mainly of γδT-cells, regulatory T-cells (T-regs), myeloid derived suppressor cells (MDSCs) and M2 macrophages [32, 37, 76, 100]. Anti-inflammatory cytokines secreted these inflammatory infiltrates play a major role in aiding the development of PDAC. Chronic pancreatitis is also an important initiator of PDAC. Chronic inflammation in the pancreas causes repeated injury to the parenchymal cells resulting in a fibrogenic response [123]. Induced by pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α [89], this inflammation causes the quiescent pancreatic stellate cells to get activated and obtain a myofibroblast-like phenotype. These myofibroblasts secrete pro-fibrogenic cytokine TGF-β and other growth factors. This further induces the fibrosis in extracellular matrix (ECM) and ultimately leading to pancreatic cancer [106]. In this chapter, we will be expanding on the various cytokines – antiinflammatory and pro-inflammatory cytokines – secreted by different cell types and how they affect the initiation and progression of PDAC.

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Cytokines in Pancreatic Cancer

Cytokines are small, low-molecular weight proteins secreted by tumor cells and surrounding cells into the TME that modulate the biological metabolic processes such as differentiation, proliferation and migration of various cells [40, 117]. Inflammatory cytokines, both pro- and anti-inflammatory cytokines, have a clinical significance as they are upregulated in pancreatic cancer and their expression has a negative effect on the clinical outcome [43, 49]. There is an interplay between the T-regs, macrophages and the tumor cells in secreting anti-inflammatory cytokines to facilitate tumor growth and invasion. Furthermore, angiogenesis and immune evasion are supported by pro-inflammatory cytokines and growth factors [13, 56, 135].

8.3

Anti-inflammatory Cytokines

8.3.1

Interleukin-10 (IL-10)

IL-10 signals through Janus Kinase (JAK) – Signal Transducer and Activator of Transcription (STAT) signaling cascade as the other members of its Interleukin family. It is the founding member of the IL-10 interleukin superfamily which also consists of Interleukin 19 (IL-19), Interleukin 20 (IL-20), Interleukin 22 (IL-22), Interleukin 24 (IL-24), Interleukin 26 (IL-26) and type III Interferons (IFNs) [17]. IL-10 is secreted by a host of immune and non-immune cells based on the type of infection or inflammation [80, 116]. The soluble IL-10 binds to the extracellular tetrameric IL-10 receptor (IL-10R) consisting of two subunits – IL-10R1 and IL-10R2, that homodimerize to initiate the signaling. The dimerized receptors activate the intracellular Tyrosine Kinase-2 (Tyk2) associated with the IL-10R1 subunit that simultaneously activates the JAK1 constitutively bound on the IL-10R2 subunit. This causes STAT3, a nuclear translocating transcription factor to induce gene expression of anti-inflammatory genes, to dock on the IL-10R components and to get phosphorylated by JAK1 and Tyk2 [164]. The phosphorylated STAT3 un-docks and dimerizes in the cytoplasm before entering into the nucleus and binding to the STAT Binding Elements (SBE), a promoter for IL-10 responsive genes, to initiate the transcription of IL-10 target genes [144] (Fig. 8.1). Suppressor of Cytokine Signaling 3 (SOCS3), a part of the 8-member SOCS family of proteins, is one of the downstream target genes is induced by IL-10 [22] that acts a negative feedback regulator of its inducer [16]. Immediately after that publication, Ding et al. in 2003, showed that another member of that family, SOCS1 is also induced by IL-10 and it controls the IL-10-mediated immune responses [39]. IL-10 functions as a prominent cytokine in regulating the inflammation in our body. Its major role is to regulate the immune cells to prevent them from causing acute tissue damage during an infection [31]. The source of IL-10 was primarily identified to be Th2 helper CD4+ T-cells in 1989 [51] but was later linked to other CD4+ T-cell types such as Th1 CD4+ T-cells and CD4+CD25+FoxP3+ T-regs, the dendritic cells (DCs) and macrophages to inhibit the activity of NK-cells, B-cells,

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Fig. 8.1 The different cascades followed by the inflammatory cytokines leading to PDAC. The pro-inflammatory cytokines activate the signaling pathways to aid in the EMT of the cancer cells and the anti-inflammatory cells facilitate this cell proliferation through development of fibrous tissue in the tumor microenvironment surrounding the tumor. EMT epithelial mesenchymal transition, IL Interleukin, TNF-α Tumor Necrosis Factor alpha, TGF-β Transforming Growth Factor beta, PSC Pancreatic Stellate Cells

CD8 T-cells and the other innate immune cells [2, 156]. This is effectively done indirectly by down-regulating the Major Histocompatibility Complex (MHC-II) expression and its co-stimulatory molecule CD80/CD86 on the antigen presenting cells (APCs) such as DCs and Macrophages [81]. IL-10 also inhibits the production of pro-inflammatory cytokines such as IL-1β, IL-5, IL-6, IL-12, TNF-α [52, 139] and other chemokines [111] by DCs and Macrophages. The APCs are also directly targeted by IL-10 through its autocrine secretion which causes inhibition of DC trafficking to lymph nodes to present the antigens to the naïve T-cells to differentiate into activated T-cells [33]. Recently, it has been identified that IL-10 production is enhanced by IL-4 secreted by Th2 cells influencing the Th1 cells towards a selfregulatory phenotype [108]. Therefore, during any infection IL-10 uses direct and indirect methods to inhibit the activation of cytotoxic cells. A context dependent, paradoxical role has been observed for IL-10 in cancer. In some cancers, it acts a tumor promoter by promoting the tumor immune evasion whereas in some conditions, it acts a tumor suppressor by inhibiting the tumor growth through increased IL-10 secretion [99]. A study showed reduced tumor growth by using mouse models injected with cells overexpressing IL-10 [61] and it was further corroborated by the requirement of IL-10 for enabling the memory of CD8 T-cells [53]. In pancreatic cancer, IL-10 acts as a tumor promoter as high levels of IL-10 was measured in serum of PDAC clinical patients, who were associated with a poor survival rate [47]. Other previous studies have also observed increased level of IL-10 in serum along with high expression levels of IL-1, IL-6, IL-8, IL-17,

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IL-23 and TNF-α [19, 157]. Mesothelin, a surface marker associated with tumor growth in multiple neoplasms including pancreatic cancer [26], is recognized better by mesothelin specific CD8 T-cells following inhibition of IL-10 in the pancreatic tumor microenvironment [12]. Mechanistically, pancreatic tumor progression is also due to the development of an immunosuppressive environment created by the infiltration of IL-10 secreting γδT-cells and blocking the efficacy of αβT-cells in the PDAC [110]. In addition to this, in vitro pancreatic cancer cells having a KRASG12D mutation converts CD4+CD25- T-cells to T-regs and activate the MEK/ERK pathway resulting in the secretion of IL-10 and TGF-β [27]. Activation of MAPK kinase pathway, especially ERK1 and ERK2, is essential for the induction of IL-10 by Th1 cells [138]. All these data indicate an essential role of IL-10 in prognosis and pathogenesis of PDAC.

8.4

Transforming Growth Factor (TGF-b)

TGF-β was initially identified in early 1980s, along with TGF-α, as a protein which had the capability to form large colonies of murine sarcoma virus-transformed 3T3 cells in soft agar [5]. When it was first discovered, the function of TGF-β was widely conflicted as it had more than one function and this was against the notion at that time that each hormone had only one function in the body. But eventually it was identified that TGF-β has a context dependent signaling in cells [102]. TGF-β superfamily consists glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic protein (BMP)/growth and differentiation factors (GDF), Activins and Inhibins, and TGF-β ligands [7, 137]. The polypeptide TGF-β ligands are constituted by TGFβ1, TGFβ2 and TGFβ3, that bind to its receptors TGFβR1 and TGFβR2, with the latter being the specific receptor required for all the three ligands [4, 130]. The production of latent inactive TGFβ is started in the Endoplasmic Reticulum (ER) consisting of the latency-associated peptide (LAP) region and TGF-β region as pre-pro-TGF-β peptide [119]. This peptide dimerizes as the low-glycosylated pro-TGF-β and enters into the Golgi complex. In the Golgi, furin binds to this 82-kDa protein and forms a high-glycosylated pro-TGF-β complex [119]. This complex could be immaturely secreted, or a more stable complex could form as high-glycosylated latent TGF-β complex that houses the TGF-β portion in between the LAP regions. This complex is secreted out as a latent TGF-β [119] . It could also be surface bound on T-regs [112]. The combination of latent TGF-β and LAP is called as the small latent complex and together with the Latent TGF-β binding proteins (LTBP 1-4) in the Extra-Cellular Matrix (ECM), it is called as large latent complex (LLC) [165]. The integrins of the cells bind to the LAP of the LLC and pull the LAP to open up the TGF-β for its receptor. The ECM and MatrixMetalloproteinases (MMP) break the TGF-β from the LLC. If the ECM is rigid, it is easier for the cytokine to get released and then MMP2 and MMP9 cleave the LAP to release TGF-β [165]. Once the active TGF-β binds to its receptors, they signal through the intracellular Serine/Threonine Kinase (Ser/Thr) domain unlike the other cytokines which signal

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via the Tyrosine Kinase domain (RTK). The TGFβR2 phosphorylates TGFβR1 of the receptor complex and classical TGFβ signaling pathway involves recruitment of the SMAD2 and SMAD3 and subsequently getting phosphorylated themselves [121]. These activated SMADs bind to the SMAD4 before translocating to nucleus and binding to the promoter regions of its target genes along with some of the other transcription factors to initiate various cellular responses [75] (Fig. 8.1). The SMAD7, co-binding with NEDD4 and Smurf1/2, is the natural regulator of the pathway [1]. The non-canonical pathway activates numerous other pathways such as JNK, P38, PI3K/AKT pathways [75, 121]. The primary function of TGF-β is to be an anti-inflammatory cytokine and suppress pro-inflammatory responses of many immune cells. The variety of immune cells regulated by TGF-β has been extensively reviewed by Mo et al. [92]. But in cancer, its role is paradoxical as it behaves as both tumor promoter and tumor suppressor in a context dependent manner. When the tumor is in its nascent stage, TGF-β tries to inhibit the growth of tumor by exerting growth arrest, causing apoptosis and preventing inflammation [67]. Initial studies also suggested the same as indicated by the absence of a functional TGF-β signaling in many human PDAC cell lines [158]. But as the tumor progresses, it turns detrimental to the body by promoting cancer through induction of epithelial-mesenchymal transition (EMT), promoting immune evasion and angiogenesis. Cancer stem cells enriched from the adjacent population of a pancreatic cell line showed enhanced ability to change from epithelial morphology to mesenchymal-like morphology on addition of TGF-β [82]. It also has pro-metastatic abilities where it modifies the TME to facilitate the extravasation of tumor cells and promotes colonization [67]. In 2003, it was shown that canonical TGF-β signaling was defective in pancreatic cancer cell lines [114]. Moreover, clinical studies have determined high levels of TGF-β in PDAC patients [77, 171]. This high level of TGF-β in the plasma heavily promotes fibrosis in the chronic pancreatitis through the increase of pancreatic stellate cells (PSCs) and induction of MMPs in ECM [46, 145]. PDAC cells secrete TGF-β to transform quiescent PSC into activated PSC that have an autocrine secretion of various cytokines including TGF-β and this activated PSC increases matrix deposition, upregulates MMPs and downregulates TIMPs ultimately resulting in fibrosis in chronic pancreatitis and cancer [21]. A cascade of MAPK signaling pathways – JNK, p38 and ERK – is activated in PSCs by TGF-β to induce fibrosis. Addition of pharma logical inhibitors of these pathways downregulated the expression of TGF-β induced fibronectin and α-SMA, thereby fibrosis [169]. Recently, it has been reported that pancreatic cancer cell-derived exosomes could induce the fibrogenic proteins mentioned above when they were added to human primary PSCs. This study identified that TGF-β was an upstream target of these cancer derived exosomes [101]. To the pancreatic cancer themselves, TGF-β downregulated a potent tumor suppressor PTEN by activating NF-κB [28]. Canonical Wnt signaling is also induced by TGF-β through production of homeobox transcription factor CUTL1, whose target gene is Wnt-5A ligand thereby promoting pancreatic cancer [131]. Another transcription factor KLF11 was inhibited by oncogenic ERK/MAPK

