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Secretory Mucin MUC5AC in Gastrointestinal Malignancies

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Secretory Mucin MUC5AC in Gastrointestinal Malignancies University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-7-2016 Secretory Mucin MUC5AC in Gastrointestinal Malignancies Shiv Ram Krishn University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Krishn, Shiv Ram, "Secretory Mucin MUC5AC in Gastrointestinal Malignancies" (2016). Theses & Dissertations. 110. https://digitalcommons.unmc.edu/etd/110 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. Secretory mucin MUC5AC in gastrointestinal malignancies by SHIV RAM KRISHN A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biochemistry & Molecular Biology Graduate Program Under the supervision of Dr. Surinder K. Batra University of Nebraska Medical Center Omaha, Nebraska April, 2016 Supervisory Committee: Dr. Michael A. Hollingsworth, Ph.D. Dr. Vimla Band, Ph.D. Parmender P. Mehta, Ph.D. Dr. Chittibabu Guda, Ph.D. Dr. Maneesh Jain, Ph.D. i Secretory mucin MUC5AC in gastrointestinal malignancies Shiv Ram Krishn, Ph.D. University of Nebraska, 2016 Advisor: Dr. Surinder K Batra, Ph.D. Secretory mucin MUC5AC is an extensively glycosylated high molecular weight protein that forms a polymeric gel layer over the epithelial layers under physiological conditions to protect these surfaces from myriad of insults. MUC5AC is known to be implicated in various malignancies including pancreatic cancer and colorectal cancer. It is overexpressed in pancreatic cancer compared to no expression in the normal pancreas. However, its functional implications and associated mechanistic basis in pancreatic cancer remain obscure. Therefore, we investigated the role of MUC5AC in initiation and progression of pancreatic cancer. Our study showed that MUC5AC expression is elevated during pancreatic cancer progression while it was not detected in the normal pancreas. To elucidate the functional role of MUC5AC in pancreatic cancer, we performed MUC5AC knockdown studies in pancreatic cancer cell lines (FG/COLO357, SW1990). Knockdown of MUC5AC decreased pancreatic cancer cell viability, tumorigenicity, metastasis, angiogenesis, and increased sensitivity to Gemcitabine. We observed that MUC5AC might impact pancreatic cancer cell growth and metastasis by its interactions with E-Cadherin and β-Catenin. To further elaborate the functional role of MUC5AC, we developed the KrasG12D; Pdx1-Cre; Muc5ac-/- mouse model by crossing Muc5ac-/- mice with pancreatic cancer mice model- KrasG12D; Pdx1-Cre mice. A delay in the onset as well as the progression of pancreatic cancer was observed in KrasG12D; Pdx1-Cre; Muc5ac-/- mice as compared to the control KrasG12D; Pdx1-Cre mice. Further, MUC5AC is not expressed in the normal colon, but it is de novo expressed during early stages and further increases during progression to advanced stages of colorectal malignancy. Despite association of increased MUC5AC levels with conventional and serrated ii neoplasia pathway of colorectal carcinogenesis, the diagnostic utility of MUC5AC for predicting future colorectal malignancy remained unexploited. Hence, we also explored the diagnostic utility of MUC5AC in conjunction with other differentially expressed mucins and mucin-associated glycans to predict colorectal malignancy. The study findings revealed an improved efficacy of combined MUC2, MUC5AC, and MUC17 expression for differentiating premalignant and/or malignant lesions from benign polyps. Furthermore, we addressed the problem of current non- availability of specific and sensitive markers to discriminate benign hyperplastic polyps from sessile serrated and tubular adenomas. We identified a combinatorial panel of mucins and glycoepitope based molecular markers (CA 19-9/MUC17/MUC5AC) that could discriminate sessile serrated adenoma/polyps and tubular adenomas from benign hyperplastic polyps with high sensitivity and specificity. Overall, our studies demonstrate that MUC5AC plays a key role in onset and progression of pancreatic cancer. Further, our findings indicate that in conjunction with other differentially expressed mucins and associated glycans, MUC5AC may significantly improve the early stage diagnosis of colorectal cancer. iii TABLE OF CONTENTS iv LIST OF FIGURES x LIST OF TABLES xii ABBREVIATIONS xiv ACKNOWLEDGEMENTS xxi iv TABLE OF CONTENTS Chapter I: Introduction Pg. No. A review on MUC5AC: Structure, and role of secretory mucin MUC5AC in benign and malignant pathologies 1. Introduction 2 2. Genomic organization, domain structure and functions 4 3. Expression