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Tgfβ/SMAD4 Signaling and Altered Epigenetics Contribute to Increased Ovarian

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Tgfβ/SMAD4 Signaling and Altered Epigenetics Contribute to Increased Ovarian TGFβ/SMAD4 Signaling and Altered Epigenetics Contribute to Increased Ovarian Cancer Severity Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniel Edward Deatherage, B.A. Molecular Cellular Developmental Biology The Ohio State University 2011 Dissertation Committee: Tim Huang, Ph.D., Advisor Amanda Toland, Ph.D. Victor Jin, Ph.D. Huey-Jen Lin, Ph.D. Copyright by Daniel Edward Deatherage 2011 Abstract Ovarian cancer is the eighth most common cancer and is the fifth most common cause of cancer related death among women. Early stage ovarian cancer is very responsive to treatments and more than 93% of patients diagnosed with early stage disease achieve a five year survival rate. By contrast less than 30% of patients who are diagnosed with late stage disease achieve a five year survival rate, yet more than 60% of all cases present as late stage. Treatment options are typically surgery followed by a combination chemotherapy regiment of a platinum-based chemotherapeutic and a taxane derivative. While this treatment plan works well for early stage disease, recurrent and late stage disease are less responsive and secondary treatment options are not nearly as beneficial. Here we present work investigating the altered epigenetics and TGFβ/SMAD4 signaling pathway in ovarian cancer in an effort to better understand the difference in disease severity at a molecular level. We have identified a microRNA hsa-mir-9-3 which is epigenetically repressed by DNA methylation in a panel of primary ovarian cancer patients. Quantitative analysis of DNA methylation in the CpG island which contains the hsa-mir-9-3 microRNA revealed significant hypermethylation in both patient samples and cell lines as compared to normal tissue samples. Although no ii significant correlation with a clinical feature could be identified, functional studies reveal that the repression of this microRNA leads to increased proliferation rates as well as a decrease in apoptosis. Despite being unable to identify a specific target gene, we believe that the hypermethylation of the hsa-mir-9-3 locus serves as a novel biomarker for ovarian cancer. TGFβ/SMAD4 signaling is commonly dysregulated in ovarian cancers while being a key growth inhibition signal for the ovarian surface epithelium during menstruation. Here we present a genome-wide profile of SMAD4 binding by ChIP-sequencing following TGFβ stimulation in the ovarian cancer cell line A2780. While A2780 displays some altered signaling by having constitutive nuclear presence of SMAD4, TGFβ stimulation has previously been shown capable of inducing additional nuclear translocation and synthesis of SMAD4. We believe this to be the first truly genome-wide profiling of SMAD4 binding using next generation sequencing approaches in an ovarian cancer model. Comparison of ChIP-seq results with previously reported ChIP-chip studies show dramatic biological and technical differences including more than 70% of all SMAD4 binding loci being more than 10kb away from the nearest transcription start site. Gene expression analysis following TGFβ stimulation revealed a group of 318 genes whose expression changed following SMAD4 binding to the distal promoter of the gene. Of those 318 genes, a