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pathway in PDAC, which had the function of terminating the inhibitory SMAD7 in the canonical TGF-β signaling in normal epithelial cells [45]. Therapeutic inhibitors at various levels of TGF-β signaling pathway in different cancers are being researched all over the world. Specifically, for the pancreatic carcinoma, E-cadherin is downregulated in cell lines of pancreatic cancer [151] but TGFβR2 targeting miR-655 has been identified a potential inhibitor which targets the EMT by inducing the expression of E-cadherin using Panc-1 cell line [63]. A kinase inhibitor, LY2109761, targeting the TGFβR1 and TGFβR2 significantly suppressed metastasis in orthotropic PC [107]. At the ligand level, BMP2 – a ligand from the TGF-β superfamily – inhibits the activation of PSCs by controlling the induction of α-SMA, fibronectin and collagen Ia [58]. From all these studies, it is evident that TGF-β is a key cytokine in promoting pancreatic cancer and targeting of its defective pathway in cancer cells would lead us in finding a solution for reduction of fibrosis in PC.

8.5

Pro-inflammatory Cytokines

8.5.1

Tumor Necrosis Factor-a (TNF-a)

TNF-α is from the TNF super family proteins that includes type II transmembrane proteins consisting of TNF homology domains and form trimers [8]. When the TNF-α is initially translated, it is membrane bound. It is in its inactive form, similar to TGF-β, is of size 26-28-kDa and is predominantly produced by macrophages and CD4+ lymphocytes, but also by eosinophils, mast cells, neurons, neutrophils and NK cells [57, 120]. Inactive TNF-α is converted to a soluble form (sTNF) by the metalloprotease called as TNF alpha converting enzyme [18]. The secreted TNF-α assumes a pyramid shape and has a smaller molecular weight of 17-kDa. Both the types of the cytokine, soluble and membrane bound TNF-α, contributes to distinct biological functions [122]. TNF-α can bind either to a 55-kDa receptor 1 (TNFR1) or a 75-kDa receptor 2 (TNFR2) [155]. The binding of the ligand to its receptors leads the receptors to trimerize. This subsequently enables the release of inhibitory protein silencer of death domain protein (SODD) and hence the adaptor protein TNF receptorassociated death domain protein (TRADD) binds to the death domain leading to the activation of three pathways [24, 160]. The first of the pathways is the activation of Nuclear Factor-κB (NF-κB) signaling cascade. NF-κB, as a transcription factor, enables the transcription of variety of proteins that aid in cell survival, proliferation and inflammatory response [60]. TRADD aids in the binding of TNF receptorassociated factor 2 (TRAF2) and Receptor Interacting Protein (RIP). TRAF2 recruits IκB kinase (IKK) to bind to itself. IKK phosphorylates the inhibitory protein IκBα, that is found to be bound to NF-κB complex. and releases NF-κB to be translocated to the nucleus for its transcriptional activity [65] (Fig. 8.1). The second pathway is the activation of MAP kinase pathway. JNK (c-Jun N-terminal kinases) are kinases that phosphorylates specific transcription domains such as c-Jun. TNF-α is found to

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be strongly activating the MAPK pathway associated with JNK phosphorylation. TRAF2 and Rac complex enables the activation of kinases of mixed-lineage protein kinases (MLK2/MLK3), MEK kinase 1 (MEKK1), TGF-β-activated kinase (TAK1), Apoptosis signal-regulating kinase 1 (ASK1). These kinases phosphorylate MAPK kinase 7 (MKK7) and in-turn activate JNK kinases [84]. The third pathway is the induction of death signaling. TNF-α-induced cell death consumes a very small portion compared to the inflammatory signaling. TRADD recruits Fas-associated protein with death domain (FADD) and this consequently binds to cysteine protease Caspase-8. Higher concentration of caspase 8 results in proteolytic activation of other caspases and results in cell apoptosis [59]. In the sequence of the pathways, there has been conflicting pathways reported to take place due to the cross talk between the components involved. These cross talks could enable differential expression of the transcription factors and thereby form a critical part in shaping the biological functions [118]. The amount of reactive oxygen species (ROS) is also found to influence and shift the balance favoring one of the three pathways [154]. TNFR1 is responsible for causing inflammation by enabling the activation of transcription factors in the NF-κB, JNK and MAPK signaling pathways [36]. Activated TNFR1 induce cell death by many signaling pathways including activation of pro-apoptotic Bcl-2 family of proteins and ROS [38]. TNFR2 is found to induce an anti-inflammatory response [44]. TNF-α which is produced by the tumor cells influences the surrounding cells presenting in the TME to produce a cascade of chemokines and other cytokines. This results in the promotion of tumor growth, angiogenesis, metastases and finally forms the immune evasive environment [9, 143, 161]. The mechanism of TNF-α causing pancreatic cancer is an increase of pro-inflammatory macrophages (M1) as compared to the anti-inflammatory macrophages (M2). With the increase in M1, there is an increase in the secretion of TNF-α. This in-turn induces DNA damage due to the upregulation in the production of reactive nitrogen species (RNS) and ROS [9]. The altered mechanism in the DNA repair fails to expand normal cells while enhancing the tumor cell expansion. In the TME, the PDAC tumor cells and the M1 macrophages produce TNF-α and this TNF-α induces the proliferation of the tumor cells and M2 macrophages and hence produces stroma and desmoplasia [9, 89]. Finally, this mechanism results in a condition called cachexia and insulin resistance. In 1993, studies indicated that use of anti-TNF-α antibodies could inhibit the progression of cancer [9]. The hypothesis of these antibodies impairing the desmoplastic tumor stroma was verified through in vitro mice experiments as there was a decrease in the number of cellular components and the amount of collagen [181]. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved five potent anti-TNF-α biologics – adalimumab, certolizumab, etanercept, infliximab, and golimumab. In vivo studies comparing infliximab and etanercept proved that the former elicits higher anti-tumor capacity [44]. The anti-TNF-α treatment has resulted in better effect in a short term, while in long term, recurrence of PDAC or an onset have also been reported in rare cases [6, 96, 163]. PDAC has been characterized by the upregulation of oncogenic miRNA

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and downregulation of tumor suppressing miRNA [64]. Studies with patients with PDAC and other diseases treated with anti-TNF-α indicated that certain miRNA such as miR-197, miR-23a, miR-221, miR-227, let-7d were found to be upregulated and associated with pancreatic cancer [23, 29, 35, 54, 88, 124, 172]. Many more therapeutic targets are still under development for PDAC.

8.5.2

Interleukin-6 (IL-6)

IL-6 is a pleiotropic cytokine that has dual inflammatory properties. It is secreted by the osteoblasts (to induce osteoclast formation) and the smooth muscle cells. It is also a potent inducer of acute response and aids in the differentiation of β-cells to Ig-secreting cells [152]. IL-6 belongs to family of cytokines that includes leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC), oncostatin M (OSM), IL-11 and ciliary neurotrophic factor (CNTF). Its structural form is a four-helix bundle [150]. The signaling pathway for IL-6 occurs via the signal transducing complex gp-130 (CD130). Upon binding to the IL-6 cytokine, the IL-6 receptor (IL-6R) and the gp-130 form a complex and thereby activate the signal transduction through JAKSTAT [66]. IL-6 receptor has been identified to be of two types – soluble (sIL-6R) and membrane bound IL-6R (mIL-6R). IL-6 either binds to the soluble IL-6 receptor (sIL-6R) to follow the trans signaling pathway [115] or to the membrane bound (mIL-6R), following the classical signaling pathway [42, 133, 134]. mIL-6R is expressed only by certain types of cells such as the hepatocytes, macrophages and neutrophils. sIL-6R has been identified to be a result of ectodomain shedding from the cell and alternate splicing. The sIL-6R interaction with IL-6 is described to cause a proinflammatory response [132, 140]. IL-6 binds to the receptor and activates JAK and Ras mediated signaling which leads to the phosphorylation of STATs (STAT2 and STAT3) and SHP2 domain containing tyrosine phosphate [113]. Activated STAT3 forms dimer and translocate to the nucleus to activate genes of the STAT3 response elements (Fig. 8.1). SHP2 connects the receptor to Ras/MAP kinase pathway, which is essential for mitogenic activity [93, 141]. The Ras mediated pathway activates the proteins downstream of MAP kinases and activates transcription factors such as NF-κB and Elk1. These transcription factors in combination with other transcription factors such as SRF (Serum Response Factor) and Activating Protein-1 (AP-1) further regulate a set of promotors [11]. IL-6 also activates the Phosphoinositide-3 kinase pathway (PI3K/ Akt/NF-κB cascade) which results in the maximum anti-apoptotic effect of IL-6 against TGF-β. This anti-apoptotic effect is due to the phosphorylation of BCL2 associated death promoter (BAD) by Akt. Termination of IL-6 signaling happens through tyrosine phosphatases, proteases and JAK kinase inhibitors (SOCS, PIAS) [170]. IL-6 associated with PDAC has been identified due to the dysregulation of JAK2STAT3 signaling pathway activation [71]. STAT3 is an anti-apoptotic regulator of a varied number of genes responsible for angiogenesis, tumor growth and survival

