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View of Breast Cancer Cell Motility…………………………………….....32 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosphy in the Graduate School of The Ohio State University By Yadwinder S Deol, MS Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Dr. Ramesh K Ganju, PhD (Advisor) Dr. Xue-Feng Bai, PhD Dr. James Waldman, PhD Dr. Sujit Basu, MD. PhD Copyright by Yadwinder S Deol, MS 2012 ABSTRACT Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα-) invasive breast cancers, its role in ERα-positive (ERα+) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα+ breast cancer cells and observed that S100A7-overexpression in ERα+ breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in a nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα+ breast cancer cells. We observed down-regulation of β- catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα+ breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β- catenin/TCF4 pathway. ii Our results also demonstrated that S100A7-overexpression in MCF7 cells increased the stability of p53 compared to vector control cells. We found p53 to be mainly localized in nucleus of the S100A7-overexpressing cells. Moreover, co- immunoprecipitation studies revealed that S100A7 binds to p53 directly and both S100A7 and p53 co-localize in the nucleus of S100A7-overexpressing cells. P53 is generally stabilized in the nucleus and we found that phosphorylation of p53 at Serine-15 residue was enhanced in S100A7-overexpressing MCF7 cells compared to vector control cells. Serine-15 is known to stabilize and activate p53 during cellular stresses. Further, real time PCR p53 array showed the increased expression of the stress related genes such as ATR and BRCA-1 along with genes involved in apoptosis and cell cycle arrest pathways. We validated the differentially regulated genes of stress pathway by western blotting and observed that S100A7-overexpression in MCF7 cells increased the expression of ATR and its downstream molecules p-Chk1 and p-Chk2 which are known to phosphorylate serine-15 residue of p53. We further evaluated the role of p53 and S100A7 in an in vivo mouse model system by generating a mouse model which was deficient in p53 expression but expressed murine S100A7 (mS100A7A10) in the mammary glands under doxycycline treatment. After two and half months of doxycycline treatment, we observed the development of spontaneous tumors in the fourth inguinal mammary gland of the mouse which strengthens our finding of association between S100A7 and p53. S100A7-overexpression in MCF7 cells also exhibited decreased actin polymerization as evident from decreased formation of migratory structures. Actin iii staining revealed F-actin expression on the edges of plasma membrane in vector control cells whereas in S100A7 overexpressing cells, actin staining was mostly intracellular. Both serum-induced and EGF-induced migration was reduced in S100A7 overexpressing cells compared to vector control cells. We further found that S100A7-overexpression reduced EGF-induced activation of EGFR, AKT and ERK. Upon further analysis of the mechanisms that regulate actin polymerization, we observed reduced activation of the Rac1 pathway and cofilin. In conclusion, our studies reveal for the first time that S100A7-overexpressing ERα+ breast cancer cells exhibit tumor suppressor capabilities through a coordinated down-modulation of the β-catenin/TCF4 and p53 pathways both in vitro and in vivo. Our results also show that S100A7 has the ability to bind to p53 and stabilize it in the nucleus which then activates the stress induced pathway. Moreover, we also show that S100A7 modulates the cytoskeleton by regulating actin polymerization. Since S100A7 has been shown to enhance tumorigenicity in ERα- cells, our studies suggest that S100A7 may possess differential activities in ERα+ compared with ERα- cells. iv DEDICATED TO My parents who always inspired me to achieve the best in whatever I do My wife, who always helped me and supported me in life and graduate school v ACKNOWLEDGEMENTS I would like to sincerely thank Dr. Ramesh K. Ganju for all kind of support and help, mentorship and for providing me with the opportunity to work in his lab and giving me the great experience in his laboratory. I am very grateful to