University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-18-2015 Regulation of the transmembrane mucin MUC4 by Wnt/β-catenin in gastrointestinal cancers Priya Pai 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 Pai, Priya, "Regulation of the transmembrane mucin MUC4 by Wnt/β-catenin in gastrointestinal cancers" (2015). Theses & Dissertations. 58. https://digitalcommons.unmc.edu/etd/58 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]. i Regulation of the transmembrane mucin MUC4 by Wnt/β- catenin in gastrointestinal cancers By PRIYA PAI 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 Department of Biochemistry and Molecular Biology Graduate Program Under the Supervision of Professor Surinder K. Batra University of Nebraska Medical Center Omaha, Nebraska November, 2015 ii Regulation of the transmembrane mucin MUC4 by Wnt/β-catenin in gastrointestinal cancers Priya Pai, PhD. University of Nebraska Medical Center, 2015 Supervisor: Surinder K. Batra, PhD. The transmembrane mucin MUC4 is a high molecular weight glycoprotein that is expressed de novo in pancreatic ductal adenocarcinoma (PDAC). MUC4 has been shown to play a tumor-promoting role in malignancies such as PDAC, ovarian cancer and breast cancer. Unlike the normal pancreas, MUC4 is ordinarily expressed by goblet and absorptive cells in the normal colonic epithelium. However, its expression/role in colorectal cancer (CRC) is not well studied. In this dissertation, the goal was to identify factor(s) that may differentially regulate MUC4 in these two disparate malignancies. Furthermore, in light of its pro-tumorigenic role in other malignancies, we analyzed the functional implications of MUC4 expression in CRC. A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites located in the proximal and distal promoters. Importantly, multiple TCF/LEF sites are typically present in the promoters of Wnt/β-catenin pathway target genes. In order to ascertain whether MUC4 was a Wnt/β-catenin target gene, we performed β-catenin knock down (KD) studies, treatment with Wnt3a ligand, as well as MUC4 promoter luciferase studies in the first section of this dissertation. In PDAC, it was observed that MUC4 transcript and protein were decreased upon β-catenin KD, WNT3a conditioned medium treatment increased MUC4 and MUC4 promoter luciferase activity was increased upon transfection with stabilized β-catenin. Furthermore, immunohistochemistry (IHC) with rapid autopsy PDAC tissues showed a positive correlation between MUC4 and β- catenin expression. Mutation of each of the three putative TCF/LEF sites showed that the sites closest to and furthest from the ATG site were critical for MUC4 promoter luciferase activity in iii the presence of stabilized β-catenin. A Chromatin immunoprecipitation assay (ChIP) confirmed that β-catenin associates with the MUC4 promoter at these two sites in PDAC. Functional studies with the β-catenin KD cells showed that migratory properties were decreased significantly upon KD, concomitant with altered levels of epithelial to mesenchymal transition (EMT) markers. We thus concluded that β-catenin up-regulates MUC4 in PDAC, and that the β-catenin-MUC4 axis likely contributes to the metastatic properties of PDAC cells. The second part of this dissertation deals with the regulation of MUC4 by β-catenin in CRC. Here, we observed that the KD of β-catenin induced an increase in MUC4 transcript and protein. This was corroborated by transient overexpression of stabilized β-catenin, which resulted in decreased MUC4. MUC4 promoter luciferase studies showed that KD of β-catenin resulted in increased promoter luciferase activity. The mutation of each of the three TCF/LEF sites and subsequent promoter luciferase assays showed that the second and third sites appeared to be repressive and that mutation of all three sites in combination caused an increase in MUC4. It was also observed that the Wnt/β-catenin pathway can indirectly repress MUC4 by suppression of Hath1, a Notch pathway target gene. Functional studies with MUC4 KD showed that MUC4 confers proliferative but not migratory properties to cells. The third part of this dissertation describes the generation of a mouse model aimed at delineating the role of Muc4 in CRC progression. Mice with colon-preferential heterozygous loss of Apc and mutant Kras, i.e. CDX2 P-NLS -Cre krasG12D/+ApcloxP/+ were crossed with the Muc4-/- mice generated in our lab. Preliminary results indicate the expression pattern of Muc4 is altered during the progression of CRC. Taken together, studies in this dissertation demonstrate the differential regulation of MUC4 by the Wnt/β-catenin pathway in PDAC and CRC, and that MUC4 expression may be pro-tumorigenic in CRC. iv TABLE OF CONTENTS CHAPTER 1A: Introduction ....................................................................................................... 1 1. Synopsis ................................................................................................................................... 3 2. Introduction to the Wnt/ β-catenin pathway ............................................................................ 4 3. The role of Wnt/β-catenin signaling in cancer ......................................................................... 5 4. Wnt/β-catenin in CRC.............................................................................................................. 6 5. Wnt/β-catenin in pancreatic cancer (PC) ................................................................................. 7 6. Mucins and their roles in cancer .............................................................................................. 9 7. The relationship between β-catenin and membrane-bound mucins in cancer, MUC1, MUC4 and MUC16 ................................................................................................................................ 11 (a) MUC1 ............................................................................................................................... 11 (b) MUC4 ............................................................................................................................... 14 (c) MUC16 ............................................................................................................................. 15 8. The relationship between β-catenin in cancer and secreted-mucins MUC2, MUC5AC, and MUC6 ........................................................................................................................................ 16 CHAPTER 1 B: MUC4 regulation and expression patterns in pancreatic ductal adenocarcinomas (PDAC) and Colorectal Cancer (CRC) ....................................................... 35 1. Synopsis ................................................................................................................................. 36 2. The regulation of MUC4 expression ...................................................................................... 37 A. The epigenetic regulation of MUC4 expression ................................................................ 37 B. Transcriptional regulation of MUC4 ................................................................................. 39 C. Post-transcriptional regulation of MUC4 .......................................................................... 42 D. Summary and conclusions ................................................................................................ 43 3. MUC4 expression patterns in PDAC and CRC ..................................................................... 44 A. MUC4 in pancreatic cancer ............................................................................................... 44 B. MUC4 in colorectal cancer................................................................................................ 45 C. MUC4 in inflammatory bowel disease: ulcerative colitis and Crohn’s disease ................ 47 D. Summary and conclusions ................................................................................................ 48 CHAPTER 1C: Dissertation General Hypothesis and Objectives .......................................... 53 1. Background and rationale ...................................................................................................... 54 v 2. Hypothesis ............................................................................................................................. 55 3. Objectives .............................................................................................................................. 55 CHAPTER 2: Materials and Methods ....................................................................................... 59 1. Cell Culture ............................................................................................................................ 60 2. Lentiviral and Retroviral Transfection ................................................................................... 60 3. Tissues specimens