subset of them was used to predict patient survival in two independent previously published patient cohorts. Together these results suggest that the loss of long distance SMAD4 gene iii regulation following TGFβ stimulation may play a key role in ovarian carcinogenesis. Additionally, we identified a novel biomarker, CLDN11, whose epigenetic repression is associated increased cisplatin resistance in a tissue culture model system. Examination of CLDN11 expression levels in a previously reported patient cohort revealed lower expression levels associated with increased tumor grade. Finally, loss of CLDN11 expression is associated with increased cellular motility. In conclusion we have investigated and correlated several different epigenetic and signaling abnormalities associated with an increase in the severity of ovarian cancer while demonstrating the importance of recent technological advances in genome-wide methodologies. Together these results are likely to aid in both future discovery methods as well as patient prognosis and treatment. iv Dedication To my wife whose love and reminders to eat and live while writing this showed me writing, living, eating, and loving can be multitasked. v Acknowledgements My thanks go out to my advisor Dr. Tim Huang for his guidance support and motivation throughout my graduate studies. I will also be eternally appreciative of the time spent by my committee members, Dr. Huey-Jen Lin, Dr. Amanda Toland, and Dr. Victor Jin. Support, insight, discussion, and challenges provided by them have made me a much better scientist. It is important to acknowledge the contributions others have made to this work beyond recommendations and advice. Specifically Brian A. Kennedy was responsible for performing the computational work presented in Chapter 3. I can only hope that my interactions with him helped improve his own computational research as much as they helped improve my own bench-work. Additionally the cisplatin resistant A2780 Round 5 cells provided by Dr. Ken Nephew‘s group (Dr. Dave Miller and Dr. Meng Li in particular) were greatly appreciated as they are central to the studies presented in Chapter 4. Finally, Pei-Yin Hsu for her assistance with performing the FACS analysis presented in Chapter 2 while I was preparing for my candidacy examination. I also wish to extend my thanks for the numerous members, including those who have moved onto other positions, of the Huang lab, and the affiliated vi labs of Dr. Huey-Jen Lin, Dr. Victor Jin, and Dr. Qianben Weng. Their daily conversations and assistance has been crucial to all the work presented herein. Special thanks are given to Dr. Pearly Yan, Dr. Michael Chan, Dr. Greg Singer, Dr. Dustin Potter, Dr. Alfred Cheng, Dr. Benjamin Rodriguez, Joseph Liu, Judy Kuo, Dr. Yi-Wen Huang, Dr. Zhengang Peng, Dr. Tao Zuo, Dr. Yu-I Weng, Ta- Ming Liu, Dr. Cenny Taslim, Dr. Shuying Sun, Sandya Liyanarachchi, Dr. Ya-Ting Hsu, Michael Trimarchi, Huang-Kai Hsu, and Pei-Yin Hsu. vii Vita 2005-2011 Graduate Research Associate The Ohio State University 2005 B.S. Biochemistry University of Evansville 2008 Graduate Travel Award OSUMC Research Day 2005 Biology award for Outstanding Senior Thesis University of Evansville Publications Yeh, Kun-Tu; Che, Tze-Ho; Yang, Hui-Wen; Chou, Jian-Liang; Chen, Lin-Yu; Yeh, Chia-Ming; Chen, Yu-Hsin; Lin, Ru-Inn; Su, Her-Young; Chen, Gary C- W.; Deatherage, Daniel E.; Huang, Yi-Wen; Yan, Pearlly S.; Lin, Huey-Jen; Nephew, Kenneth P.; Huang, Tim