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[176]. STAT3 dysregulation is found to be a key in cancer progression. A role of STAT3 in pancreatic lesion development and pancreatic intraepithelial neoplasia (PanIN) has been identified [30, 55]. In addition, IL-6 was also identified to be involved in the pancreatic cancer cell migration by the activation of a specific GTPase CDC42 [128]. Absence of IL-6 in lesions of PanIN’s resulted in the decreased activation of MAPK, Akt and STAT3 signaling mechanisms and hence had a reduced proliferation rate [179]. A link between pancreatic cancer development and inflammation has been long established [97, 109, 123]. IL-6 was examined to be over expressed in tissues of pancreatic cancer compared to the controls [14]. The serum level of the IL-6 is used as a biomarker of malignancy but the specificity and sensitivity of this test are highly variable [85]. Mesothelin, a pancreatic cancer marker, has been found to influence the IL-6 expression in PDAC [79]. Second molecule which influences this protein’s expression is the receptor for advanced glycation end products (RAGE) [24]. KRAS plays an important role in pancreatic tumorigenesis [83]. Zinc transporter ZIP4 is also associated with pancreatic cancer progression [178]. IL-6 expression was also observed to be increased in cases of hypoxia induced by miR-21, a pro-tumorigenic miRNA [10, 50]. IL-6 promotes angiogenesis and invasion by increasing the expression of Vascular Endothelial Growth Factor (VEGF) and Matrix metalloproteinase-2 (MMP-2) [72, 103, 153]. IL-6 enhances the expression of IL-5, IL-7, IL10, IL-13 and granulocyte macrophage-colony stimulating factor (GM-CSF), thereby providing a pro-tumorigenic environment in pancreatic cancer [50]. IL-6 increased the expression of neuropilin-1, which subsequently increased the MAPK signaling, thereby inducing chemo-resistance and anoikis in pancreatic cancer [50, 162]. KRASG12D model system was found to be the ideal model system to understand the initiation and development of PDAC [69]. IL-6 was also found to predominantly expressed in the infiltrating immune cells (F4/80+ macrophages) as compared to the acinar cells [91]. Chimeric anti-IL-6 monoclonal antibody called siltuximab was able to stabilize disease in ovarian cancer [173]. A humanized antibody, Tocilizumab, has successfully demonstrated to treat cancer cachexia and suppressed the growth of human squamous cell carcinoma [3, 146]. Unfortunately, no clinical trials identifying antiIL-6 for treatment of PDAC has been performed. Treating KPCY mice having PanIN with anti-IL-6 was able to reduce the count of precursor lesions [179]. Adjuvant and palliative inhibition of classical signaling by tocilizumab and trans signaling by sgp130Fc reduced tumor growth of pancreatic tumor cells in SCID mice [71]. AntiIL-6R antibodies targeted LY6Chi monocytes in mouse were able to inhibit the STAT3 activation and decreased tumor cell progression in vivo. IL-6R blockade coupled with chemotherapy was able to induce tumor apoptosis, regression and increased the survival [95]. Bazedoxifene was found to inhibit JAK1 binding and STAT3 phosphorylation. It also inhibited cell proliferation, viability and glycolysis of pancreatic cancer cells [25]. Suppression of IL-6 by sh-RNA increased cell apoptosis and increased the antitumor effect of gemcitabine [168]. Promising studies on the therapeutic targets for IL-6 in PC has been done in mouse models but clinical translations are yet to come.

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Interleukin-17 (IL-17)

IL-17, a pro-inflammatory cytokine, is homo-dimeric and scereted by a CD4+ T helper cell subgroup called as T helper 17 cells (Th17 cells) in response to stimulation with IL-23 [70]. IL-17 is also produced by other adaptive immune cells such as γδ T-cells [62]. IL-17 can be further classified as six different proteins, from IL-17A to IL-17F, based on its amino acid sequence homology [74, 90, 136, 175]. IL-17A and IL-17F signal through the heterodimeric IL-17RA/RC receptors. The effect of IL-17A signaling is higher than the IL-17F signaling [182]. The IL-17R receptor complex undergoes a conformational change when the cytokine binds. This conformational change aids in homotypic interactions between the SEF/IL-17R domain (SEFIR) and signal adaptor Act1 [125]. This could cause the initiation of the canonical pathway, wherein the E3 Ubiquitin (Ub) ligase activity of Act1 could aid in the Lys-63 mediated ubiquitylation of the TRAF6 [142]. Furthermore, the canonical NF-κB, c/EBPβδ and MAPK pathways are activated [174]. This enables transcriptional activation of genes involved in the release of chemokines, proinflammatory cytokines and antimicrobial peptides. Non-canonical pathway is initiated by the phosphorylation of Act1 at the amino acid residue 311 by the IκB kinase (IKKi) and TBK-1 [20, 126]. TRAF2 and TRAF5 are recruited to the site of receptor complex and could sequester the effect of the RNA destabilizing factor ASF/SF2. On the contrary, mRNA stabilizing factor, HuR, is recruited that increases the half-life of mRNAs [68, 149]. IL-17E is found to signal through a heteromeric receptor, similar to 17A or 17F but the heterodimeric complex consists of IL-17RB, instead of IL-17RC, to bind to IL-17RA [129]. Similar to 17A canonical signaling cascade, intracellular portion of IL-17RA/RB complex also enables Act1 to recruit TRAF6, thereby activating the MAPK ad NF-κB pathways [98]. However, in contrast, Act1 could recruit TRAF4 aiding in further recruitment of E3 Ub ligase SMURF2. Ubiquitylation and degradation of IL-17RB inhibitor DAZAP2 helps in the subsequent IL-17E mediated signaling [177]. Act 1 independent activation of STAT5 could also initiate Th2 response [166]. The downstream signaling of IL-17B, C and D remains unclear. IL-17B is found to engage in proinflammatory secretion to enhance inflammation in pancreatic and breast cancer [73]. IL-17C is identified to signal through a heterodimeric IL-17RA/E complex [127] and activates the MAPK and NF-κB pathway [148]. The role of IL-17 as a pro-inflammatory cytokine in pancreatic cancer initiation and progression has not been investigated clearly and there are only a limited number of studies elucidating the factors contributing to it. IL-17, produced by the Th17 cells, have been found to be actively involved in causing the chronic inflammation leading to tumorigenesis [86, 167]. Human pancreatic cancer lesions have been found to be infiltrated by Th17 cells with overexpression of IL-17RA. Oncogenic KRAS and chronic pancreatitis populate Th17 cells and IL-17+ γδT cells at the tumor microenvironment. Mouse study with increase in expression of IL-17A indicated an increase in the occurrence of panIN [104]. REG-3β has been illustrated to promote KRAS mediated PDAC initiation, cell proliferation and inhibits

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apoptosis. REG-3β was found to increase the phosphorylation dependent activation of JAK2-STAT3 [30, 94]. IL-17 has been studied to increase pancreatic cancer stemness through the overexpression of couple of known cancer stemness genes (POU2F3 and DCLK1) along with ALDH1A1 [180]. Neutralizing antibodies against IL-17 and IL-17R effectively reduced the initiation and the progression of PanIN, indicating this cytokine as a therapeutic target for pancreatic cancer [104]. Monoclonal antibodies against IL-17 (Secukinumab and Ixekizumab) and IL-17R (Brodalumab-human monoclonal antibody) have already been employed in phase III trials to treat psoriasis. More potent antibodies to target IL-17 and better targets of IL-17 mediated pancreatic cancer could be developed with the better understanding of the underlying IL-17 signaling mechanism in this cancer [105].

8.6

Conclusion

Cytokines play an important role in the progression of pancreatic cancer. Several studies have reported the presence of different cytokines in the blood, tissue, pancreatic fluid. The PDAC patients have exhibited high amounts of IL-2, IL-6, IL-8 and IL-10 in serum. Cytokine signaling cascades are dysregulated in pancreatic cancer, causing inflammation leading to neoplasm. Both pro-inflammatory and antiinflammatory cytokines aid in the developing a tumor-promoting microenvironment helping the tumor to progress. So therapeutic strategies against these cytokines and their mechanisms are being investigated and developed. Current fundamental studies have showed positive results targeting cytokines like IL-6. Translating these findings in murine models to human studies are the next step to realize the goal of diagnosing the pancreatic cancer at an earlier stage.

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Perspectives and Molecular Understanding of Pancreatic Cancer Stem Cells L. Saikrishna, Prameswari Kasa, Saimila Momin, and L. V. K. S. Bhaskar

Abstract

Pancreatic cancer (PC) is a lethal, malignant cancer that bears high mortality rates. Due to its lack of noticeable symptoms, it is often diagnosed late and current pancreatic cancer therapies are ineffective with poor prognosis. Despite the high mortality and poor survival of PC patients, there is limited information on factors propagating resistance. Resistance to standard therapies in pancreatic cancer patients is partly associated with the presence of a subpopulation of highly plastic “stem-like” cells (pancreatic cancer stem cells: paCSC) in tumors. In this connection, it is important to have a strong understanding of the paCSC population, especially its distinct characteristics, in order to engineer new therapies to target these resistant cells. Therefore, the purpose of this investigation is to highlight and discuss PaCSC and their specific surface markers. Overall, in this study, we searched MEDLINE, EMBASE, the Cochrane Library, Web of Science, and ISI Proceedings for observational studies relating to the PaCSC and PC. Pancreatic cancer stem cells exhibit specific immune characteristics on their surface. The CD133, CD44, CD24, ALDH1, c-Met, DCLK1, CXCR4, EpCAM and ABCG2 are prominent Pa-CSC markers. As PaCSCs drive tumorigenesis and metastasis, their manipulation approaches would have widespread clinical implications and hence improve outcomes in pancreatic cancer. L. Saikrishna Department of Zoology, Visvodaya Government Degree College, Venkatagiri, India P. Kasa Dr. LV Prasad Diagnostics and Research Laboratory, Hyderabad, Telangana, India S. Momin Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA L. V. K. S. Bhaskar (*) Guru Ghasidas Vishwavidyalaya, Bilaspur, India e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_9

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Keywords

Cancer stem cells · Pancreatic cancer · Stem cell markers · Signaling cascades

Abbreviations 15-PGDH 5-FU ABCG2 ADLH ALCAM CSCs CXCR4 DCLK1 EMT EpCAM HA HGF mRNA nab-paclitaxel NETs NOD/SCID PaCSCs PC PDAC PDX1 SP TME

9.1

15-Hydroxyprostaglandin dehydrogenase 5-fluorouracil ATP-binding cassette subfamily G member 2 Aldehyde dehydrogenase leukocyte cell adhesion molecule Cancer stem cells C-X-C chemokine receptor type 4 Doublecortin-like kinase 1 Epithelial mesenchymal transition Epithelial cell adhesion molecule hyaluronic acid Hepatocyte growth factor Messenger RNA albumin-bound paclitaxel Neuroendocrine tumors nonobese diabetic/severe combined immunodeficiency Pancreatic cancer stem cells Pancreatic cancer pancreatic ductal adenocarcinoma pancreatic and duodenal homeobox 1 side-population Tumor microenvironment

Introduction

Pancreatic cancer (PC) is considered to be one of the most lethal cancers and is currently the seventh leading cause of global cancer deaths [69]. It is the most aggressive and hazardous malignancy with high rates of mortality [1]. Over the past years, the incidence rates have increased for pancreatic cancer. The individuals with risk factors such as tobacco usage, obesity, diabetes and pancreatitis, and positive family history were more susceptible to the risk of PC [55]. About 5–10% of cases with PC are genetic with about 80% penetrance [4, 14]. Although the exact reason for the differences in the incidence of PC among global populations is not known, the variations in certain risk factors such as tobacco smoking, dietary style and obesity could account for these differences [26, 38]. Furthermore, the incidence and mortality of PC in global populations also correlated with increasing age

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[12]. Considerable variations (30-fold) in the mortality rates for PC in the global populations include: highest mortality in European countries and lowest in Eastern African and South-Eastern Asian countries [12]. Survival rates of PC are determined by the stage at the time of diagnosis, tumor size, healthcare facilities available and completeness of follow-up [92]. However, over the past 35 years, the survival rate of PC has increased from 3% to 6.8% [5]. Standard treatments currently for PC include 5-fluorouracil (5-FU), gemcitabine, FOLFIRINOX and nab-paclitaxel (albuminbound paclitaxel). However, these treatments only extend the survival of patients up to a year and often fail to eradicate tumors. Additionally, pancreatic cancer is highly resistant to both radiotherapy and chemotherapy and has the ability to quickly spread to other organs, causing irreparable damage. Current studies and advancements have definitely contributed to our understanding of the pathophysiology of pancreatic cancer, specifically on the causes of cancer metastasis, PC relapse, and resistance towards chemotherapy Along these lines, a subset of cancer cells with stem-like properties that coincide with other cellular units/factors of tumor microenvironment (TME) have been identified. These cells are described as cancer stem cells (CSCs), the key drivers of tumor initiation and progression.

