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Stat5a Regulation by Serine Phosphorylation in Breast Cancer

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Stat5a Regulation by Serine Phosphorylation in Breast Cancer Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2021 STAT5A REGULATION BY SERINE PHOSPHORYLATION IN BREAST CANCER Alicia E. Woock Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Disease Modeling Commons, and the Neoplasms Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/6537 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. ©Alicia Woock, March 2021 All Rights Reserved. i STAT5A REGULATION BY SERINE PHOSPHORYLATION IN BREAST CANCER A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University by ALICIA E. WOOCK B.S., Chemistry Appalachian State University, 2012 Director: Charles V. Clevenger, M.D., Ph.D., Chair, Department of Pathology Virginia Commonwealth University Richmond, Virginia March, 2021 ii Abstract The neuroendocrine hormone prolactin (PRL) and its cognate receptor (PRLr) have been implicated in the pathogenesis of breast cancer. PRL signaling relies on activating kinases such as the tyrosine kinase Jak2 and serine/threonine kinases ERK1/2, NEK3, PI3K, and AKT. In the canonical pathway of PRL signaling, JAK2 phosphorylates the transcription factor STAT5a at tyrosine residue 694 (pY694-STAT5a), preceding STAT5a nuclear translocation and transcriptional activity. However, STAT5a exists with functional duality as a transcription factor, having both pro-differentiative and pro-proliferative target genes. Other STAT family members (STATs 1, 3, and 6) have been shown to have transcriptional activity in the un-tyrosine- phosphorylated (upY) state, distinct from that of pY-STAT activity. This distinction (upY vs. pY) may underlie the duality of STAT5a, coupled with additional regulatory non-canonical post- translational modifications. Within this notion, STAT5a contains two serine residues, S726 and S780, whose phosphorylation are necessary for hematopoietic transformation. However, their functions in PRL-mediated breast cancer pathogenesis have not been examined. We hypothesize that STAT5a serine phosphorylation regulates STAT5a nuclear activity in a non-canonical fashion, contributing to its role in mammary oncogenesis, specifically in luminal breast cancer. As shown in a tissue microarray (TMA), human breast cancer tissues express both pS726- and pS780- STAT5a. Nuclear Allred score for pS726-STAT5a increases two-fold with increasing tumor grade, with no difference in staining associated with estrogen or progesterone receptor (ER, PR) status, nor other clinical characteristics. Likewise, patient derived xenograft (PDX) tumors of various molecular subtypes express pS726- and pS780-STAT5a. Phosphorylation of S726-STAT5a is PRL-responsive in vitro. To examine the functional significance of STAT5a serine phosphorylation in vitro, we have performed STAT5a knockdown (KD) in the breast cancer cell iii line MCF7. Following STAT5a KD, cells were rescued with phospho-site specific STAT5a mutant constructs targeting Y694, S726 and S780. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. Further, PRL-inducible gene expression changes on the global transcriptomic level showed differential effects of the STAT5a mutations compared to wild type STAT5a, specifically enrichment of different oncogenic signatures and transcription factor programs. Mechanistic studies examine the ability of these STAT5a mutant proteins to undergo nuclear translocation and their regulatory role on phosphorylation of STAT5a tyrosine residue 694. Collectively, these studies provide novel insights into the role of the non-canonical pathway of STAT5a activation in breast cancer pathogenesis. iv Acknowledgments “Great things are not done by impulse, but by a series of small things brought together.”-Vincent van Gogh Completion of this thesis would not have been possible without the help of others. First, I’d like to acknowledge my committee members, Dr. Larisa Litovchick, Dr. Chuck Harrell, Dr. Joyce Lloyd, and Dr. Devanand Sarkar, for their support of my project and for pushing my science and understanding further. I’d also like to acknowledge my co-authors on my manuscript submitted February 5, 2021, who provided tissue samples, performed analyses, and gave constructive feedback on the manuscript. Drs. Michael Idowu and Patricija Zot provided tissues for the tissue microarray and analysis. Dr. Chuck Harrell provided PDX samples, which were prepared by either Tia H. Turner or Mohammad A. Alzubi. Amy L. Olex performed the RNA-seq quality control and