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Gain-Of-Function Effects of Mutant P53 Explored Using a Three Gain-of-Function Effects of Mutant p53 Explored Using a Three- Dimensional Culture Model of Breast Cancer William A. Freed-Pastor Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2011 William A. Freed-Pastor All Rights Reserved ABSTRACT Gain-of-Function Effects of Mutant p53 Explored Using a Three-Dimensional Culture Model of Breast Cancer William A. Freed-Pastor p53 is the most frequent target for mutation in human tumors and mutation at this locus is a common and early event in breast carcinogenesis. Breast tumors with mutated p53 often contain abundant levels of this mutant protein, which has been postulated to actively contribute to tumorigenesis by acquiring pro-oncogenic (“gain- of-function”) properties. To elucidate how mutant p53 might contribute to mammary carcinogenesis, we employed a three-dimensional (3D) culture model of breast cancer. When placed in a laminin-rich extracellular matrix, non-malignant mammary epithelial cells form structures highly reminiscent for many aspects of acinar structures found in vivo. On the other hand, breast cancer cells, when placed in the same environment, form highly disorganized and sometimes invasive structures. Modulation of critical oncogenic signaling pathways has been shown to phenotypically revert breast cancer cells to a more acinar-like morphology. We examined the role of mutant p53 in this context by generating stable, regulatable p53 shRNA derivatives of mammary carcinoma cell lines to deplete endogenous mutant p53. We demonstrated that, depending on the cellular context, mutant p53 depletion is sufficient to significantly reduce invasion or in some cases actually induce a phenotypic reversion to more acinar-like structures in breast cancer cells grown in 3D culture. Additionally, using stable overexpression of a panel of tumor-derived p53 mutants in non-malignant mammary epithelial cells, we found that mutant p53 is sufficient to disrupt normal acinar morphogenesis. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates revealed that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on mammary tissue architecture. We then showed that mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed mevalonate pathway genes in human breast tumors and elevated expression of the mevalonate pathway correlates with a poor prognosis in breast cancer. We also queried a number of pathways/proteins that had previously been implicated in breast cancer and shown to be sufficient to bring about a phenotypic reversion in 3D culture to search for additional mechanisms by which mutant p53 might contribute to mammary carcinogenesis. Using this approach, we identified integrin β4 as a novel target of mutant p53 in breast cancer cells and demonstrated that stable knockdown of integrin β4 is sufficient to dramatically reduce invasive processes in breast cancer cells grown in 3D culture. We also show that mutant p53 associates with the promoter of ITGB4, the gene encoding integrin β4. Finally, we demonstrated that inhibition of NF-κB, a downstream mediator of integrin β4signaling, can mimic the phenotypic effects of mutant p53 depletion. These findings contribute to our understanding of breast carcinogenesis and may offer novel prognostic indicators and therapeutic targets for tumors bearing mutations in p53. TABLE OF CONTENTS CHAPTER 1: INTRODUCTION....................................................................................... 1 THE DISCOVERY OF THE P53 “PROTO-ONCOGENE” ............................................................... 2 P53 AND TUMOR SUPPRESSION ............................................................................................. 3 P53 AND THERAPEUTIC APPROACHES ................................................................................... 5 FUNCTION OF WILD-TYPE P53............................................................................................... 7 GAIN-OF-FUNCTION HYPOTHESIS.......................................................................................... 9 Pro-Oncogenic Roles of Mutant p53.......................................................................... 12 Mutant p53 interacting partners ................................................................................ 14 The p53 family: p63 and p73...................................................................................... 14 Mutant p53 and transcription .................................................................................... 17 What can we learn from mutational spectra of TP53 ................................................ 22 Mutant p53: One name, many proteins...................................................................... 24 Stabilization of Mutant p53 in Tumors....................................................................... 25 P53 AND METABOLISM ....................................................................................................... 27 TP53 AND BREAST CANCER................................................................................................. 28 MODELING THE MAMMARY GLAND: FROM 2D TO 3D CULTURE ........................................... 30 MODELING BREAST TUMORIGENESIS IN 3D CULTURE.......................................................... 32 Luminal Clearance..................................................................................................... 33 DRIVING TUMORIGENESIS: EFFECTS ON MAMMARY TISSUE ARCHITECTURE........................ 35 Extracellular Factors ................................................................................................. 35 Cell Adhesion Molecules............................................................................................ 38 Receptor Tyrosine Kinases......................................................................................... 41 i Intracellular Signal Transduction.............................................................................. 43 Nuclear Proteins ........................................................................................................ 47 3D CULTURE AND METABOLISM ......................................................................................... 49 THE HISTORY OF CHOLESTEROL.......................................................................................... 50 THE MEVALONATE PATHWAY (ISOPRENOID/STEROL BIOSYNTHETIC PATHWAY) ................. 51 Non-sterol products of the mevalonate pathway........................................................ 53 Regulation of the mevalonate pathway ...................................................................... 54 MEVALONATE PATHWAY INHIBITORS ................................................................................. 57 THE MEVALONATE PATHWAY AND CANCER........................................................................ 59 CONCLUDING REMARKS ..................................................................................................... 60 REFERENCES ..................................................................................................................... 62 FIGURE LEGENDS ............................................................................................................... 90 FIGURES............................................................................................................................. 91 TABLES............................................................................................................................... 93 CHAPTER 2: MUTANT P53 DISRUPTS MAMMARY ACINAR MORPHOGENESIS VIA THE MEVALONATE PATHWAY......................................................................... 98 SUMMARY........................................................................................................................ 100 INTRODUCTION ................................................................................................................ 101 RESULTS .......................................................................................................................... 103 DISCUSSION ..................................................................................................................... 118 EXPERIMENTAL PROCEDURES .......................................................................................... 124 ACKNOWLEDGEMENTS..................................................................................................... 137 REFERENCES .................................................................................................................... 138 ii FIGURE LEGENDS............................................................................................................. 149 SUPPLEMENTAL FIGURE LEGENDS ................................................................................... 159 FIGURES........................................................................................................................... 168 SUPPLEMENTAL FIGURES ................................................................................................. 175 SUPPLEMENTAL TABLES .................................................................................................. 182 CHAPTER 3: MUTANT P53 REGULATES INTEGRIN
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