9.2

Cancer Stem Cells

Stem cells are described as subpopulations of cancer cells with distinct properties such as self-renewal and multi-lineage differentiation. Formation of CSC and tumor development are driven mainly by the reactivation of certain embryonic signaling pathways in cancer cells [44, 95]. Although, epigenetic and metabolic alterations in cancer cells are highly intertwined, CSCs are both epigenetically and metabolically different from non-CSCs of the surrounding niche environment. Hence effective recognition and characterization of CSCs is possible through the expression of specific cell surface markers. The existence of specific CSC clones depends on several extrinsic factors such as hypoxia, chemotherapy, and even nutrient deprivation [86]. Hence, the heterogeneous cell populations derived from these CSCs determine tumor heterogeneity [66]. Further mutations in stem cells were found to increase cancer risk by causing interruptions in cell division or preventing differentiation, which led to a significant increase in stem cell proliferation [99]. Understanding of CSCs may improve existing therapies and help us in finding new therapeutic methods against CSCs. It would seem that the new manipulation approaches of cancer stem cells would have widespread clinical implications and hence improve outcomes in pancreatic cancer.

9.3

Markers of PC Stem Cells

The presence of CSCs in various solid tumors was demonstrated after two decades of the identification of CSCs in human acute myeloid leukemia [11, 54, 80]. Subsequent studies identified various cell surface markers expressed by pancreatic cancer stem

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Fig. 9.1 Prominent pancreatic cancer stem cell markers

cells (PaCSCs). Some markers are of specific functional relevance to PC (Fig. 9.1). The prominent Pa-CSC markers include CD133, CD44, CD24, ALDH1, c-Met, DCLK1, CXCR4, EpCAM and ABCG2 (Table 9.1).

9.3.1

CD133

CD133 (prominin-1) is a glycoprotein that serves as a CSC marker in a large variety of human malignancies [28, 41]. The CD133 is encoded by PROM1 gene on human chromosome 4. Transcription of this gene is carefully regulated in a tissue-specific manner; in other words, expression is only found in certain tissues [21]. According to studies on CD133 mRNAs, these tissues include pancreas, testis, kidney, placenta, mammary gland and digestive tract [23]. Additionally, CD133 is associated with the Notch pathway and Hedgehog signaling, such that when these pathways are inhibited (i.e. Sonic hedgehog, Shh, is inhibited), there is an increased sensitivity of CD133+ glioma cells towards temozolomide therapy [84]. CD133 plays a strong role in motility, invasiveness and epithelial mesenchymal transition (EMT) due to their location in internalization prone membrane domains and involvement in multiple signaling pathways. Hypoxia induces the expansion of CD133+ that leads to tumor aggressiveness of pancreatic cancer cells in the presence of hypoxiainducible factor 1-alpha (HIF-1α) [29]. Pancreatic cancer tissue was shown to have CD133+, highly tumorigenic and gemcitabine resistant migrating cells. Depletion of CD133+ and CXCR4+ pancreatic cancer cells significantly decreased the tumorigenic potential of PC [30]. CD133 is localized to areas rich in cadherin and associated with the signaling pathways regulating the EMT program. Downregulation of endogenous CD133 in the Capan1M9 cells contributed towards Slug

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Table 9.1 Markers of pancreatic cancer stem cells Cell surface marker CD44

Characteristics Non-kinase transmembrane glycoprotein

Function Cell adhesion molecule, receptor for hyaluronic acid

CD24

Glycosyl phosphatidylinositol (GPI)-linked sialoglycoprotein

Cell adhesion molecule

CD133

Penta-span transmembrane glycoprotein.

Organizer of cell membrane topology.

ALDH1

Nicotinamideadenine dinucleotide phosphate–positive (NAD(P)+)dependent enzymes

Cellular detoxifying enzyme

c-met

Tyrosine-protein kinase met

Hepatocyte growth factor receptor (HGFR)

DCLK1

Microtubuleassociated kinase

Involved in the epithelialmesenchymal transition (EMT).

Significance Cell adhesion, migration, drug resistance and apoptosis. Knockdown of CD44 expression by its shRNA in pancreatic cancer cells led to decreased cellular proliferation and migration ability. Enhanced clonogenic capacity, multilineage potential, invasiveness, high proliferation and poor prognosis. Self-renewal pathway signaling, increased tumorigenicity, tumor progression, and metastasis. Display stem-like features, such as selfrenewal, clonogenic growth, tumorinitiating capacity. Knockdown of ALDH1B1 caused a 35% reduction in cell growth in the high ALDH1B1expressing cell lines. Signaling induces growth and invasion. Cells with increased c-met has tumorigenic potential. DCLK1 is essential for the invasive and metastatic properties of CSC. Overexpression promotes the tumor progression, sphere formation and migration of PC cells. Knockdown of

Reference [49, 97]

[53, 62, 71]

[28, 34, 61]

[75, 82, 87]

[31, 47, 93]

[36, 50]

(continued)

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Table 9.1 (continued) Cell surface marker

Characteristics

Function

CXCR4

Seven transmembrane Gprotein-linked CXC chemokine receptor

It is an alphachemokine receptor specific for stromalderived-factor-1 (SDF-1 or CXCL12)

EpCAM

Transmembrane glycoprotein

Mediate Ca2+ independent homotypic cell-cell adhesion in epithelia.

ABCG2

ATP-binding cassette (ABC) transporter

Drug efflux pump

Significance DCLK1 suppressed liver metastasis of pancreatic CSCs. Involved in the occurrence of metastasis in PC. CXCR4/ CXCL12 axis involved in cell proliferation, migration, and angiogenesis. Cell signaling, cellcell adhesion, proliferation, tumorigenesis, differentiation, metastasis and migration. ABCG2 overexpression lead to fast progression of malignancy, unsuccessful chemotherapy and poor prognosis.

Reference

[13, 30, 89]

[52, 64]

[88, 94]

suppression and a decrease in migration/motility and invasion [19]. In patients who have tumors that are not excisable, the ascites-derived exosomes of these patients suggested that highly glycosylated CD133 is a possible biomarker for improved prognosis of patients [70]. Furthermore, an overlap of 10–40% between CD133+ and CD44+, CD24+, EpCAM+ was reported in PDAC cells [30].

9.3.2

CD44

CD44 is a cell surface glycoprotein that mediates interactions between neighboring cells, cellular adhesion and cellular migration. CD44 gene produces a variety of isoforms (CD44s and CD44v) that are expressed differently in both normal and cancer cells. Analysis of CD44 isoforms in primary and metastatic human pancreatic adenocarcinomas revealed a novel CD44(v6) isoform in metastatic lesions [67]. Furthermore, CD44v6 expression was more prevalent in human tumor tissues of advanced stages of metastatic pancreatic carcinomas [48]. Pancreatic cancer cells (MiaPaCa-2) are involved in hyaluronic acid (HA) and HA oligosaccharide

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generation. Tumor-derived HA oligosaccharides can increase CD44 intracellular cleavage and motility of tumor cells; when this interaction is inhibited it can lead to a halt in tumor cell motility [77]. Knockdown of CD44 expression reduces cellular expansion and migration in PC cells when there is a simultaneous reduction in p-ERK and p-AKT concentrations [49]. Overexpression of CD44 in pancreatic cancer cell lines and pancreatic tumors indicating the CD44 plays a major role in carcinogenesis and progression of pancreatic cancer [43]. Additionally, correlation of clinicopathological factors with CD44 expression demonstrated that high CD44 expression predicts early recurrence of tumors in PC patients undergoing radical surgery [42]. The ionophore antibiotic gramicidin reduced the expression levels of CD133, CD44, and CD47 and suppressed cancer cell proliferation in synergism with gemcitabine [90]. In addition, BRM270 also inhibits metastasis and stem-like traits in CD44+ pancreatic ductal adenocarcinoma (PDAC) through the Shh signaling pathway [33].

9.3.3

CD24

CD24 is a heat stable antigen and a biomarker in both healthy and malignant stem cells [38, 81]. CD24 is a surface marker for pancreatic and duodenal homeobox 1 (PDX1) positive pancreatic progenitors derived from human embryonic stem cells [39]. Furthermore, CD24 is one of the cancer stem cells primarily found in both pancreatic ductal adenocarcinomas and intestinal ductal adenocarcinomas [35, 37, 39]. Furthermore, CD24 expression was strongly associated with poor glandular differentiation, high proliferation and poor prognosis [62]. Analysis of CD24 expression in intestinal and pancreatic neuroendocrine tumors (NETs) revealed that the CD24 has little to no expression in pancreatic and duodenal NETs [71]. A direct link between WNT/β-catenin pathway and CD24 expression was established in the context of PDAC differentiation. The presence of CD24 expression was associated with differentiated tumors [53]. Additionally, studies in human pancreatic cancer xenografts of mice with nonobese diabetic/severe combined immunodeficiency (NOD/SCID) exhibited that the co-expression of CD44+, CD24+, and ESA+ in a subpopulation of malignant cells plays a key role in self-renewal and multidifferentiation potential [46].

9.3.4

CD166

CD166 is described as an activated leukocyte cell adhesion molecule (ALCAM). Elevated expression of CD166 mediates migratory and tumorigenic activity of pa-CSCs and serves as an independent marker for prognosis and tumor relapse [40]. However, expression of ALCAM in the PC lesions is not associated with clinical or pathological data [79]. Subsequent studies delineated that the highly

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tumorigenic tendency of CD166+ cells was due to the overexpression of TSPAN8 and BST2, while stronger invasive and migratory activities CD166- cells was due to overexpression of BMP7 and Col6A1 [25].

9.3.5

CD326 or EpCAM

Epithelial cell adhesion molecules (EpCAM) are primarily expressed in rapidly proliferating tumors of epithelial origin [83]. EpCAM’s functions include cell signaling, cell-cell adhesion, proliferation, tumorigenesis, differentiation, metastasis and migration [52, 64]. The overexpression of EpCAM was found in many malignant tumors including PC [64, 85]. Compared to the EpCAM- PC cells, EpCAM+ CSCs show an increased tumorigenic potential by a 100-fold [45]. Furthermore, in PC patients, the expression of EpCAM was higher in the cytoplasm but lower in the membranes. However, the EpCAM overexpression suggested a worse survival rate in patients with advanced stage of carcinomas [57]. In contrast to this, Ep-CAM expression was strongly correlated with the suppression of PC, leading to a better prognosis in patients undergoing the resection [2]. In addition, there are no differences in the pathological features in between patients with low and high concentrations/expression of Ep-CAM [2]. Hence, further studies are necessary to validate the clinical significance of EpCAM in PC.

9.3.6

ALDH1

ALDH1 is an intracellular cytosolic enzyme, which plays a primary role in cellular detoxification and differentiation. ALDH1 activity has been associated with both normal stem cells and progenitor cells [59]. The expression of ALDH1 is regulated by various mechanisms including stem cell protein Piwil1 [15], long non-coding RNA HOTTIP [24] and acetylation of lysine 353 [96]. Although increased expression of ALDH-1 in healthy pancreatic tissues disqualifies ALDH1 as a suitable marker for CSCs in humans, (OPTIONAL: investigations show that) ALDH1 is associated with tumorigenic cells in PDAC [17, 42]. Pancreatic CSCs expressing aldehyde dehydrogenase (ADLH) showed an enhanced clonogenic growth and reduced OS of PC patients [68]. Studies in direct xenograft tumors, showed that the cell populations with high ALDH activity alone could enhance tumorigenic potential with efficiency in tumor-initiation [42]. A current study showed that the ALDH1 expression is inversely associated with the expression of 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and is associated with poor outcomes in PDAC patients [6]. Down-regulation of the expression of ALDH1 significantly reduced the expression of downstream genes and also decreased the growth of PDAC cells [6].