and functions of MUC5AC 7 3.1 MUC5AC in physiological conditions 7 3.2 MUC5AC in Benign pathologies 8 3.2.A Allergic Rhinitis 8 3.2.B Chronic Rhinosinusitis 9 3.2.C Otitis media 9 3.2.D Chronic airway diseases 10 3.2.E Barrett’s esophagus 11 3.2.F Helicobacter pylori infections 12 3.2.G Pancreatitis 13 3.2.H Inflammatory bowel disease 13 3.3 MUC5AC and malignant diseases 14 3.3.A Salivary gland carcinomas 15 3.3.B Lung cancer 15 3.3.C Breast cancer 17 3.3.D Gastric cancer 17 3.3.E Gallbladder adenocarcinoma 18 3.3.F Cholangiocarcinoma 19 3.3.G Pancreatic cancer 20 v 3.3.G.a Pancreatic cancer diagnosis 22 3.3.G.b Pancreatic cancer therapy 23 3.3.H Colorectal cancer 24 3.3.I Ovarian cancer 26 3.3.J Cervical cancer 27 3.3.K Prostate cancer 28 3.3.L Urothelial bladder cancer 28 3.3.M Extramammary Paget’s disease 28 3.3.N Pseudomyxoma peritonei 29 4. Functional and mechanistic implications of MUC5AC in cancer 29 5. Muc5ac mice models 31 6. Regulation of MUC5AC expression 35 7. Hypoxia and MUC5AC 36 8. Smoking and MUC5AC 37 9. Conclusions and perspectives 39 References 48 General Hypothesis and objectives 71 Chapter II: Materials and Methods 73 1. Molecular basis of MUC5AC-mediated cell cycle regulation, metastatic properties, angiogenesis and chemoresistance of pancreatic cancer 74 1.A Immunohistochemistry 74 1.B Cell culture and transfections 75 1.C Immunoblotting 75 1.D Immunofluorescence/Confocal microscopy 76 1.E RNA extraction, RT-PCR, Q-PCR 77 vi 1.F MTT Assay 77 1.G Anchorage independent proliferation assay 78 1.H Apoptosis assay 78 1.I Cell cycle analysis 79 1.J Motility assay 79 1.K Cell adhesion assay 80 1.L Angiogenesis tube formation assay 80 1.M Co-immunoprecipitation 80 1.N Orthotopic implantation 81 1.O Subcellular fractionation 82 1.P Statistical analyses 82 2. Loss of Muc5ac mediated impact on pancreatic cancer progression in KRasG12D; Pdx1- Cre mouse model 83 2.A Generation of KrasG12D; Pdx1-Cre; Muc5ac-/- mouse model 83 2.B Immunohistochemistry (Mouse tissues) 83 2.C c-DNA isolation and genotyping 84 3. Mucins and associated glycan signatures in colon adenoma-carcinoma sequence: Prospective pathological implication(s) for early diagnosis of colon cancer 85 3.A Tissue specimens 85 3.B Immunohistochemistry (IHC) 85 3.C Statistical Analysis 86 4. Combinatorial assessment of CA 19-9/MUC17/MUC5AC expression discriminates sessile serrated adenoma/polyps and tubular adenoma from hyperplastic polyps 87 4.A Tissue specimens and patient demographics 87 4.B Immunohistochemistry (IHC) 87 vii 4.C Statistical analysis 88 References 95 Chapter III: Molecular basis of MUC5AC mediated cell cycle regulation, metastatic properties, angiogenesis and chemoresistance of pancreatic cancer cells 96 1. Synopsis 97 2. Background and rationale 98 3. Results 99 3.A MUC5AC is overexpressed in PC tissues and PC cell lines 99 3.B MUC5AC silencing decreased cell viability, anchorage-independent growth and induces apoptosis 100 3.C MUC5AC silencing induces G1/S phase cell cycle arrest 101 3.D MUC5AC confers resistance to Gemcitabine in pancreatic cancer cells 101 3.E MUC5AC knockdown reduces cell motility and adhesion to extracellular matrix proteins 103 3.F MUC5AC exists in a complex with E-Cadherin and β-Catenin 104 3.G Impact of MUC5AC expressing pancreatic cancer cells on endothelial cell viability and tube formation capacity 105 3.H Inhibition of MUC5AC in pancreatic cancer cells results in decreased tumorigenicity and metastasis in vivo 106 4. Discussion 106 References 131 Chapter IV: Loss of Muc5ac mediated impact on pancreatic cancer progression in KRasG12D; Pdx1-Cre mouse model 134 1. Synopsis 135 2. Background and rationale 136 viii 3. Results 137 3.A Breeding strategy and establishment of PDAC mice model with knockout 137 of Muc5ac 3.B Progression of PC in the KrasG12D; Pdx1-Cre; Muc5ac -/- mouse model 137 4. Discussion 139 References 150 Chapter V: Mucins (MUC5AC, MUC2, MUC17) and associated glycans (Tn/STn-MUC1) signatures in colon adenoma-carcinoma sequence: Prospective pathological implication(s) for early diagnosis of colon cancer 153 1. Synopsis 155 2. Background and rationale 156 3. Results 157 3.A Expression of transmembrane mucins 157 3.B Expression of secretory mucins 159 3.C Expression of mucin-associated glycans 160 3.D Multivariate analyses to evaluate biomarker combinations as predictors of malignancy 162 4. Discussion 163 References 184 Chapter VI: Combinatorial assessment of CA 19-9/MUC17/MUC5AC expression discriminates sessile serrated adenoma/polyp and tubular adenoma from hyperplastic polyps 189 1. Synopsis 190 2. Background
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