Deciphering the Functions of Ets2, Pten and P53 in Stromal Fibroblasts in Multiple

Deciphering the Functions of Ets2, Pten and p53 in Stromal Fibroblasts in Multiple Breast Cancer Models DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Julie Wallace Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2013 Dissertation Committee: Michael C. Ostrowski, PhD, Advisor Gustavo Leone, PhD Denis Guttridge, PhD Dawn Chandler, PhD Copyright by Julie Wallace 2013 Abstract Breast cancer is the second most common cancer in American women, and is also the second leading cause of cancer death in women. It is estimated that nearly a quarter of a million new cases of invasive breast cancer will be diagnosed in women in the United States this year, and approximately 40,000 of these women will die from breast cancer. Although death rates have been on the decline for the past decade, there is still much we need to learn about this disease to improve prevention, detection and treatment strategies. The majority of early studies have focused on the malignant tumor cells themselves, and much has been learned concerning mutations, amplifications and other genetic and epigenetic alterations of these cells. However more recent work has acknowledged the strong influence of tumor stroma on the initiation, progression and recurrence of cancer. Under normal conditions this stroma has been shown to have protective effects against tumorigenesis, however the transformation of tumor cells manipulates this surrounding environment to actually promote malignancy. Fibroblasts in particular make up a significant portion of this stroma, and have been shown to impact various aspects of tumor cell biology. Although the contributions of these stromal fibroblasts to tumor progression have been well studied, the role of specific signaling pathways important for these tumor associated functions remain elusive. ii In the current studies, we examine the fibroblast specific roles of the protooncogene Ets2 as well as the tumor suppressor genes Pten and p53 in tumorigenesis using conditional knockout mouse models. Importantly, the functions of these genes are assessed in multiple breast cancer models to account for the heterogeneity observed in breast cancer. Additionally, gene expression analysis is used to decipher potential mechanisms by which these genes function in the stroma. Deletion of Ets2 in fibroblasts in both PyMT and ErbB2 driven tumorigenesis slowed the progression of these tumors without significantly effecting the development of the normal mammary gland. Gene expression profiling revealed Ets2 controls a tumor specific transcription program in fibroblasts that promotes angiogenesis in both breast cancer models. Interestingly, Ets2 seems to target a portion of the same genes in both models, thereby implicating it as a “master regulator” in these tumor associated cells. Additionally, loss of fibroblast Ets2 impacts gene expression in surrounding endothelial cells. Both the PyMT and ErbB2 derived Ets2 dependent signatures were represented in the stroma of human breast cancer tissue, and could also predict patient outcome in independent whole tumor data. Using a similar strategy, we also examined the function of Pten in the stromal fibroblast compartment. Interestingly, loss of this tumor suppressor gene promoted ErbB2 driven tumorigenesis. Gene expression profiling determined that loss of Pten promotes inflammation and ECM remodeling, which is manifested by increased macrophage proliferation and deposition of collagen. The transcription factor Ets2 was shown to be a downstream target of Pten signaling in fibroblasts, which was at least in iii part mediated by regulation of miR-320. To our surprise, Pten loss in stromal fibroblasts did not impact Ras mediated tumorigenesis. To determine if this collaboration was specific to the Pten tumor suppressor, we also conditionally deleted p53 in mammary fibroblasts in the context of ErbB2 and Ras epithelial drivers. Conversely to what we observed upon Pten deletion, loss of p53 did not affect ErbB2 driven tumorigenesis, but significantly impacted Ras mediated transformation. Through gene expression arrays, we determined crosstalk between these epithelial cells and fibroblasts induces changes in a specific collaborative fashion. Loss of Pten or p53 promoted the hyperproliferation of surrounding epithelial cells, however deletion of these genes alone was not sufficient to cause tumor development. In summary, we have shown Ets2 to be an important transcription factor driving angiogenesis from the tumor associated fibroblast compartment in both PyMT and ErbB2 breast cancer models. Alternatively, Pten was shown to have strong tumor suppressor function in the stroma in ErbB2 mediated tumorigenesis, however its function was dispensible during Ras induced tumorigenesis. The opposite was observed in the context of fibroblast p53 function which was important in Ras driven tumorigenesis but had modest effects in the context of ErbB2. Understanding this complex communication between tumor cells and the microenvironment is a critical step as we move forward in the battle against cancer. iv Dedication This dissertation is dedicated to my grandma, a breast cancer survivor and my motivation to make a difference for people with this disease. Also to my parents for believing in me and their unending love and support. And finally to my husband for keeping me going when I wanted to give up. vAcknowledgments I owe a debt of gratitude to my advisor Dr. Michael C. Ostrowski for his support and encouragement thoughout my time here. I would