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9.3.7

165

c-Met

c-Met is a receptor of the tyrosine kinase family that serves as a proto-oncogene and is activated by its ligand hepatocyte growth factor (HGF) [58]. As an important marker for pa-CSCs, its signaling induces growth and invasion, and cells that express increased c-Met has tumorigenic potential [47, 78]. c-Met inhibitors given alone or with gemcitabine, reduce tumor growth and prevent the development of metastases [47]. Additionally, inhibiting c-Met via XL184 halts self-renewal capacity in pancreatic CSCs and prevents the development of metastases [31]. Furthermore, targeting the HGF-c-MET pathway inhibits local tumor growth and metastasis [65]. A recent study showed that the HGF-c-MET mediates tumor metabostemness by modulating YAP/HIF-1α [93]. Furthermore, complete suppression of the c-Met signaling pathway with gemcitabine chemotherapy acts as a more effective therapeutic option for PDAC [60]. Some phytochemicals, such as withaferin A and carnosol, are used to suppress c-Met kinase domain to treat in c-Met-dependent cancers [3].

9.3.8

DCLK1

Although the microtubule regulator doublecortin-like kinase 1 (DCLK1) is expressed predominately in invasive and metastatic CSCs, previous studies have noted that it is a normal stem cell marker in the gut [27, 56]. The expression of DCLK1, in recent studies, is described as a biomarker for pa-CSCs [8]. Manipulation DCLK1 and other co-expressed markers (ATAT1, HES1, HEY1, IGF1R, and ABL1) demonstrated a reduction in the clonogenic potential of PDAC cell lines [8]. The expression of DCLK1 was positively correlated with intrahepatic metastasis and poor disease-free survival in patients with hepatocellular carcinoma [20]. The DCLK1 gene was also associated with H3K4me3 and H3K27me3 histone modifications in pancreatic CSCs with invasive and metastatic potential [36]. Overexpression of microRNA-195 inhibited cellular proliferation, cellular migration and invasion of PC cells by targeting DCLK1 [98]. Furthermore, we found that DCLK1 silencing could inactivate/suppress epithelial-mesenchymal transition in cancer cells by downregulating Bmi-1, Snail and Vimentin [50]. Overall these studies revealed that the human pancreatic CSCs showed DCLK1 overexpression and inhibition, suggesting that regulation of DCLK1 expression offers a novel approach in treating pancreatic cancer cells.

9.3.9

CXCR4

Expression C-X-C chemokine receptor type 4 (CXCR4 or CD184) was found on tumor cells of various cancers [9]. Overexpression of CXCR4 has been linked with lung and liver metastases of murine pancreatic cancer [72]. CXCR4 expression has been identified in the stem cell population of various cancers and is linked with

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aggressiveness and the promotion of metastasis [16, 18, 22, 63]. Furthermore, studies showed that the paCSC with CXCR4+/CD133+ is essential for the metastasis of PC [30]. Additionally, the drug resistance caused by the activation of CXCR4 in pancreatic cancer cells indicates that CXCR4 is a novel therapeutic target for PC therapy [74]. Hypoxia-inducible factor 1 is a strong activator of CXCR4 and CXCL12 expression and the interaction of CXCR4/CXCL12 contributes heavily in cancer cell progression, cellular expansion, invasion, as well as metastasis of PC [73, 89]. Currently CXCR4 antagonists have shown the most striking response in tumor growth delay; thus, a randomized clinical trial is currently under development to study the effectiveness of applying a combination of immune-checkpoint inhibitors with CXCR4 antagonists (NCT03193190).

9.3.10 ABCG2 The ATP-binding cassette subfamily G member 2 (ABCG2) is a cancer stem cell marker in various cancers [76, 88]. The ABCG2 gene is located on chromosome 4q22, which leads to the coding of 655-amino-acid breast cancer resistance protein BCRP [7]. Overexpression of Gastrin and ABCG2 were found in PC cell lines as well as cancer tissues. ABCG2 transporter expression in side-population (SP) cells derived from the human PDAC cells indicates its role in determining SP phenotype [10]. Furthermore, gastrin induced ABCG2 expression by activating NF-κB and thereby alters invasion activity and metastasis in PC [88]. Verapamil, a inhibitor of ABCG2 transporter, increased the responsiveness of PDAC SP cells to the vinca alkaloid vincristine [10]. Furthermore, the drugs that are able to overcome ABCG2 resistance make them an effective therapeutic option against stem-like cancer cell populations in pancreatic tumors [91]. The FL118, an ABCG2 non-substrate anticancer agent, may be able to bypass ABCG2 -mediated drug resistance of human PC [51].

9.4

Conclusions

Pancreatic cancer is a highly aggressive tumor type with dismal prognosis. Current treatments of PC such as surgery, radiotherapy, and chemotherapy are not effective in eliminating the disease. Although some promising drug candidates with antitumor activity against PC have been identified, recent trials of these drugs have shown limited to no benefit. The primary reason for the continual appearance tumors despite radiation or chemotherapy is due to the presence of self-renewing CSCs. Pancreatic cancer CSCs are involved in aggressive, chemo-resistant and metastatic nature of this cancer. This understanding raised the possibility to stratify cancer patients based

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on surface markers and the subsequent discovery of novel treatment options for each subtype of PC. The agents targeting the tumor microenvironment and intrinsic signaling pathways targeting CSC include hedgehog, Wnt/ß-catenin, and notch. Conflict of Interest There are no conflicts of interests.

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The Role of Hypoxia Inducible Factor-1a in Pancreatic Cancer and Diabetes Mellitus

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Saimila Momin and Ganji Purnachandra Nagaraju

Abstract

Hypoxia inducible factor-1α, abbreviated as HIF-1α, is a protein which is encoded by the HIF1A gene and is primarily responsible for the modulation of cellular feedback regarding hypoxia, which results from the lack of proper delivery of oxygen to cells. When HIF-1α is unregulated or over-expressed due to hypoxia, it can lead to irregular cellular metabolism and pancreatic cancer (PC) progression. There seems to be a strong, positive correlation between up regulation of HIF-1α and cancers, specifically PC. Understanding the regulation, expression, and interaction of HIF-1α will allows us to further understand the role this pathway can play in deadly diseases such as PC. In this chapter we will explore the role of the HIF-1α pathway in PC, and in metabolic disorders, such as diabetes mellitus, and studies that have been conducted in order to further holistically understand the role of HIF-1α. Keywords

Hypoxia inducible factor 1-α · Pancreatic cancer · Diabetes mellitus · Diseases · Signaling pathway

Abbreviations ARNT bHLH-PAS HIF-1α HSP90

aryl hydrocarbon nuclear translator Basic helix-loop-helix – per-arnt-sim domain Hypoxia inducible factor-1α heat shock protein 90

S. Momin · G. P. Nagaraju (*) Department of Hematology and Medical Oncology, Winship Cancer Institute, School of Medicine, Emory University, Atlanta, GA, USA e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_10

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KRAS NAD+/NADH PC pVHL RACK1 SWI-SNF TAD TGF-β

10.1

Kirsten rat sarcoma viral oncogene homolog Nicotinamide adenine dinuclotide Pancreatic cancer von Hippel-Lindau protein complex Receptor for activated C kinase 1 Switch/sucrose non-fermentable topologically associated domain Transforming growth factor beta

Introduction

10.1.1 Pancreatic Cancer Pancreas is a gastrointestinal organ that is primarily responsible for releasing hormones and enzymes, also known as digestive juices, in order to aid in the digestion of foods [1]. The pancreas has both an endocrine and exocrine function because it secretes and releases digestive enzymes into both the bloodstream and into ducts, using its exocrine glands [2]. The pancreas primarily releases the following enzymes: lipase to digest fats, trypsin and chymotrypsin to digest proteins, and amylase to digest carbohydrates [2]. Overall, in the digestive system, digestive juices are released initially into the pancreatic ducts and then the duodenum of the small intestine in order to digest fats, carbohydrates, and proteins, with the additional help of bile, a digestive juice produced in the liver [2]. As mentioned above, along with the exocrine function, the pancreas also has a endocrine function which plays a key role in blood glucose regulation [2]. Islet cells of the pancreas generate and release insulin and glucagon in order to regulate blood glucose levels [3]. Insulin is released to lower blood glucose levels and glucagon is released to increase blood glucose levels. Maintaining blood glucose levels is critical as organ function and overall metabolic function heavily depends on it [3]. Abnormal function of the pancreas can lead to deadly diseases such as pancreatic cancer and diabetes mellitus [4]. Pancreatic cancer (PC) results when cells within this gland organ begin to abnormally divide and grow due to mutations [5]. These mutations arise due to either or both genetic and environmental factors, which include diets, obesity, and substance use [5]. Additionally, even diabetes can increase the likelihood of developing PC [5]. Even though PC is considered to be a deadly and unresolved medical disease, there are current investigations being conducted in order to understand in the underlying pathophysiology and potential treatment plans to tackle the disease. The current treatment plan for PC includes a combination of chemotherapy, interventional radiology, and radiation therapy, which is to attack tumors and decrease their presence [6]. However, there are ongoing investigations studying the molecular mechanisms in order to attack the tumor and overall progression of cancer at its core. These investigations include but are not limited to: AKT inhibitors [7],

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Hypoxic environment

Growth factors

HIF-1β

PI3K HIF-1α

P300

HIF-1α

HIF-1β

HRE

Cell metabolism Cell division Cell growth Cell survival

Nucleus Fig. 10.1 The pathway begins with growth factors that ultimately leads to HIF-1α and HIF-1β subunits with p300 binding to HRE to lead to metabolism, cell survival and cellular proliferation in a hypoxic environment PIK3: phosphoinositide 3-kinase (enzymes); HIF-1α/β: hypoxia inducible factor-1 alpha/beta; HRE: hypoxia response element Masoud GN, Li W (2015) HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B 5(5): 378–389

integration of albumin-bound paclitaxel (nab-paclitaxel) plus gemcitabine [8], and other studies in chemotherapy and radiation therapy. Additionally, even though the pathophysiology of PC is still not fully understood, ongoing integrated genomic analyses have revealed 32 mutated genes and 10 pathways that are strongly associated with the increased likelihood of PC [9]. These include KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signaling, G1/Stransition, SWI-SNF, chromatin alteration, DNA repair function and RNA generation. Along with these identified mutated genes and molecular pathways, hypoxia-inducible factor-1α (HIF-1α) has also been considered to be strong associated with PC along with other cancers [10]. Additionally, by understanding HIF-1α, specifically its role and function, interaction in the body, and how it can be controlled, we can get one step closer to potentially learning how to treat PC and even other cancers that are affected and/or associated with HIF-1α (Fig. 10.1).

10.1.2 Diabetes Mellitus Diabetes is a metabolic disorder that negatively affects how the body digests food and obtains energy for healthy organ and cellular function [11]. Glucose is the primary source of energy in the body and is obtained by foods digested in the

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body and used by all cells in order to properly function. Diabetes generally develops due to the abnormal production of insulin in the body by the pancreas [11]. Insulin is required in order to transport glucose into the cells, which is then converted into energy [12]. Individuals diagnosed with diabetes generate little to no insulin and therefore cellular function decreases and the level of blood glucose increases [13]. In order to compensate for the increased blood glucose levels, the body filters the blood and eliminates the excess glucose. Diabetes can be broken down into two types, type 1 diabetes and type 2 diabetes. Type 1 diabetes is described as the following [14]: a chronic condition in which the islet cells of the pancreas are unable to generate insulin because the immune system is attacking the islet cells of the pancreas. On the other hand, type 2 diabetes is also a chronic condition but it is not an auto-immune disease and develops gradually in which the hyperglycemia leads to insulin resistance. Contributing factors include genetics and environmental factors, such as obesity, unhealthy and high glucose and/or carbohydrate diets, and inactive lifestyle.