my advisory committee members, Dr. Sujit Basu, Dr. James Waldman and Dr. Xue-Feng Bai for their valuable time and guidance. I would like to thank all members of the Dr. Ganju lab who have been a great help throughout my stay in the lab. I would especially like to thank Dr. Mohd Nasser who helped me with the set up of the project and also Dr. Anand Appakuddal and Dr. Nagaraja Tirumuru who helped me when I had questions in the lab. I also thank Michelle Van-Fossen for her help in everyday things. vi VITA 1998................................................................Dasmesh Public High School, India 2002................................................................BS, Punjab Agricultural University, India. 2006................................................................MS, The Ohio State University, USA 2007 to present ..............................................PhD, The Ohio State University, USA PUBLICATIONS 1. Nasser MW, Qamri Z, Deol YS, Shilo K, Leone G, Bai X-F, Zou X, Wolf R, Yuspa S and Ganju RK. 20112. S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways. Cancer Research, 72(3):604-15. 2. Deol YS, Nasser MW, Yu L, Zou X, Ganju RK. 2011. Tumor suppressive effects of psoriasin (S100A7) are mediated through β-catenin/TCF4 pathway in estrogen receptor positive breast cancer cells. Journal of Biological Chemistry, 286(52):44845- 54. 3. Nasser MW, Qamri Z, Deol YS, Smith D, Shilo K, Zou X, Ganju RK. 2011. Crosstalk between Chemokine Receptor CXCR4 and Cannabinoid Receptor CB2 in Modulating Breast Cancer Growth and Invasion. PLoS ONE 6(9): e23901. doi:10.1371/journal.pone.0023901 4. Anand AR, Prasad A, Bradley RR, Deol YS, Nagaraja T, Ren X, Terwilliger EF, Ganju RK. 2009. HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2, p38 MAP kinase, and LSP1. Blood. Oct 22; 114 (17):3588-600. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology Area of Emphasis: Pathology of Breast Cancer vii TABLE OF CONTENTS Abstract………………………………………………………………………………........ii Dedication…………………………………………………………………………….......iv Acknowledgments……………………………………………………………………....…v Vita……………………………………………………………………………………......vi List of figures………………………………………………………………………........viii List of Tables…………………………………………………………………………......ix CHAPTERS CHAPTER 1: INTRODUCTION……………………………………………………........1 1.1 Breast Cancer………………………………………………………......1 1.2 S100 Family of Proteins…………………………………………..…...6 1.3 Psoriasin (S100A7) ………………………………………………........9 1.4 S100A7 and Breast Cancer……………………………………….......14 1.5 β-Catenin/TCF 4 Pathway…………………………………………....20 1.6 β-Catenin/TCF4 Pathway and Tumorigenesis…………………..........23 1.7 Expression of p53 and its Role in Cancer………………………....….26 1.8 Actin Cytoskeleton and its Role in Cancer ……………………....…..31 viii 1.9 Objectives of the Study…………………………………………….....34 CHAPTER 2: MATERIAL AND METHODS……………………………………..........36 2.1 Cell Culture, Reagents and Antibodies………………………….........36 2.2 Constructs and Transfections…………………………………....……37 2.3 Proliferation Assay……………………………………………….......38 2.4 Chemotaxis Assay…………………………………………………....38 2.5 Wound Healing Assay……………………………………………......39 2.6 Western Blotting………………………………………………..….....39 2.7 Co-Immunoprecipitation……………………………………….….....40 2.8 Confocal Microscopy…………………………………………..….....40 2.9 TCF4 Luciferase Reporter Assay………………………………….....41 2.10 Microarray Analyses……………………………………………......41 2.11 Quantitative Real Time Polymerase Chain Reaction (qRT- PCR)……………………………………………………………………...42 2.12 Xenograft Mouse Model………………………………………....…43 2.13 Immunohistochemistry (IHC)…………………………………....…43 2.14 Rac1 Activation Assay………………………………………….......44 2.15 Real Time p53 PCR Array……………………………………….....44 2.16 Generation of Transgenic Mice…………………………………......45 ix 2.17 Whole-mount Analysis of Mammary Glands………………...…......45 2.18 Statistical Analyses……………………………………………….....46 CHAPTER 3: RESULTS…………………………………………………………….......47 3.1 S100A7-overexpression decreases growth in ERα+ cells both in vitro and in vivo…………………………………………………….....…47 3.1.1 S100A7-overexpression reduced proliferation in MCF7 and T47D cells in vitro…………………..………………………………..................47 3.1.2 S100A7-overexpression inhibits tumor growth in vivo.......…….....48 3.2 Mechanisms of growth inhibition by S100A7-overexpression
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