also like to thanks my committee members Dr. Gustavo Lenoe, Dr. Denis Guttridge and Dr. Dawn Chandler for their helpful insights and for giving me an opportunity to prove my research abilities. All other members of my lab, current and past, as well as members of the Leone lab have been invaluable resources for stimulating conversation. You are friends as well as co-workers. I am grateful for the help of our biostatisticians Soledad Fernandez and Lianbo Yu. I am also thankful to the histology core, in particular Lisa Maysoon Rawahneh for timely processing and encouragement. And extra thanks to our bioinformatics expert Thierry Pecot for helping with data analysis and heatmap generation. vi Vita 26 August 1982..............................................Born – Cincinnati, Ohio May 2004 .......................................................B.S. Zoology, Miami University September 2006 – December 2006................Teaching Assistant, The Ohio State University September 2005 – December 2010 ...............Research Assistant, The Ohio State University January 2011 – December 2012.....................Graduate Fellow, The Ohio State University January 2012 – Present ..................................Research Assistant, The Ohio State University Publications 1. Bronisz A, Godlewski J, Wallace JA, Merchant AS, Nowicki MO, Mathsyaraja H, Srinivasan R, Trimboli AJ, Martin CK, Li F, Yu L, Fernandez SA, Cory S, Hallett M, Park M, Piper MG, Marsh CB, Yee LD, Jimenez RE, Nuovo G, Lawler SE, Chiocca EA, Leone G, Ostrowski MC. (2011) Reprogramming of the tumor microenvironment by stromal Pten-regulated miR-320. Nat Cell Biology 14, 159-67. 2. Wallace JA, Li F, Leone G, Ostrowski MC. (2011) Pten in the breast tumor microenvironment: modeling tumor-stroma coevolution. Cancer Res 71, 1203-1207. 3. Li F, Wallace JA, Ostrowski MC. (2010) ETS transcription factors in the tumor microenvironment. The Open Cancer Journal 3, 49-54. 4. Kim Y, Wallace JA, Li F, Ostrowski MC, Friedman A. (2010) Transformed epithelial cells and fibroblasts/myofibroblasts interaction in breast tumor: a mathematical model and experiments. J Math Biol. 61, 401-421. vii 5. Trimboli AJ, Cantemir-Stone CZ, Li F, Wallace JA, Merchant A, Creasap N, Thompson JC, Caserta E, Wang H, Chong JL, Naidu S, Wei G, Sharma SM, Stephens JA, Fernandez SA, Gurcan MN, Weinstein MB, Barsky SH, Yee L, Rosol TJ, Stromberg PC, Robinson ML, Pepin F, Hallett M, Park M, Ostrowski MC, Leone G. (2009). Pten in stromal fibroblasts suppresses mammary epithelial tumors. Nature 461, 1084-1091. 6. Chen CL, Hsieh FC, Lieblein JC, Brown J, Chan C, Wallace JA, Cheng G, Hall BM, Lin J. (2007) Stat3 activation in human endometrial and cervical cancers. Br J Cancer 96(4), 591-599. Fields of Study Major Field: Molecular, Cellular and Developmental Biology viii Table of Contents Abstract............................................................................................................................... ii Dedication........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita.................................................................................................................................... vii Table of Contents............................................................................................................... ix List of Tables ................................................................................................................... xix List of Figures.................................................................................................................. xxi Chapter 1: Introduction....................................................................................................... 1 1.1 Mammary Gland Biology ............................................................................................. 1 1.1.1 Human Breast Development...................................................................................... 2 1.1.2 Mouse Mammary Gland Development...................................................................... 4 1.1.3 Stromal Contribution to Breast Development............................................................ 8 1.2 Malignancies of the Breast............................................................................................ 9 1.2.1 Origin of Breast Cancer Cells.................................................................................... 9 1.2.1.1 Clonal Evolution Theory......................................................................................... 9 1.2.1.2 Cancer Stem Cell Hypothesis ............................................................................... 11 ix 1.2.2 Heterogeneity of Breast Cancer............................................................................... 14 1.2.2.1 Intertumor Heterogeneity...................................................................................... 16 1.2.2.2 Intratumor Heterogeneity...................................................................................... 19 1.2.2.3 Heterogeneity of Tumor Stroma........................................................................... 21 1.2.3 Targeted Therapies for Treatment of Breast Cancer................................................ 23 1.2.4 Modeling Breast Cancer in Mice............................................................................. 26 1.2.4.1 MMTV-PyMT....................................................................................................... 29 1.2.4.2 MMTV-ErbB2 ...................................................................................................... 