University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2012 Matrix Metalloproteinase genes are transcriptionally regulated by E2F transcription factors: a link between cell cycle control and metastatic progression Jacqueline Lea Johnson University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons, Biology Commons, Molecular Biology Commons, and the Oncology Commons Scholar Commons Citation Johnson, Jacqueline Lea, "Matrix Metalloproteinase genes are transcriptionally regulated by E2F transcription factors: a link between cell cycle control and metastatic progression" (2012). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/4092 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Matrix Metalloproteinase genes are transcriptionally regulated by E2F transcription factors: a link between cell cycle control and metastatic progression By Jacqueline L. Johnson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Program in Cancer Biology Department of Cell Biology, Microbiology, and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Srikumar Chellappan, Ph.D. Said Sebti, Ph.D. W. Douglas Cress, Ph.D. Eric Haura, M.D. Philip Hinds, Ph.D. Date of Approval: February, 20 th , 2012 Keywords: cancer, retinoblastoma, Raf-1, colonization, lung Copyright © 2012, Jacqueline L. Johnson Dedication To my caring husband Jan, who patiently listens to my frustrations, and enthusiastically celebrates my successes. Thank you again, and I promise to only get one Ph.D. .for your sake. To my supportive family, who taught me what it meant to work hard and never give up—because without hard work, there can only be limited play. To my incredible “friend-family”, I am so lucky to have found you all: my soul mates. I must have had some great karma stored up from something. To all—this is truly a product of your unconditional love and encouragement. Acknowledgements Firstly, I would like to thank my mentor, Dr. Srikumar Chellappan, who remained supportive and invested in my success as a graduate student from my first day in the lab. I would also like to thank my committee members Drs. Said Sebti, Douglas Cress, and Eric Haura for always asking important questions and offering guidance over the years. Thanks also go to Dr. Pedro Santiago at the Ponce School of Medicine for his support during my summer research rotation. I would also like to thank Philip Hinds, Ph.D., Tufts Medical Center, for serving as my external chairperson for my dissertation defense. I should not forget to mention the people who were crucial to making my experience in the Cancer Biology program a success; Cathy Gaffney and Dr. Ken Wright have been instrumental. I am deeply indebted to Dr. Smitha Pillai for spending countless hours training me at the bench, in addition to always being available for scientific discussion. I would also like to thank my friends in the Cancer Biology Ph.D. program, lab mates, and colleagues that have aided, inspired, and influenced me on this journey. It has been quite an experience. Table of Contents List of Tables .......................................................................................................iv List of Figures....................................................................................................... v Abstract ...............................................................................................................vii Chapter 1: Introduction ......................................................................................... 1 1. The Retinoblastoma gene...................................................................... 1 1.1 Rb pocket proteins family members.......................................... 2 1.2 Rb interacting proteins.............................................................. 5 1.2.1 Rb inactivation upon mitogenic and apoptotic stimulation....................................................................... 5 1.2.2 Rb inactivation by Raf-1..............................................8 1.2.3 Rb inactivation upon nicotine stimulation................... 10 1.3 Downstream mediators of Rb function: the E2F family ....................................................................................... 13 1.4 Rb and E2Fs regulate the cell cycle........................................ 16 1.5 Additional E2F target genes.................................................... 21 1.5.1 E2Fs regulate apoptosis............................................ 21 1.5.2 E2Fs regulate autophagy.......................................... 23 1.6 Rb and E2F knockout mouse models ..................................... 24 1.7 Alterations of the Rb-E2F pathway in cancer.......................... 26 1.8 Rb and E2Fs in angiogenesis and tumor progression.............................................................................. 28 2. Regulation of tumor progression.......................................................... 30 2.1 Metastasis as a multistage process ........................................ 32 2.2 Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) ............................. 33 2.3 Mechanisms of invasion.......................................................... 34 2.4 Circulating tumor cells............................................................. 37 2.5 Metastatic colonization............................................................ 38 2.5.1 Metastasis to bone.................................................... 39 2.5.2 Metastasis to lung ..................................................... 42 2.5.3 Metastasis to brain.................................................... 44 3. Matrix Metalloproteinases in cancer and tissue remodeling................. 45 3.1 Matrix Metalloproteinase family members............................... 46 3.2 MMP substrates and functions................................................ 49 3.2.1 MMPs regulate signaling molecules.......................... 49 3.2.2 MMPs regulate apoptosis.......................................... 50 i 3.2.3 MMPs regulate angiogenesis....................................51 3.2.4 MMPs regulate invasion and metastasis................... 51 3.3 Regulation of MMP activity...................................................... 53 4. Summary ............................................................................................. 55 Chapter 2. Materials and Methods...................................................................... 58 Chapter 3. Regulation of matrix metalloproteinase genes by E2F transcription factors: Rb-Raf-1 interaction as a novel target for metastatic disease Abstract....................................................................................................74 Introduction.............................................................................................. 75 Results..................................................................................................... 77 MMP9, MMP14, and MMP15 promoters recruit E2F1 and Rb...... 77 MMP9, MMP14, and MMP15 promoters are responsive to E2F1, Rb, and Raf-1................................................................ 83 MMP9, MMP14, and MMP15 are induced by E2F1-5................... 85 E2F1, E2F3, and E2F5 are required for MMP gene expression ............................................................................... 87 Depletion of E2Fs inhibits collagen degradation ........................... 89 E2F1 overexpression can rescue MMP-luciferase activity when E2F3 is depleted ............................................................ 91 Depletion of E2F1 or E2F3 reduces cell migration independent of proliferation ..................................................... 93 Depletion of E2F1 or E2F3 reduces cell invasion.......................... 95 Rb-Raf-1 disruptor, RRD-251, inhibits invasion and migration in vitro ...................................................................................... 97 Rb-Raf-1 disruptor, RRD-251 inhibits MMP transcription and collagen degradation ............................................................... 99 RRD-251 inhibits metastatic lung colonization in vivo................. 101 Discussion ............................................................................................. 103 Chapter 4: Differential regulation of MMP2 by E2F1 involves a p53-KAP1- HDAC1-dependent mechanism Abstract.................................................................................................. 106 Introduction............................................................................................ 107 Results................................................................................................... 111 MMP2 promoter is transcriptionally regulated by E2F transcription factors ............................................................... 111 E2F represses MMP2 independent of Rb, BRG1, or other known corepressors............................................................... 114 c-MYC and ID1 depletion enhances MMP2-luciferase activity.... 117 c-MYC directly binds and colocalizes with ID1...........................