10.1.3 Hypoxia Inducible Factor 1 (HIF-1) Hypoxia inducible factor 1 is a heterodimeric transcription factor, which is constructed by two key subunits, an α and a β subunit, which belong to the bHLH-PAS protein family [15]. HIF-1α is an oxygen dependent or sensitive unit which is primarily associated with hypoxia; in other words, when the body undergoes a hypoxia, the α subunit is activated [16]. On the other hand, HIF-1β, which is referred to the aryl hydrocarbon nuclear translator or ARNT acts as a ligand which binds to the aryl hydrocarbon receptor or AhR [15]; upon binding of these two entities, it leads to the translocation to the nucleus. The subunits have domains such as, b, HLH, PAS and TAD, are able to self-regulate their expression [15]. As mentioned before, the HIF-1α plays a key role in aerobic metabolism in order to modulate hemostasis, primarily in cellular oxygen levels. In mammalian bodies, under normal conditions, HIF-1α is transcribed and expressed continuously but eliminated via degradation with a half-life of approximately 5 min [17]. However, when the body undergoes a state of hypoxia, multiple pathways are activated in order to maintain the stability of the α subunit; functions that become activated include: hydroxylation, acetylation, phosphorylation reactions, and ubiquitination, which is primarily mediated by the von Hippel-Lindau protein [15, 18]. Hypoxia can cause from a catastrophe at any step in the transport of oxygen to cells. That leads to decreased limited pressures of oxygen, difficulties with distribution of oxygen in the lungs, inadequate oxygen in hemoglobin [19]. In this case hypoxia, more specifically reaction oxygen species, including nitric oxide, induces the α subunit of the HIF-1. Overall, HIF-1 is responsible for oxygen delivery to cells through the process of angiogenesis [20], which is the process of generating new blood vessels from pre-existing blood vessels and adapting to a hypoxic environment through the process of glycolysis, the first metabolic step to ultimately convert glucose into energy. PC growth and metabolic disorders are strongly correlated to the HIF-1

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expression, specifically over-expression of HIF-1α [21, 22]. Over-expression of this transcription factor leads to PC progression, as tumor cells are able to activate survival pathway to adjust and adapt to the hypoxic environment [18, 23]. Understanding the molecular mechanism and the connection to the pathophysiology of PC and diabetes mellitus can potentially lead to advance treatments for such diseases and others.

10.2

Hypoxia Inducible Factor 1 (HIF-1): Molecular Pathway, Interactions and Regulation

10.2.1 HIF-1 and Pancreatic Cancer As discussed, HIF-1 is a transcription factor that, under hypoxic conditions, primarily modulates the transcription of other genes in order to regulate oxygen levels within the body [24]. The primary subunit which plays an essential role in the control of oxygen levels is the α subunit. The α subunit is modulated by the O2-dependent hydroxylation of proline residue 402, 564, or both, by prolyl hydroxylase domain protein 2 [25]. This leads to the binding and activation of the von Hippel-Lindau protein [25]. Upon binding other processes such as proteasomal degradation and ubiquitination begin to follow, along with oxygen-dependent hydroxylation of asparagine residue 803 [18]. These series of reactions lead to the use of oxygen and α-ketoglutarate to produce carbon dioxide and succinate. As a result of the low levels of oxygen and high levels of products of the tricarboxylic acid cycle, which include isocitrate, succinate, and fumarate, hydroxylase activity is halted [25]. This leads to competitive binding of HIF-1α between the receptor for C kinase 1 or RACK1 and heat shock protein 90 (HSP90). The receptor for C kinase 1 modulates proteasomal degradation and ubiquitination [25]. Overall, under hypoxic conditions, HIF-1α transactivation domains interact with co-activators and leads to the production of reactive oxygen species [24, 25]. Under normal conditions, HIF-1α is hydroxylated by HIF-1α-specific prolyl hydroxylases, which are sensitive to oxygen levels [24]. The process of hydroxylation leads to ubiquitination and proteasomal degradation via the von Hippel-Lindau protein (pVHL) complex. Additionally, HIF-1α is also a substrate a hydroxylase factor that inhibits HIF-1α known as asparaginyl hydroxylase [25]. Since this hydroxylase factor is also sensitive to oxygen levels, it has the ability to interrupt the interaction between HIF-1α transactivation domains and co-activators, which ultimately results in the inhibition of HIF transcriptional activation [25]. There is a strong correlation between the increased concentration of HIF-1α and generation of tumor and PC progression. It is highly suggested that tumor cells are able to adapt to hypoxic conditions and resist apoptosis due to this over-expression of HIF-1α subunit. An experimental study revealed that 13 of 19 diverse tumor types, HIF-1α was severely over-expressed and was also strongly correlated to cellular proliferation [26]. When the body is under hypoxic conditions, processes such as glycolysis and neovascularization are dramatically increased, leading to the

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production of tumors and metastasis [26]. The HIF-1 pathway has the ability to transcribe genes that are responsible for transportation of glucose and such activity is dominated by the HIF-1α unit [26]. Overexpression of HIF-1α leads to impairment in the overall HIF-1 pathway [27], which ultimately leads to tumors cells being able to adapt to such hypoxic conditions and allowing them to thrive in such conditions; whereas, normal cells are unable to properly function in hypoxic conditions. The strong connection between the HIF-1 pathway and HIF-1α subunit to PC progression serves as an attractive topic to explore for therapeutic options [28]. Theoretically, decreasing or down-regulating the HIF-1 pathway can stop tumor progression. Investigations are currently exploring how activating hydroxylase can decrease the activity of the HIF-1 pathway [23]. Other studies are exploring how proteins such as co-activators of the HIF-1α can be altered or deleted in order to prevent transcription [23]. Further exploration and investigation into the manipulation and alteration of such small molecules will lead to decrease of HIF-1 activity and may suppress tumor progression in individuals diagnosed with PC.

10.2.2 HIF-1 and Diabetes Individuals diagnosed with diabetes are often associated with another condition known as pseudohypoxia. Pseudohypoxia is described as having an increased concentration of NADH compared to NAD+ which is primarily because of the increased blood glucose levels (through the polyol pathway [29, 30]. Specifically, studies have shown that diabetic tissues show increased levels of pimonidazole, which is a strong marker for hypoxia, suggesting that diabetic individuals have high levels of hypoxia [30–32]. Additionally, there was also increased expression of HIF-1α [30]. Recall, that the structure of HIF-1α can be easily stabilized by hypoxia and the process of glycolysis. However, individuals with diabetes have decreased transcriptional activity of HIF-1 due to an impairment of transactivation of HIF-1 expression, primarily because of the p300 co-activator [30]. Please note that this impairment does not negatively affect the HIF-1α structural stability. Furthermore, increased glucose levels have the ability to lead to signal transduction (HIF-1 mediated) by ChREBP, a binding protein [30]. Ultimately, experiments have shown that high levels of glucose lead to inactivation and destruction of the HIF-1α subunit and overall impairment of the HIF-1 pathway. Ultimately, cells in the body are unable to properly adapt and respond to the hypoxic conditions, which can lead to immediate cellular dysfunction and other long-term, negative consequences.

10.3

Current Experiments and Investigations

Recently, researchers used triptolide, which is a diterpenoid epoxide extracted from Tripterygium wilfordii and is well-known for its anti-tumor properties, and treated subjects with it to observe the effects it may have on the HIF-1α protein [33]. Upon

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exposure to triptolide, the concentration of HIF-1α increased and the hypoxic response decreased [33]. Additionally, HIF-1 activity was also decreased due to the decrease of the co-activator p300 caused by triptolide [33, 34]. And additionally, triptolide also decreased oncogenic signaling pathways [33]. This substance definitely stands as a strong treatment option because it has the ability to manipulate the HIF-1α at the molecular level and decrease the hypoxic environment. Another study showed that the deletion of pancreas-specific HIF-1α leads to PC and increased concentration of B lymphocytes [35]. However, deletion of the B lymphocytes led to the decrease of PC progression [35]. This suggests that HIF-1α may have a strong and necessary role in preventing tumor progression. Specifically, in this this study, researchers used a KrasG12D-driven murine model and tumors from humans to show that hypoxia and the structural stabilization of HIF-1α are present during the preliminary stages of PC [35]. When the HIF-1α is deleted in pancreas-specific cells, neoplasia is significantly increased, suggesting that HIF-1α plays a key role in regulating and possibly suppressing PC progression.

10.4

Conclusion

Cancer is one of the most intricate mysteries known to the human body, there are many molecular mechanisms and cellular processes that govern and modulate optimal function and the HIF-1α pathway is a particularly relevant mechanism that plays a major, yet complex role in PC and diabetes. Even though the subunit, HIF-1α’s role is understood, it is critical to understand how this subunit interacts with other molecules and if it plays additional roles in other body mechanisms. Possible experimental studies include: exploring interactions of HIF-1α with other organs and other molecular and cellular functions and pathways, identify contributing factors in the pathway that can inhibit or activate HIF-1α pathway, manipulation of the pathway and integrate such changes in live subjects, exploring activation of hydroxylases, which are able to decrease overall HIF-1 activity, targeting the HIF-1 complex for eventual degradation, studying the interaction of the p300, the co-activator of HIF-1α, and observing specific molecules that can serve as inhibitors. Once the implications and side effects of these relatively novice treatment plans and preliminary research studies are properly understood, then these treatment options can undergo clinical trials and studies with human patients, which can ultimately lead to permanent solutions to PC. By further examining this mechanism and studying specifically how the pathway can be manipulated may lead to new therapeutic treatments benefitting those diagnosed with cancers and metabolic disorders. Even though, thus far, as a scientific community, we have not been able to successfully manipulate and alter a transcription factor in a live subject, this method still remains to be a strong therapeutic option; if achieved and applied properly, it could potentially treat PC and other cancers.