31 1.2.4.3 Tet-o-KrasG12D and MMTV-rtTA ......................................................................... 32 1.2.4.4 Breast Tumor Cell Lines and Injection Models.................................................... 34 1.3 Breast Tumor Microenvironment ............................................................................... 34 1.3.1 Tumor Associated Fibroblasts ................................................................................. 35 1.3.1.1 Fibroblasts Contribute to Epithelial Tumor Growth............................................. 36 1.3.1.2 Important Signaling Pathways in Fibroblasts ....................................................... 37 1.3.1.3 microRNAs in Tumor Associated Fibroblasts...................................................... 40 1.3.2 Fibroblast Effects on Other Cellular Heterogeneity of the Microenvironment ....... 41 1.3.2.1 Fibroblasts Important in “Angiogenic Switch”..................................................... 41 1.3.2.2 Fibroblasts and Inflammation ............................................................................... 42 1.3.2.3 Fibroblasts and the Extracellular Matrix............................................................... 43 x 1.4 Ets2 as a Member of the Ets Transcription Factor Family ......................................... 44 1.4.1 Misregulation of Ets Factors in Cancer ................................................................... 45 1.4.2 Biological Functions of Ets2.................................................................................... 47 1.4.3 Ets Factors in Tumor Stroma................................................................................... 48 1.5 Pten and p53 as Tumor Suppressors ........................................................................... 49 1.5.1 Pten and Cancer ....................................................................................................... 50 1.5.2 Tumor Suppressor Function of PTEN ..................................................................... 50 1.5.2.1 Molecular Mechanism of Tumor Suppression...................................................... 50 1.5.2.2 In Vivo Analysis of Pten Function ........................................................................ 52 1.5.3 p53 Mutations and Cancer ....................................................................................... 54 1.5.4 Tumor Suppressor Function of p53 ......................................................................... 54 1.5.5 In Vivo Analysis of p53 Functions........................................................................... 56 1.6 Hypothesis................................................................................................................... 58 Chapter 2: Materials and Methods.................................................................................... 59 2.1 Mouse Colony Maintenance ....................................................................................... 59 2.1.1 Animal Care............................................................................................................. 59 2.1.2 Transgenic Mouse Lines.......................................................................................... 59 2.1.3 Mouse Genotyping................................................................................................... 60 2.1.3.1 Tail DNA Preparation........................................................................................... 60 xi 2.1.3.2 Genotyping Primers and PCR Conditions ............................................................ 60 2.1.4 Animal Procedures................................................................................................... 61 2.1.4.1 Mammary Gland Dissection and Tissue Harvesting ............................................ 61 2.1.4.2 Mammary Tissue Transplantation ........................................................................ 62 2.1.4.3 Mammary Fat Pad Injection.................................................................................. 62 2.1.4.4 Matrigel Plug Injections........................................................................................ 63 2.1.4.5 Xenograft Assays.................................................................................................. 63 2.1.4.6 Intraperitoneal Injection........................................................................................ 63 2.1.4.7 Doxycycline Administration in Food.................................................................... 64 2.2 Tissue Culture............................................................................................................. 64 2.2.1 Primary Fibroblast Isolation .................................................................................... 64 2.2.2 Primary Epithelial Cell Isolation ............................................................................. 65 2.2.3 Establishment of Wild Type, Pten Null and Pten/Ets2 Double Knockout (DKO) Mouse Mammary Fibroblast Cell Lines ........................................................................... 66 2.3 Flow Cytometry/FACS............................................................................................... 66 2.3.1 Isolation of YFP+ Cells from Transgenic Mice....................................................... 66 2.3.2 Isolation of Endothelial Cells and Macrophages using CD31 and F4/80 Antibodies ........................................................................................................................................... 