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The 8P11-P12 Amplicon Oncogenes ASH2L and NSD3 Regulate Cell

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The 8P11-P12 Amplicon Oncogenes ASH2L and NSD3 Regulate Cell Medical University of South Carolina MEDICA MUSC Theses and Dissertations 2017 The 8p11-P12 Amplicon Oncogenes ASH2L and NSD3 Regulate Cell Cycle Progression via Epigenetic Alterations and Result in Overexpression and Estrogen-Independent Activation of ERα in Breast Cancer Jamie Nicole Mills Medical University of South Carolina Follow this and additional works at: https://medica-musc.researchcommons.org/theses Recommended Citation Mills, Jamie Nicole, "The 8p11-P12 Amplicon Oncogenes ASH2L and NSD3 Regulate Cell Cycle Progression via Epigenetic Alterations and Result in Overexpression and Estrogen-Independent Activation of ERα in Breast Cancer" (2017). MUSC Theses and Dissertations. 335. https://medica-musc.researchcommons.org/theses/335 This Dissertation is brought to you for free and open access by MEDICA. It has been accepted for inclusion in MUSC Theses and Dissertations by an authorized administrator of MEDICA. For more information, please contact [email protected]. TABLE OF CONTENTS Acknowledgements…………………………………………………………….…...... ii List of Figures…………………………………………………….………………....... iii List of Tables.…………………………………………………………………….…… iv List of Appendices……………………………………………………………...…….. v List of Abbreviations………………………………………………………………….. vi ABSTRACT.…………………………………………………………….….…………. viii CHAPTER 1: Introduction and Review of Literature…………….…………..……. 2 CHAPTER 2: NSD3 amplification and overexpression results in overexpression and estrogen-independent activation of the estrogen receptor in human breast cancer……………………………………………………………… 55 CHAPTER 3: ASH2L regulates gene expression via H3K4me3 in promoters and knockdown of ASH2L reduces expression of NSD3, ERα, and other genes important in cell proliferation processes………………….......................... 70 CHAPTER 4: Discussion and Future Directions….………….…….…………..…. 94 CHAPTER 5: Materials and Methods…………………………………………….... 124 REFERENCES CITED……………………………………………………………….. 136 Appendix Materials………………………………………………………………….... 162 Page | i ACKNOWLEDGEMENTS First and foremost, I would like to thank Steve Ethier for his mentorship. I appreciate all he taught me, the time and energy he invested in me, and the guidance he gave while also allowing me the freedom to explore scientific thought. Steve always challenged me and I will always be grateful to have had him as my mentor. I would also like to thank the members of the Ethier lab for support and advice through this process. It has been a pleasure working with Brittany Turner-Ivey, Christy Kappler, and Ericka Smith. Brittany has been a wonderful mentor and friend and I am thankful for all she taught me. Thank you to Jon Irish, Steve Guest, Ginny Davis, members of adjacent labs, and the Department of Pathology and Laboratory Medicine as a whole for your friendship and for acting as invaluable resources in my training process. I would like to thank my committee members, Melissa Cunningham, John Wrangle, Victoria Findlay, and Amanda LaRue for taking time from their busy schedules to invest in my scientific development. Perry Halushka, Amy Connolly, and my colleagues from the MSTP at MUSC have been an amazing source of support, as have the MUSC core facilities and personnel. Thank you to my clinical mentors and the faculty at MUSC as a whole for participating in my training. Finally, I would like to thank my friends and family. My husband, Ian Mills, always supported me and I could not have accomplished this without him. Thank you to my extended family, especially my mom, Diane Lingenfelter, and grandma, Vel Helton. I am grateful for support from my friends that have become family, especially Gerry Barnard, Renee Vick, Dawn Simmons, and the members of Redeemer Presbyterian Church. Last but not least, I have to thank my daughters, Gwendolyn and Eleanor. More than anyone, they have challenged me and shaped me in ways I never thought possible and I hope they continue to do so at every stage of life and career. Page | ii LIST OF FIGURES Figure 1.1. Schematic representation of ERα structure. Figure 1.2. Breast cancer intrinsic subtype proportions and characteristics of luminal tumors. Figure 1.3. Schematic representation of the 8p11-p12 amplicon. Figure 1.4. Frequencies of ASH2L and NSD3 alterations in primary tumors (TCGA). Figure 1.5. Illustration of major histone modifications and effects on chromatin. Figure 1.6. Hierarchical representation of histone lysine methyltransferase enzyme relationships. Figure 1.7. Representation of the three isoforms of NSD3. Figure 2.1. NSD3 is overexpressed in 8p11-p12 amplicon-bearing cell lines and is a verified oncogene. Figure 2.2. NSD3 knockdown results in knockdown of estrogen receptor alpha (ERα). Figure 2.3. NSD3 