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Role of Heat Shock Protein 90 in Diabetes and Pancreatic Cancer Management

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Pinninti Santosh Sushma, Saimila Momin, and Gowru Srivani

Abstract

Heat shock protein 90 (Hsp90) plays a key role in the activation of client proteins, which implicate Hsp90 in various biological processes, specifically coordinating regulatory mechanisms in order to control their activity. One of the key regulators responsible for the upregulation of Hsp90 is heat shock factor (HSF1), whose primary role is to bind heat shock elements (HSEs) with Hsp90 promoters. HSF1 functions by interacting with the transcriptional programming of Hsp90 and with integrate biological signals to regulate levels of Hsp90, especially during times of stress. Furthermore, not only are these Hsp90 protein chaperones upregulated but they can also be released from pancreatic beta cells during pro-inflammatory circumstances. Additionally, Hsp90 interferes with survival and metastatic pathways that are associated with pancreatic cancer (PC) progression. Future investigations on protein chaperons that are associated with Hsp90 may lead to the identification of biomarkers for diseases such as diabetes and PCs and potentially lead to therapeutic strategies in management of these chronic diseases. Keywords

Heat shock proteins · Diabetes · Pancreatic cancer

P. S. Sushma (*) Department of Biotechnology, Dr. NTR University of Health Sciences, Vijayawada, Andhra Pradesh, India e-mail: [email protected] S. Momin Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India Department of Biology, Emory University, Atlanta, GA, USA G. Srivani Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_11

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Abbreviations 17-DMAG ATP CTA DBD FAK HIF-1a HSEs HSF Hsp90 HSR IGF-IR IL-6 NK cells NTA PBMC PC T1D VEGF

11.1

17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin Adenosine triphosphate C-terminal transactivation DNA-binding domain Focal adhesion kinase Hypoxia-induced factor-1α Heat shock elements Heat shock factor Heat shock protein 90 Heat shock response Insulin-like growth factor-1 receptor Interleukin-6 Natural killer cells N-terminal transactivation Peripheral blood mononuclear cell Pancreatic cancer Type 1 diabetes Vascular endothelial growth factor

Introduction

Hsp90 (heat shock protein 90) constitutes nearly 2% of cellular proteins and 6% of cellular proteins among stressed cells [1–4]. Hsp90 levels usually depend on a master HSR (heat shock response) regulator and HSF1 (heat shock factor 1), subjected to regulatory processes. Hsp90, which is regulated by various mechanisms, influences transcription and is subjected to post-translational modification as well as modulation via co-chaperones. The human cells usually contain both Hsp90β and heat-inducible Hsp90α [5]; these molecules have an 86% amino acid sequence identity [6]. Despite the fact that these two molecules are highly evolutionarily conserved, these proteins exhibit a variety of different functions [7]. Surprisingly, Hsp90α may not even be necessary in mammals; however, on the other hand, Hsp90β participates in maintaining the viability. Hsp90α is involved in adaptive roles [8]. Hsp90 triggers the maturation of prominent signaling proteins like regulatory kinases [9–11], transcription factors, and hormone receptors [14]. Hsp90 is also associated with protein complexes [12] that suppress phenotypic variations [13–16]. Hsp90α and Hsp90β both interact with eukaryotic proteomes [17], which represents approximately 2000 proteins, among which around 725 interactions have been proven by protein to protein interaction in experimental results. This suggests that Hsp90 is highly necessary in a range of mechanisms in

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functional regulation. This review aims at examining the role of HSPs in diabetes and pancreatic cancer (PC).

11.2

Structure of Hsp90

Hsp90 contains a N-terminal dimerization domain which is primarily responsible for binding ATP to the middle domain through an unstructured charged linker. The N-terminal domains participate in the transient dimerization process by binding with ATP [18]. Then the C-terminal domain is responsible for protein dimerization. ATP binding promotes the lid segment moment in N-terminal domains over the bound ATP [19]. These lid movements expose the surface residues, which play a key role in the dimerization of the N-terminal domains within Hsp90. Activity of ATPase in Hsp90 is initiated when the middle domain catalytic loops relocate to the open active state [20]. These loops possess an arginine residue which interacts with γ -phosphate and therefore leads to the promotion of ATP hydrolysis by Hsp90. The catalytic loop confirmation may be modulated by binding with co-chaperones, such as Aha1, which activates the activity of ATPase [21]. The conformational changes that occur represent the rate-limiting step of the Hsp90 chaperone cycle. The molecular details regarding Hsp90 chaperone cycle activation and maturation are yet to be investigated.

11.3

Activation of HSF1 and the Heat Shock Response

HSF proteins regulate HSR [22–24], which can be induced by various stimuli like elevated temperature levels, viral and bacterial infections, and even oxidative stress [25]. Being a primary regulator of HSR and Hsp90 client protein, examining the functions of HSF1 is necessary in Hsp90 regulation. HSF proteins contain four essential components: DBD (DNA-binding domain), a trimerization domain, HR-C region, and CTA (C-terminal transactivation) [22, 23, 26–28]. Yeasts possess an NTA (N-terminal transactivation) domain [29]. The DNA-binding domain of these HSFs binds to HSEs (heat shock elements) that contain numerous nGAAn units [30]. The arrangement of these units leads to the promotion of cooperativity within the binding of HSF trimers. The trimerization domain in HSF constitutes of a couple of heptad repeats (HR-A and HR-B), forming a triple-stranded α-helical coil [31]. In Drosophila as well as mammals, HSF1 third repeat (HR-C) is known for intramolecular interactions to aid in maintaining HSF1 in monomeric form, which can be counteracted by stress [32– 34]. Trimerization is triggered by DBD intermolecular interactions, such as tryptophan and phenylalanine residues [35], and mammals contain two cysteine residues, which allows it to form disulfide bonds in response to stress [35, 36]. Elevated temperature levels result in hyperphosphorylation of particular proteins. This increases transcriptional programming when HSF trimer binds to the nGGAn units [37, 38]. When HSF1 trimer binds to HSEs that contain four phosphorylation sites at

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Ser303, Ser307, as well as Ser363, HSF1 becomes repressive to transcription, but this process can be overwritten during stress conditions [39, 40], suggesting that this system is stress sensitive [26]. In yeast, an unstructured NTA domain [41] mediates stimulation of HSF1, suggesting its role as a negative regulator of the CTA domain; in other words, it is unable to activate stress-mediated HSF1 [42, 43]. The CTA domain encourages increased concentrations of promoter activity 15–33  C. On the other hand, transient activity, which is modulated by the NTA domain, is generally activated between 34.5 and 39  C [44]. During stress conditions, the levels of denaturation in proteins increases [28], triggering cytoplasmic non-DNA-binding HSF1 conversion to a homotrimer, which increases DNA-binding activity. Thus, HSF1 that is dispensed in association with Hsp90 [45–47] undergoes homotrimerization [34] as well as translocation to the nuclear region upon binding to HSE [48–50]. In the next phase, series of phosphorylation processes transform HSF1 trimers to activate transcription factors [26, 27], which results in upregulation of Hsp90 along with other chaperones including Hsp70, Hsp27, and Hsp40 [51].

11.3.1 HSPs and Diabetes Type 1 diabetes (T1D) is an autoimmune disease mediated by T-cell responses, in which pancreatic β cells, which produce insulin, are destroyed by the immune system. The shortage of identifiable biomarkers and disease-modifying therapies have become an obstacle in the management of T1D [52]. Studies have primarily focused on β cell stress pathways, like stress associated with endoplasmic reticulum as well as oxidative stress, which can potentially play a major role in further stimulating the immune system and accelerating T1D progression [53, 54]. The activation of such pathways precedes the advances in clinically detectable hyperglycemia [55], making the study of β cell health-based biomarkers and their application in diabetes an attractive option in both the prediction and therapeutic strategies of T1D. Islet Hsp90 levels found in experiments, conducted with NOD mice before hyperglycemia onset [56], were elevated. The expression of intracellular Hsp90 was also found to be upregulated, especially in response to environmental factors like reactive oxygen species, hypoxia, and irradiation [57]. Additionally, cadaveric human islets as well as pancreatic β cell lines discharged higher levels of Hsp90 during pro-inflammatory cytokine treatment [58, 59]. Pediatric populations with T1D exhibited higher serum levels of Hsp90 (alpha cytoplasmic form) in comparison with age- and gender-matched controls [56]. Furthermore, increased levels of circulating anti-Hsp90 autoantibodies were recognized in T1D patients [60]. Protein markers associated with ER stress were found to be increased in islets among individuals diagnosed with T1D [61]. ER stress promotes T1D development in NOD mice [62], whereas ER stress mitigation with chemical chaperones prevents T1D development in NOD mice [63]. In the pancreas, pro-inflammatory cytokines are released by macrophages, NK cells, as well as T cells during insulitis [59]. Ex vivo therapy of cadaveric islet cells as well as β cell lines with cytokines led to ER

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stress [64]. In contrast, previous studies have reported that pro-inflammatory cytokines released from β cells induce Hsp90 [58], and therefore the expression of the Hsp90 protein becomes increased among islets within prediabetic NOD mice when ER stress conditions arise. Investigations revealed blood Hsp90 levels also increased among pediatric patients during T1D diagnosis [56]. The elevated Hsp90 during diagnosis of T1D needs to be tested in order to establish whether it serves as a strong biomarker of T1D. Hsp90 is found to be expressed in several tissues, specifically immune cells. Studies have also demonstrated elevations in serum Hsp90 levels during both autoimmune and inflammatory conditions. In a cohort study, individuals diagnosed with bullous pemphigoid, along with an increase of peripheral blood mononuclear cells (PBMC), had elevated Hsp90 levels compared to the control group [65]. PBMC Hsp90 levels were also elevated in individuals diagnosed with systemic lupus erythematosus. Additionally, elevated PBMC Hsp90 levels are also strongly correlated with ascending levels of IgG autoantibodies to Hsp90 [66]. The elevations of serum Hsp90 levels promote a generalized inflammation condition within patients that have β cell autoimmunity. Studies were also conducted to investigate whether there is an association between Hsp90 levels with demographic and clinical variables. Future longitudinal analysis is needed to test this approach. Surprisingly, there is a negative correlation between Hsp90 levels and age in autoantibody-positive subjects. Additionally, there was also a negative trend with respect to age among progressors to T1D. These correlations are similar with previous studies that have investigated associations between serum concentrations and age. Hsp90 levels recorded in children, within the age group of 4–15 years, with recent onset of T1D, [56] served as an indication of aggressive B cell autoimmunity among this age group [67, 68]. A strong association between age and inflammation is well-established [69]. In a study, Hsp90 inhibitors exhibited therapeutic efficacy, playing the role as a senolytic agent in rodents to extend their lifespan [70]. The elevated serum Hsp90 levels in older individuals suggest that the elevated inflammation connected with aging, whereas the absence of age-associated increases is reporting Hsp90 as potential biomarker in detecting pathological inflammatory progressions among younger persons. The process of targeting oncogenic signaling pathways can lead to improvements in PC therapy. Hsp90 is recognized as a crucial component for the proper functioning of oncogenic kinases as well as signaling intermediates [71–73]. The transcription factors like HIF-1α and STAT3 are identified as client proteins of Hsp90 [73, 74]. Hsp90 is highly expressed among cancer cells [75]; studies indicate that Hsp90 inhibitors exhibit the highest affinity of binding to Hsp90 in malignant cells, in comparison with healthy cells [76]. The inhibition of Hsp90 may target signaling intermediates, including transcription factors, which can lead to reductions in PC growth and angiogenesis. To this date, geldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are considered to be the best inhibitors of Hsp90. These inhibitors are still being investigated but are involved in phase I/II trials of clinical studies [77–79]. However, current synthetic inhibitors that are being developed also have promising results as for cancer treatment [80, 81].

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The Hsp90 inhibition interferes with IGF-I/IGF-IR signaling within cancer cells. During initial experiments, cells pretreated with 17-AAG inhibited IGF-I-mediated activation of both Erk1/2 and Akt signaling [82]. The STAT3 phosphorylation decreased IGF-I activation, suggesting STAT3 is not associated with the IGF-IR signaling cascade. IRS-1, which is upstream of Erk and Akt, is found to respond to 17-AAG during phosphorylation impairment. According to subsequent experiments, Akt and Erk1/2 inhibition by 17-AAG was dose-dependent, in which a dose of 100 nmol/L can lead to a reduction in phosphorylation substrates. These experiments strongly suggest that inhibiting Hsp90 impairs downstream signaling of the IGF-IR signaling pathway in PC. Inhibiting Hsp90 may also disrupt the IL-6/STAT3 pathway in PC [83, 84]. This pathway is activated by Hsp90 and is involved in metastasis of PC [85]. IL-6mediated STATs activation with treatment of 17-AAG significantly decreased STAT3 activation and phosphorylation of STAT5. IL-6 stimulation leads to Erk1/ 2 activation, which is regulated by Hsp90 in malignant cells. Similarly, PI3K/Akt inhibition via inhibitors such as LY290004 did not affect phosphorylation of STATs upon IL-6 exposure. Therefore, Hsp90 inhibition can directly disrupt IL-6- mediated activation in STAT3 and STAT5 in PC cells.

