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Elucidating the Role of Smad7 in Pancreatic Cancer

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Elucidating the Role of Smad7 in Pancreatic Cancer ELUCIDATING THE ROLE OF SMAD7 IN PANCREATIC CANCER THROUGH IN VIVO STUDIES Sudha Satish Savant Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University September 2017 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _____________________________ Murray Korc, MD, Chair _____________________________ Ronald C. Wek, PhD Doctoral Committee _____________________________ Lawrence A. Quilliam, PhD June16, 2017 _____________________________ Jing Wu Xie, PhD ii Dedication I dedicate this dissertation to my parents, Monica and Satish Savant, and to my husband, Rajesh Sardar, as without their unconditional love and support this would have not been possible. iii Acknowledgments I want to thank my advisor, Murray Korc, and my committee, Ronald Wek, Lawrence A. Quilliam and Jing Wu Xie for their guidance and support during my graduate studies. I would like to thank all the past and current members of the Korc lab for their feedback and scientific discussions in the lab. I would like to thank all the faculty, staff and students of the Biochemistry and Molecular Biology Department and of the Simon Cancer center for their support throughout my graduate studies. Additionally, I would like to thank the Graduate division, especially Randy Brutkiewicz, Tara Hobson-Prater and Brandy Wood for their support throughout graduate school and for providing opportunities and nurturing my professional career. I am grateful to Barry Muhoberac, Melissa Fishel, Lakshmi Prabhu, Eshaani Mitra and my parents for their suggestions and support with this thesis. I would like to thank my brother, family and friends for their continued support and love throughout the graduate journey. iv Sudha Satish Savant Elucidating the role of Smad7 in pancreatic cancer through in vivo studies Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death with a mere 9% survival rate. PDACs harbor KRAS (92-95%) and CDKN2A (90%) mutations, overexpress tyrosine kinase receptors, their ligands, and transforming growth factor-β (TGF-β) isoforms. Canonical TGF-β signaling is mediated via Smad2, Smad3 and Smad4, whereas inhibitory Smad6 and Smad7 attenuate TGF-β signaling. Smad7 is overexpressed in PDAC and blocks TGF-β-mediated growth inhibition in vitro. However, the exact role of Smad7 in PDAC is not known. We have established a genetically engineered mouse model of PDAC in which conditional expression of Smad7 and oncogenic KrasG12D are driven in the pancreas by Pdx1-Cre. These LSL- KrasG12D;SMAD7;Pdx1-Cre(KS7C) mice exhibit accelerated progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC by comparison with LSL-KrasG12D;Pdx1-Cre (KC) mice harboring the KrasG12D mutation alone, whereas in the absence of oncogenic Kras, pancreatic histology remains normal in spite of a 9- fold increase in Smad7 mRNA levels. KS7C pancreata exhibit increased PanIN and pancreatic cancer cell proliferation, and these changes are recapitulated in a tetracycline (tet) inducible mouse model of Smad7 (KtetS7C). In both models, pre-neoplastic lesions and PDACs exhibited increased levels of anterior gradient 2 (AGR2), hyper-phosphorylated retinoblastoma protein (p-pRb) and p-Smad2, but low levels of p-Smad3 and p21. Smad7 overexpression in human pancreatic cancer cells (hPCCs) results in downregulated p21 and upregulated AGR2 mRNA and protein levels, and decreased binding of Smad3/4 complex to the AGR2 promoter. Smad3 silencing in hPCCS also resulted in downregulated p21 mRNA and upregulated AGR2 mRNA levels. These findings indicate that Smad7 blocks TGF-β pathways, in part, by preferentially decreasing Smad3 v phosphorylation and enhancing AGR2 expression in PDAC, and suggest that targeting Smad7 may constitute a novel therapeutic approach in PDAC. Murray Korc, MD, Chair vi Contents List of Tables ............................................................................................................... xi List of Figures .............................................................................................................. xii Abbreviations .............................................................................................................. xv 1 Introduction 1.1 Pancreatic cancer .................................................................................................. 1 1.2 Risk factors for PDAC ............................................................................................. 4 1.3 Pancreas ................................................................................................................ 5 1.4 Exocrine pancreas .................................................................................................. 6 1.5 TGF-β signaling ...................................................................................................... 7 1.6 Smad7 .................................................................................................................... 9 1.7 Cell cycle .............................................................................................................. 10 1.8 KRAS mutations in PDAC ..................................................................................... 12 1.9 Need for understanding Smad7 in pancreatic cancer ............................................ 14 1.10 Pancreatitis, TGF-β and Smad7 .......................................................................... 16 2 Materials and Methods ............................................................................................. 17 2.1 LSL-SMAD7 Transgene generation ...................................................................... 17 2.2 ES Cell Electroporation, Selection, Injection and Screening ................................. 17 2.3 Generation of SMAD7 overexpressing transgenic mice ........................................ 18 2.4 Mouse Maintenance and Genotyping.................................................................... 20 2.5 Generation of KS7C, KtetS7C and KR+/-S7C mouse models ................................ 20 2.6 DNA extraction from tail ........................................................................................ 21 2.7 Genotyping ........................................................................................................... 21 2.8 RNA isolation from murine pancreatic tissue and cell lysates and conversion to cDNA ...................................................................................................................... 22 2.9 DNA extraction from cells ..................................................................................... 22 2.10 Gene expression analysis ................................................................................... 22 2.11 Protein isolation and quantification ..................................................................... 22 2.12 Immunoblotting and quantification of bands ........................................................ 23 2.13 Histology, Immunohistochemistry and Immunofluorescence ............................... 23 vii 2.14 IF quantification .................................................................................................. 24 2.15 Statistics ............................................................................................................. 24 2.16 Cell culture .......................................................................................................... 24 2.17 Tetracycline inducible SMAD7 overexpressing hPCCs ....................................... 25 2.18 Constitutive overexpression of SMAD7 in hPCCs ............................................... 26 2.19 Knockdown of SMAD3 in COLO-357 .................................................................. 26 2.20 SMAD3 CRISPR of hPCCs ................................................................................. 26 2.21 Isolation of mouse cells from tumor..................................................................... 27 2.22 Immunocytochemistry ......................................................................................... 27 2.23 Cell proliferation assays ...................................................................................... 28 2.24 3-Dimentionsional tissue culture ......................................................................... 28 2.25 Orthotopic injections ........................................................................................... 28 2.26 Induction of Acute Pancreatitis ............................................................................ 29 2.27 Chromatin Immunoprecipitation Assays .............................................................. 29 2.28 Gene Set Enrichment Analysis ........................................................................... 29 2.29 Composition of buffers used ............................................................................... 30 2.30 Primers used ...................................................................................................... 31 2.31 PCR reactions .................................................................................................... 34 2.32 PCR cycling conditions ....................................................................................... 34 2.33 List of antibodies used .......................................................................................
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