overexpression results in overexpression of estrogen receptor alpha (ERα). Figure 2.4. SUM-44 cells are estrogen-independent despite high-level ERα expression. Figure 2.5. SUM-44 cells are ERα-dependent. Figure 3.1. ASH2L is overexpressed in ER+, 8p11-p12 amplicon-bearing breast cancer. Figure 3.2. Overall H3K4 tri-methylation patterns in SUM-44 cells are similar between control and ASH2L knockdown on a global scale. Figure 3.3. ASH2L is responsible for H3K4me3 in the promoters of genes involved in breast cancer tumorigenesis. Figure 3.4. ASH2L knockdown reduces expression of genes with decreased promoter H3K4me3 levels. Figure 3.5. ASH2L, NSD3, and ERα influence the expression of similar suites of genes related to cell cycle progression. Figure 3.6. Knockdown of ASH2L results in decreased proliferation and clonogenicity in luminal B breast cancer. Figure 3.7. Knockdown of ASH2L alters response to palbociclib. Figure 4.1. Summary model of NSD3 functions. Figure 4.2. Summary model of ASH2L functions. Figure 4.3. Summary model. Page | iii LIST OF TABLES Table 2.1. Genes with reduced transcript expression following knockdown of NSD3-S in SUM-44 cells. Table 2.2. Biological processes to which genes with altered expression following knockdown of NSD3-S and ESR1 were annotated. Table 3.1. Genes with promoter H3K4me3 ChIP-seq peaks called in control but not ASH2L knockdown datasets. Table 3.2. Genes with reduced transcript expression following knockdown of ASH2L in SUM-44 cells. Table 3.3. Summary of genes identified by ASH2L knockdown H3K4me3 ChIP-seq, ASH2L knockdown RNA-seq, NSD3-S knockdown array, or ESR1 knockdown array. Table 3.4. Genes annotated to palbociclib response in ToppFun. Page | iv LIST OF APPENDICES Appendix A. NSD3-S and ESR1 knockdown microarray results. Appendix B. H3K4me3 ChIP-seq quality control and enrichment reports. Appendix C. H3K4me3 ChIP-seq peak datasets: LacZ control and ASH2L knockdown. Appendix D. Gene lists: overall H3K4me3 ChIP-seq peaks. Appendix E. Gene lists and ToppFun analysis: promoter only H3K4me3 ChIP-seq peaks. Appendix F. ASH2L knockdown RNA-seq results. Appendix G. Gene lists and ToppFun analysis: ASH2L knockdown RNA-seq downregulated genes. Appendix H. Gene lists and ToppFun analysis: ASH2L knockdown H3K4me3 ChIP-seq and RNA-seq. Appendix I. Gene lists and ToppFun analysis: ASH2L, NSD3-S, and ESR1 knockdown expression analysis and ASH2L knockdown H3K4me3 ChIP-seq overlap. Page | v LIST OF ABBREVIATIONS 4OH-tamoxifen – hydroxytamoxifen HDAC(i) – histone deacetylase (inhibitor) AF – activating function (ERα domain) HER2 – human epidermal growth factor receptor 2 AI – aromatase inhibitor HOX – homeobox gene family AML – acute myeloid leukemia HMT – histone methyltransferase ASH2L – Absent, Small, or Homeotic disc 2-Like HMTi – histone methyltransferase inhibitors/inhibition BET – bromodomain and extraterminal domain hPTM – histone post-translational modification CCND1 – cyclin D1 gene IGV – integrated genome viewer CDK – cyclin-dependent kinase IHC - immunohistochemistry CHD – chromodomain IP - immunoprecipitation ChIP – chromatin immunoprecipitation IPTG – isopropyl β-D-1- – copy number alteration CNA thiogalactopytanoside CTD – comparative toxicogenomics K – lysine database LBD – ligand binding domain DBD – DNA binding domain LSCC – lung squamous cell carcinoma DNMT – DNA methyltransferase MLL – mixed lineage leukemia EGFR – epidermal growth factor receptor NLS – nuclear localization signal ER/ERα – estrogen receptor (alpha) NS – non-significant ERE – estrogen response element NSD – Nuclear-receptor binding SET ESR1 – estrogen receptor alpha gene Domain-containing FDR – false discovery rate NSD3-L – long isoform of NSD3 GISTIC – genomic identification of NSD3-S – short isoform of NSD3 significant targets in cancer NSD3-T – total NSD3 (both isoforms) GO – gene ontology PBS – phosphate buffered saline GRH – gonadotrophin releasing hormone PCA – principal component analysis H – histone PcG – polycomb group HAT – histone acetyltransferase PHD – plant homeodomain HBSS – Hank’s buffered saline solution Page | vi PR – progesterone receptor PRC – polycomb repressive complex PWWP – proline-tryptophan-tryptophan- proline R – arginine RT – room temperature RT-PCR – reverse transcription polymerase chain reaction SAC – SET-associated cysteine-rich SERD – selective estrogen receptor degrader SERM – selective estrogen receptor modulator SET – Suppressor of variegation 3-9, Enhancer of zeste and Trithorax -seq – high throughput sequencing shRNA – short hairpin RNA TAD – transcription activating domain TBST – tris-buffered saline + Tween 20 TCGA – the cancer genome atlas TNBC – triple negative breast cancer trxG – trithorax group WAR – WDR5-ASH2L-RbBP5 sub- complex WRAD – WDR5-RbBP5-ASH2L-DPY-30 sub-complex WHSC1L1 – Wolf-Hirschhorn Syndrome Candidate 1-Like 1 Page | vii ABSTRACT
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