alignment and performed/provided guidance on RNA-seq analysis. She was extremely helpful throughout the process of analyzing the RNA-seq. Finally, Jackie M. Grible: not only a co-author and former lab member, but a friend. Jackie helped with technical aspects of my research, performing and analyzing the soft agar assay. But she was also a sounding board for ideas, and I appreciate her letting me hang out with her dogs, Tommy and Sammy, when I needed dog cuddles. I’d also like to acknowledge our laboratory technicians, Shannon E. Hedrick and Madhavi Puchalapalli for their technical support. Shannon’s knowledge of molecular biology, and her time, were so appreciated. Her technical support on my project ranged from performing the IHC for the TMA, to teaching me how to clone, run PCR, and analyze DNA gels. I would not be the scientist I am today without her. v I’d like to acknowledge my mentor and Principal Investigator, Dr. Charles Clevenger. His guidance and support of my project got it to where it is today. In my time in his lab I have learned so much about science and the process of research. His mentoring allowed me to become an independent researcher, and for that I am thankful. Lastly, I’d also like to acknowledge my family and friends, who supported me, kept encouraging me, and helped balance me. Our use of FaceTime has been impressive. vi Abbreviations (v/v) Volume per volume ratio (w/v) Weight per volume ratio A Alanine AA Amino acid ABL Abelson AKT Protein kinase B AML Acute myeloid leukemia ANOVA Analysis of variance APC Adenomatous polyposis coli APS Ammonium persulfate AR Androgen receptor ATCC American Type Culture Collection bcl6 B-cell lymphoma 6 BLAST Basic local alignment search tool BLG β-lactoglobulin gene BME β-mercaptoethanol bp Base pairs BRCA Breast cancer BSA Bovine serum albumin C Celsius C- Carboxy c-Src Cellular-Src CBP CREB-binding protein CC3 Cleaved caspase 3 CC7 Cleaved caspase 7 CCND1 Cyclin D1 vii cDNA Complementary deoxyribonucleic acid ChIP Chromatin immunoprecipitation CISH Cytokine-inducible SH2-containing protein CML Chronic myeloid leukemia CMV Cytomegalovirus Co-IP Co-immunoprecipitation CO2 Carbon dioxide CTCF CCCTC-binding factor CypA Cyclophilin A d Day DAPI 4',6-Diamidino-2-Phenylindole, Dihydrochloride DCIS Ductal carcinoma in situ DEG Differential gene expression Dex Dexamethasone dH2O Distilled water DMEM Dulbecco’s modified eagle’s medium DMSO Dimethylsulfoxide DN Dominant-negative DNA Deoxyribonucleic acid DNase Deoxyribonuclease DPBS Dulbecco’s phosphate-buffered saline E2 Estrogen ECL Enhanced chemiluminescence ECM Extracellular matrix EDTA Ethylene diamine tetra acetic acid EGFR Epidermal growth factor receptor EGTA Ethylene glycol-bis (aminoethylether) N,N,N',N'-tetraacetic acid viii EMSA Electrophoretic mobility shift assay EMT Epithelial-to-mesenchymal transition EPO Erythropoietin ER Estrogen receptor ERE Estrogen response element ERK Extracellular signal-related kinase ES Enrichment score EV Empty vector EZH2 Enhancer of zeste homolog 2 F Phenylalanine FAK Focal adhesion kinase FBS Fetal bovine serum FDR False discovery rate FL Full length For Forward g Gram GAS Gamma-activated sequence GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GH Growth hormone GHR Growth hormone receptor Gly Glycine GM-CSF Granulocyte-macrophage colony-stimulating factor GR Glucocorticoid receptor GSEA Gene set enrichment analysis h Hour H3K27me3 Histone H3 tri-methylation of lysine 27 ix HCl Hydrochloric acid HDAC6 Histone deacetylase 6 HEK293T Human embryonic kidney HMGN2 High-mobility group N2 HP1 Heterochromatin protein 1 HRP Horseradish peroxidase HUVEC Human umbilical vein endothelial cell IB Immunoblotting ICS-2 Interferon consensus sequence 2 IF Immunofluorescence IFN Interferon IGF Insulin-like growth factor IgG Immunoglobulin G IHC Immunohistochemistry IL Interleukin IP Immunoprecipitation IPA Ingenuity pathway analysis IRF Interferon regulatory factor Jak Janus kinase kb Kilobases KD Knockdown KDa Kilo Dalton KiPIK Kinase inhibitor profiling to identify kinases KO Knockout LB Luria-Bertani broth LSP Leu-Ser-Pro motif LTE2 Long-term estrogen independence x M Molar mAb Monoclonal antibody MAPK Mitogen-activated protein kinase MEF Mouse embryonic fibroblast MEK Mitogen-activated protein kinase kinase mg Milligram MGT Mammary gland tumor min Minutes ml Milliliter mM Millimolar MMTV-PyMT Mammary tumor virus-polyoma middle tumor-antigen mRNA Messenger ribonucleic acid MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N- amino NaCl Sodium chloride NEB New England Biolabs
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