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Identification of the Rassf3 Gene As a Potential Tumor Suppressor

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Identification of the Rassf3 Gene As a Potential Tumor Suppressor Clemson University TigerPrints All Dissertations Dissertations 12-2006 Identification of the Rassf3 Gene as a Potential Tumor Suppressor Responsible for the Resistance to Mammary Tumor Development in MMTV/ neu Transgenic Mice Isabelle Jacquemart Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Microbiology Commons Recommended Citation Jacquemart, Isabelle, "Identification of the Rassf3 Gene as a Potential Tumor Suppressor Responsible for the Resistance to Mammary Tumor Development in MMTV/neu Transgenic Mice" (2006). All Dissertations. 26. https://tigerprints.clemson.edu/all_dissertations/26 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. IDENTIFICATION OF THE Rassf3 GENE AS A POTENTIAL TUMOR SUPPRESSOR RESPONSIBLE FOR THE RESISTANCE TO MAMMARY TUMOR DEVELOPMENT IN MMTV/neu TRANSGENIC MICE A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Microbiology by Isabelle C. Jacquemart December 2006 Accepted by: Dr. Wen Y. Chen, Committee Chair Dr. Charles D. Rice Dr. Lyndon L. Larcom Dr. Lesly Temesvari i ABSTRACT The MMTV/neu transgenic mouse line is a well-documented animal model for studying HER2/neu-related breast cancer. It has been reported that a small percentage, approximately 20%, of the virgin female MMTV/neu mice seems resistant to the development of mammary gland adenoma, despite the overexpression of the neu oncogene. To identify the factors that are responsible for the tumor resistance in these MMTV/neu female transgenic mice, comparative genetic profiling was used to screen the alterations in gene expression in the mammary gland. A novel gene named the RAS Association domain (RalGDS/AF-6) Family 3 (Rassf3), which belongs to a family of RAS effectors and tumor suppressor genes was identified in this study. Data presented in this dissertation show: 1) that the Rassf3 gene is overexpressed in the mammary gland of the tumor-resistant MMTV/neu mice compared to their tumor-susceptible MMTV/neu transgenic littermates or age-matched non-transgenic FVB mice, and 2) that the Rassf3 gene is significantly up-regulated in neu-specific mouse mammary tumors compared to adjacent normal tissues. To further confirm the role of the Rassf3 gene in mammary carcinogenesis, a series of in vitro and in vivo experiments were explored. The results show that overexpression of RASSF3 inhibits cell proliferation in HER2 positive human and mouse breast cancer cell lines. The inhibitory effect of RASSF3 seems to be through induction ii of apoptosis. In addition, co-transfection of the Rassf3 gene with the activated H-RAS gene in SKBR3 human breast cancer cells decreased H-RAS protein level, suggesting that RASSF3 protein can indirectly interact with H-RAS protein. A novel MMTV/Rassf3-neu bi-transgenic mouse line, overexpressing both Rassf3 and neu genes in mammary glands, was also established. The mammary tumor incidence in virgin female bi-transgenic mice was delayed compared to their MMTV/neu+/- littermates. Together, these data suggest that Rassf3, a RAS effector, is a candidate gene that may influence the mammary tumor incidence in virgin female MMTV/neu transgenic mice. iii ACKNOWLEDGEMENTS Several individuals have played a major role in the development of this dissertation. My advisor, Dr. Wen Y. Chen, has been of great assistance and guidance throughout my four years of study at Clemson. His depth of knowledge and experience in the field of Molecular Biology has sparked many conversations and ideas that are shown through my work. I am very grateful for the role he has played in my education. I would also like to recognize the support received from my friends and colleagues, Jang P. Park, John F. Langenheim and Seth Tomblyn at the Molecular Biology Lab of the Oncology Research Institute in Greenville Hospital System. Special thanks go to my friends and colleagues Michele L. Scotti for her encouragement and friendship throughout my program and Alison E. Spring for her support and assistance in the preparation of this dissertation. In addition, I would like to thank Ms. Margaret Nicholson, the Executive Director of the Fulbright Program in Belgium and Dr. Calvin L. Schoulties, the Dean of the College of Agriculture, Forestry and Life Sciences at Clemson University for their sponsor and support throughout my program at Clemson University. Finally, I would like to thank my family. There have been countless conversations of motivation that have kept my morale up and pushed me to complete this work. Their love, understanding and encouragement are what brought me to Clemson and helped me achieved my goals during these four years of study. iv TABLE OF CONTENTS Page TITLE PAGE ......................................................................................................... i ABSTRACT ........................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................... iv LIST OF TABLES ................................................................................................. viii LIST OF FIGURES ............................................................................................... ix ABBREVIATIONS ............................................................................................................. xii CHAPTER 1. INTRODUCTION ................................................................................... 1 1.1. Cancer ........................................................................................ 1 1.2. Oncogenes, Tumor Suppressors and Stability Genes ........................................................................ 3 1.3. Hallmarks of Cancer .................................................................. 6 1.4. Breast Cancer ............................................................................. 10 1.5. HER2/neu Oncogene .................................................................. 23 1.6. RAS Oncogene ........................................................................... 36 1.7. The Use of Transgenic Mouse Technology in Cancer Research ...................................................................... 47 1.8. The Use of Gene Expression Profiling in Cancer Research ...................................................................... 53 2. HYPOTHESIS AND OBJECTIVES....................................................... 57 3. MATERIAL AND METHODS............................................................... 58 3.1. Animal Model ............................................................................ 58 3.2. RNA Isolation ............................................................................ 58 3.3. Microarray Analysis ................................................................... 59 3.4. Reverse Transcription-PCR (RT-PCR) ...................................... 62 3.5. Cloning and Plasmid Construction ............................................ 65 v Table of Contents (Continued) Page 3.6. RASSF3 Protein Production ...................................................... 68 3.7. Custom Antibody Design and Production ................................. 69 3.8. Cell Culture and Reagents ......................................................... 72 3.9. Protein Isolation and Western Blot Analysis ............................. 73 3.10. Transient Transfection ............................................................. 76 3.11. Cell Proliferation MTS/PMS Assay ......................................... 76 3.12. Apoptosis Assay ....................................................................... 77 3.13. Generation and Study of Transgenic Mice .............................. 78 3.14. Mammary Gland Whole Mounts ............................................. 80 3.15. Statistical Analyses .................................................................. 81 4. RESULTS ............................................................................................... 82 4.1. Comparison of the Mammary Gland Morphology of the MMTV/neu Transgenic Mice ....................................... 82 4.2. Identification of Differentially Expressed Genes by cDNA Microarray Analysis ............................................... 84 4.3. Analysis of the Differentially Expressed Candidate Genes ..................................................................... 90 4.4. Functional Studies of the Rassf3 Gene in HER2/neu-initiated Breast Cancer .......................................... 102 4.5. Generation and Analysis of Novel Transgenic Mouse Lines: The MMTV/Rassf3 Transgenic Mice and MMTV/Rassf3-neu Bi-Transgenic Mice ................................................................ 114 5. DISCUSSION ......................................................................................... 125 5.1. HER2/neu Human Breast Cancer and its Mouse Models ..................................................................................... 125 5.2. Identification of the Genes Responsible for the Tumor Resistant Phenotype in MMTV/neu Transgenic Mice by cDNA Microarray Analysis ................................................................................... 129 5.3. Analysis
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