H-M.; Lai, Hung-Cheng; Chan, Michael. Aberrant TGFβ/SMAD4 Signaling Contributes to Epigenetic Silencing of a Putative Tumor Suppressor, RunX1T1 in Ovarian Cancer. Epigenetics 6 2011. Zuo, Tao; Liu, Ta-Ming; Lan, Xun; Weng, Yu-I; Shen, Rulong; Gu, Fei; Huang, Yi- Wen; Liyanarachchi, Sandya; Deatherage, Daniel E.; Hsu, Pei-Yin; Taslim, Cenny; Ramaswamy, Bhuvaneswari; Shapiro, Charles L.; Lin, Huey-Jen L.; viii Cheng, Alfred SL.; Jin, Victor; Huang, Tim H-M. Epigenetic Silencing Mediated through Activated PI3K/AKT Signaling in Breast Cancer. Cancer Research 71 1752-762 2011 Chou, Jian-Liang; Su, Her-Young; Chen, Lin-Yu; Liao, Yu-Ping; Hartman-Frey, Corinna; Lai, Yi-Hui; Yang, Hui-Wen; Deatherage, Daniel E., Kuo, Chieh-Ti; Huang, Yi-Wen; Yan, Pearlly S.; Hsiao, Shu-Huei; Tai, Chien-Kuo; Lin, Huey- Jen L.; Davuluri, Ramana V.; Chao, Tai-Kuang; Nephew, Kenneth P.; Huang, Tim H-M.; Lai, Hung-Cheng; Chang, Michael W-Y.. Promoter Hypermethylation of FBXO32, a novel TGF-β/SMAD4 Target Gene and Tumor Suppressor, is Associated with Poor Prognosis in Human Ovarian Cancer. Laboratory Investigation 90: 414-425, 2010. Hsu, Pei-Yin; Hsu, Hang-Kai; Singer, Gregory A.C.; Yan, Pearlly S.; Rodriguez, Benjamin A.T.; Liu, Joseph C.; Weng, Yu-I; Deatherage, Daniel E.; Chen, Zhong, Pereira, Julia S.; Lopez, Ricardo; Russo, Jose; Wang, Qianben; Lamartiniere, Coral A.; Nephew, Kenneth P.; Huang, Tim H-M.. Estrogen- Mediated Epigenetic Repression of Large Chromosomal Regions through DNA Looping. Genome Research 20: 733-744, 2010. Weng, Yu-I; Hsu, Pei-Yin; Liyanarachchi, Sandya; Liu, Joseph; Deatherage, Daniel E.; Huang, Yi-Wen; Zuo, Tao; Rodriguez, Benjamin; Lin, Ching-Hung; Cheng, Ann-Lii; Huang, Tim H-M.. Epigenetic Influences of Low-Dose Bisphenol A in Primary Human Breast Epithelial Cells. Toxicology and Applied Pharmacology 248: 111-121, 2010. Deatherage, Daniel E.; Potter, Dustin; Yan, Pearlly S.; Huang, Tim-H.-M; Lin, Shili. Methylation Analysis by Microarray. Methods in Molecular Biology 556: 117-139, 2009 Yan, Pearlly S.; Potter, Dustin; Deatherage, Daniel E.; Huang, Tim H.-M; Lin, Shili. Differential Methylation Hybridization: Profiling DNA Methylation with a ix High-Density CpG Island Microarray. Methods in Molecular Biology 507 (Part III): 89-106, 2009. Huang, Yi-Wen; Liu, Joseph C.; Deatherage, Daniel E.; Luro, Jingqin; Mutch, David G.; Goodfellow, Paul J.; Miller, David S.; Huang, Tim-H.M. Epigenetic Repression of microRNA-129-2 Leads to Overexpression of SOX4 Oncogene in Endometrial Cancer. Cancer Research 69: 9038-9046, 2009. Hsu, Pei-Yin; Deatherage, Daniel E.; Rodriguez, Benjamin, A.T.; Liyanarachchi, Sandya; Weng, Yu-I; Zuo, Tao; Liu, Joseph; Cheng, Alfred S.L.; Huang, Tim H-M.. Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells. Cancer Research 69: 5936-5945, 2009 Lin, Huey-Jen L.; Zuo, Tao; Lin, Ching-Hung; Kuo, Chieh Ti; Liyanarachchi, Sandya; Sun, Shuying; Shen, Rulong; Deatherage, Daniel E.; Potter, Dustin; Asamoto, Lisa; Lin, Shili; Yan, Pearlly S.; Chen, Ann-Lii; Ostrowski, Michael C.; Huang, Tim H-M.. Breast Cancer-Associated Fibroblasts Confer AKT1- Mediated Epigenetic Silencing of Cystatin M in Epithelial Cells. Cancer Research 68: 10257-10266, 2008 Chan, Michael WY.; Huang, Yi-Wen; Hartman-Frey, Corinna; Kuo, Chieh-Ti; Deatherage, Daniel E.; Qin, Huaxia; Cheng, Alfred SL.; Yan, Pearlly S.; Davuluri, Ramana V.; Huang, Tim H-M.; Nephew, Kenneth P.; Lin, Huey-Jen L.
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