67 2.3.3 In vivo BrdU Flow Assay......................................................................................... 68 xii 2.4 RNA Analysis and Microarrays.................................................................................. 69 2.4.1 RNA Extraction ....................................................................................................... 69 2.4.2 cDNA Preparation.................................................................................................... 69 2.4.3 Quantitative Real Time PCR ................................................................................... 70 2.4.4 Microarray Analysis................................................................................................. 71 2.4.5 microRNA Profiling................................................................................................. 71 2.5 Western Blotting......................................................................................................... 71 2.6 Chromatin Immuoprecipitation................................................................................... 72 2.7 Histology/Immunostaining ......................................................................................... 73 2.7.1 Whole Mount Staining of Mammary Gland ............................................................ 73 2.7.2 Immunohistochemistry (IHC) Staining Protocol..................................................... 73 2.7.3 Immunoflourescence (IF) Staining Protocol............................................................ 74 2.8 Bioinformatic Analysis............................................................................................... 75 2.8.1 Gene Set Enrichment Analysis (GSEA) .................................................................. 75 2.8.2 Generating Human Stroma Heatmaps Using Gene Expression Data from Mouse Models............................................................................................................................... 75 2.8.2.1 PyMT Derived Ets2 Fibroblast Gene List............................................................. 75 2.8.2.2 PyMT Derived Ets2 Dependent Endothelial Cell Gene List................................. 76 2.8.2.3 Pten Null Fibroblast Gene List ............................................................................. 76 xiii 2.8.3 Kaplan-Meier Survival Analysis.............................................................................. 76 2.9 Statistical Tests ........................................................................................................... 77 2.9.1 Ets2 Tumor Study and Immunostaining .................................................................. 77 2.9.2 Pten/p53 Tumor Study and Immunostaining ........................................................... 78 Chapter 3: The Role of Ets2 in Fibroblasts During Mammary Tumor Progression......... 79 3.1 Introduction................................................................................................................. 79 3.1.1 Cre Mediated Deletion of Ets2 Using FspCre......................................................... 81 3.2 Results......................................................................................................................... 84 3.2.1 Normal Development of Mammary Glands Lacking Fibroblast Ets 2..................... 84 3.2.2 Ets2 Deletion in Fibroblasts Slows Progression of PyMT Driven Mammary Tumors ........................................................................................................................................... 86 3.2.3 Regulation of PyMT Tumor Specific Gene Expression by Ets2.............................. 90 3.2.4 Ets2 Directly Binds and Induces Expression of MMP9 ........................................ 100 3.2.5 Ets2 in Fibroblasts Promotes Angiogenesis Through VEGFR2 Signaling in Endothelial Cells............................................................................................................. 103 3.2.6 Gene Expression Changes in Endothelial Cells Induced by Ets2 Signaling in Fibroblasts....................................................................................................................... 108 3.2.7 PyMT Derived Ets2 Fibroblast Gene Signature is Represented in Human Breast Cancer Stroma................................................................................................................. 112 xiv 3.2.8 Ets2 Fibroblast and Endothelial Gene Signatures Predict Patient Outcome in Whole Tumor Datasets ............................................................................................................... 115 3.2.9 Fibroblast Ets2 Function Critical in ErbB2 Induced Tumorigenesis..................... 117 3.2.10 ErbB2 Drives Similar Tumor Specific Ets2 Dependent Gene Signature in Fibroblasts....................................................................................................................... 121 3.2.11 Ets2 in ErbB2 Associated Fibroblasts Promotes Tumor Angiogenesis............... 130 3.2.12 ErbB2 Driven Ets2 Dependent Signature Represented in Human Stromal Data and Predicts Patient Outcome................................................................................................ 132 3.3 Discussion................................................................................................................. 135 Chapter 4: The Role of Pten in Fibroblasts During Mammary Tumor Initiation and Progression...................................................................................................................... 137

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