11.3.2 Effect of Hsp90 Inhibition on IL-6 and Hypoxia-Mediated Induction of HIF-1a HIF-1α is a transcription factor known to be a key promoter of cancer growth as well as angiogenesis in many cancers, including PC [82, 86, 87]. HIF-1α is necessary for complex formation with STAT3, in order to ultimately facilitate target gene expression such as VEGF [88, 89]. Hence, Hsp90 inhibition affects the function of HIF-1α either through direct inhibition or by inhibition of STAT3. Furthermore, studies showed that 17-AAG decreased the levels of nuclear HIF-1α protein in PC cells. HIF-1α induction by hypoxia was prevented by 17-AAG. Similarly, activation with IL-6 elevated nuclear HIF-1α content. The response decreased via Hsp90 inhibition with17-AAG. Hsp90 inhibition also decreased HIF-1α levels by decreasing its expression, which was experimentally identified by real-time PCR. Hsp90 inhibitor 17-AAG decreased VEGF-A expression and increased IGF-1 expression under hypoxic conditions, as well as IGF-I stimulation [82]. So Hsp90 inhibitors can be used in the reduction of VEGF-mediated angiogenesis in PC. Previous studies concluded that HIF-1α function may be inhibited by blocking Hsp90. Additionally, HIF-1α regulates cell motility in cancers [90]. Focal adhesion kinase (FAK), the chief mediator of cell motility, is a client protein of Hsp90. 17-AAG therapy can inhibit FAK phosphorylation in PC. Additionally, IGF-Imediated cancer cell motility was found to be impaired by 17-AAG. So Hsp90 inhibition can reduce PC metastasis by blocking invasive and migratory pathways.

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11.3.3 Effect of Hsp90 Blockade on Growth of PC A s.c. xenograft model of PC (HPAF-II) was used to evaluate the effects of Hsp90 inhibition on cancer growth. When treated with 17-DMAG, cancer growth was inhibited. Treatment with 17-DMAG upregulated Hsp70 in cancer samples. The changes in expression and in FAK phosphorylation were not detectable. The growth inhibitory as well as antiangiogenic properties of 17-DMAG were studied in an orthotopic pancreatic tumor model. After treatment with 17-DMAG for 21 days, data analyses showed a decrease in orthotopic cancer growth. Similarly, STAT3 activation was diminished in tumor cells treated with 17-DMAG. Analyses suggested that IGF-IR as well as STAT3 could be prominent targets of Hsp90 blocking in PC. Studies additionally recognized IL-6/STAT3/HIF-1, a novel autocrine activation loop in pancreatic tumor cells, can also cause disruptions by blocking Hsp90. Studies using orthotopic tumor models suggested that targeting Hsp90 with inhibitors such as 17-DMAG can reduce cancer growth as well as tumor vascularization. These inferences can lead to a multifactorial approach in using Hsp90 inhibitors in PC therapy. Overall, IGF-IR turned out to be a remarkable target in cancer therapy. Hsp90 inhibitors can directly affect IGF-IR, by decreasing receptor phosphorylation upon ligand stimulation. Inhibition leads to decreased expression of IGF-IR, when given in the appropriate time frame with the correct dose. Thus, Hsp90 elicits acute and chronic inhibitory effects on IGF-IR. Hsp90 inhibition also leads to downregulation of IGF-IRh mRNA followed by slight increase in IGF-IRh mRNA, thereby suggesting a potential response. Additionally, increase of this precursor also results in Hsp90 inhibitor-mediated damage of pro-convertase furin/PC5 [91]. The effect of downregulation of IGF-IR via Hsp90 inhibitors was described in a few other studies; however, its effect on phosphorylation was not discussed. Terry et al. [92] revealed geldanamycin-derivate 17-AAG decreased the IGF-IR expression in synovial carcinoma cell lines and concluded that cancer cells that overexpress IGF-IR are virtually nonreactive to Hsp90-inhibiting therapy. These findings suggest that Hsp90 inhibitors can serve as strong therapeutic options for PC, especially since IGF-IR is overexpressed and active in this cancer [82, 93, 94]. Additionally, Hsp90 inhibitors affect PC growth and angiogenesis through another mechanism besides IGF-IR. This mechanism involves IL-6, which is a cytokine that can increase angiogenesis by increasing the expression of VEGF-A [84, 95, 96]. Tang et al. [83] discussed IL-6-mediated VEGF upregulation in PC cell lines. Masui et al. [84] showed a strong correlation between IL-6 receptor expression and VEGF expression in human pancreatic cancer models. Furthermore, IL-6 signaling in malignant cells requires STAT3 activation [97, 98]. IL-6-mediated STAT3/5 activation may be inhibited in pancreatic tumor cells through 17-AAG. An IL-6 autocrine activation loop was identified because both a hypoxic environment and recombinant IL-6 led to the increase in levels of IL-6 mRNA in pancreatic malignant cells. This autocrine (IL-6/STAT3/ HIF-1α) loop contributes to PC. Thus,

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targeting Hsp90 may disrupt the IGF-IR system as well as the IL-6/STAT3/HIF-1α autocrine activation loop within PC. Additionally, 17-AAG inhibits STAT5 phosphorylation [98, 99]. The growth inhibitory as well as antiangiogenic impact of Hsp90-targeted therapy was experimentally tested in a s.c. xenograft model and orthotopic cancer model in pancreatic tumor cells. Hsp90 inhibitors like 17-DMAG reduced growth as well as vascularization of PCs. The main regulators of pancreatic tumor cell growth and angiogenesis are IGF-IR and STAT3. Both IGF-IR and STAT3 can be successfully inhibited by Hsp90 inhibitors. These results are critical for clinical trial research. The efficacy of Hsp90-targeted treatment is examined by evaluating and measuring client proteins in FNAC biopsies from PC subjects [100]. The antineoplastic effect of Hsp90 inhibitors on cancers, other than pancreatic malignancies, is half of the maximum tolerated dose. Effectively targeting oncogenic pathways can be accomplished without high-dose Hsp90 inhibitors. Currently, Hsp90 inhibitors and their role in metastasis are still unclear. Price et al. [101] showed that geldanamycin, in a breast cancer model, promoted bone metastases. In PC, the Hsp90 inhibitors block pro-migratory molecules and weaken migration of pancreatic tumor cells [102]. A daily regimen of 17-DMAG therapy can be more efficacious. Mice that were given 17-DMAG exhibited decreased lymph node metastasis, thereby suggesting that metastatic features of pancreatic tumor cells can be functionally impaired in vivo. In conclusion, Hsp90 inhibition impairs functions of IGF-IR and disrupts the IL-6/HIF-1α/STAT3 autocrine activation loop within PC. These concepts are strongly connected with reduction in cancer growth as well as vascularization in orthotopic pancreatic tumor models. Hsp90 inhibitors therefore serve as valuable assets for molecular therapies in PC treatment.

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Insulin Resistance Is a Common Core Tethered to Diabetes and Pancreatic Cancer Risk

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Henu Kumar Verma and L. V. K. S. Bhaskar

Abstract

The association between type 2 diabetes mellitus (T2DM) and pancreatic cancer (PC) is complex bidirectional. Recent epidemiological data suggest that about 80% of PC patients have impaired glucose tolerance (IGT) or new onset of diabetes. Obesity, smoking, family history, genetic factors, insulin resistance (IR), and chronic pancreatitis are significant risk factors for PC. Several lines of evidences demonstrated that the production of insulin increases with increasing IR in diabetes patients. Hyperinsulinemia and IR are involved in the development of PC. Majority of studies suggested that risk ratio for diabetes in associated PC was higher in patients with small periods of diabetes as compared those with long periods of diabetes. Several studies also reported that antidiabetic drugs decrease risk for PC and increase survival in diabetes patients. In this chapter we describe the link between insulin resistance and pancreatic cancer risk. Keywords

Diabetes · Obesity · Insulin resistance · Biomarkers · Pancreatic cancer

Abbreviations ADA ADM AGE BMI

American Diabetes Association Adrenomedullin Advanced glycation end product Body mass index

H. K. Verma Stem Cell Laboratory, Institute of Endocrinology and Oncology, Naples, Italy L. V. K. S. Bhaskar (*) Guru Ghasidas Vishwavidyalaya, Bilaspur, India e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 G. P. Nagaraju, A. BM Reddy (eds.), Exploring Pancreatic Metabolism and Malignancy, https://doi.org/10.1007/978-981-32-9393-9_12

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CI CRP DPP-4 DZ FBG GCKR GI GLP-1 GWAS HbA1c IGF IGF-1 IGF-2 IGFBP-1 IGFBP-3 IGT IR ObR OR PC PCCC PDAC PPARG RAGE RR SHH sTNFR1 sTNF-R2 T2DM WHO

12.1

Confidence interval C-reactive protein Dipeptidyl peptidase-4 Thiazolidinedione Fasting blood glucose Glucokinase regulator gene Glucose intolerance Glucagon-like peptide-1 Genome-wide association studies Glycated hemoglobin Insulin-like growth factor Insulin-like growth factor 1 Insulin-like growth factor 2 Insulin-like growth factor-binding protein 1 Insulin-like growth factor-binding protein 3 Impaired glucose tolerance Insulin resistance Leptin receptor Odds ratio Pancreatic cancer Pancreatic Cancer Cohort Consortium Pancreatic ductal adenocarcinoma Peroxisome proliferator-activated receptor gamma Receptor for advanced glycation end products Risk ratio Sonic hedgehog Soluble tumor necrosis factor receptor 1 Soluble tumor necrosis factor receptor 2 Type 2 diabetes mellitus World Health Organization

Introduction

Pancreatic cancer (PC) is the fourth most common cause of cancer-related deaths in the United States [1]. PC is commonly diagnosed at advanced stages with a 5-year overall survival rate of only ~ 5–8% [2, 3]. It is expected that PC becomes the second most significant cause of cancer deaths by the year 2020 [4]. As there are no effective chemo- and radiotherapy treatment strategies and a silent feature of the disease, majority of the individuals diagnosed with PC die of this lethal disease [5]. Potential risk factors for PC include chronic pancreatitis, age (usually >40 years), gender (more prevalent in males), genetic factors, family history, smoking, excessive drinking, obesity, and diabetes [6–11]. In addition, epidemiological studies have associated type 2 diabetes mellitus (T2DM) with the PC risk [12, 13]. Some of the early symptoms of PC are nonspecific and overlap with the T2DM [14]. Quantitative

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analysis of the timing of the genetic evolution of PC indicated a broad 20-year time window from a precursor lesion to develop distant metastases [15]. Previous studies suggested increased incidence of PC in diabetes patients. A retrospective case– control study suggested that about 68% of patients with PC had diabetes and 40% developed diabetes 3 years prior than the diagnosis of PC [16]. Like T2DM patients, patients with progressive PC express several metabolic abnormalities such as inhibition of glucose production, deregulation of hepatic glucose metabolism, and insulin resistance (IR). The progression of diabetes in patients with PC is expected as a secondary factor due to failure of pancreatic β-cell function and subsequent to the progression to peripheral IR [17]. Several epidemiological studies reported high incidence of PC with T2DM with a comparative risk ratio (RR) ranging from 1.6 to 2.0 [12, 18]. Hence in 74% of PC patients with diabetes, the diagnosis of diabetes ensued