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1 Characterizing the Role of GREB1 in Regulation of Breast Cancer Characterizing the role of GREB1 in regulation of breast cancer proliferation Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Corinne Nicole Haines Graduate Program in Molecular, Cellular, and Developmental Biology The Ohio State University 2019 Thesis Committee Craig J. Burd PhD., Advisor Sharon L. Amacher, PhD. Anne M. Strohecker, PhD. Philip N. Tsichlis, PhD. 1 Copyrighted by Corinne Nicole Haines 2019 2 Abstract Breast cancer is the most frequently diagnosed malignancy in women. The vast majority (>70%) of breast cancer patients are diagnosed with the estrogen receptor-positive subtype. These cancers express the transcription factor estrogen receptor α (ER) and depend on its activation and subsequent regulation of estrogen-responsive genes for survival and proliferation. Patients diagnosed with ER-positive breast cancer are typically given endocrine therapies that target the activity of the ER. Although endocrine therapies are initially effective, patients invariably develop resistance to currently available treatments, underscoring the need to better characterize the mechanism by which ER-target genes drive proliferation in order to develop new and innovative therapies. One gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation of ER-positive breast cancer cells. The GREB1 gene encodes three distinct protein isoforms: GREB1a, GREB1b, and GREB1c. Unfortunately, few studies have investigated the mechanism by which GREB1 regulates proliferation and no studies have investigated the isoform specific molecular functions of GREB1. Here, I investigate the role of the GREB1 isoforms in modulation of ER activity and proliferation. I show that all three isoforms interact with ER through their homologous amino terminus. Analysis of ii isoform-specific regulation of ER activity reveals that none of the GREB1 isoforms are potent regulators of ER activity. Interestingly, exogenous expression of GREB1a, but not GREB1b, was associated with increased expression of some ER-target genes and this function was independent of ER activity. While these data demonstrate diverging roles for GREB1a and GREB1b in modulation of gene expression, exogenous expression of either GREB1a or GREB1b resulted in similar patterns of growth repression in breast cancer cell lines, independent of ER expression. Analysis of GREB1 isoform expression in cell lines and patient samples reveals that while GREB1a is the predominant isoform, there appears to be an increase in the expression of both GREB1b and GREB1c in malignant patient samples compared to normal patient samples. Taken together, these data suggest that GREB1a has an isoform-specific function in transcriptional regulation while all three isoforms share an ER-independent function to modulate proliferation. Despite the clear relationship between GREB1 expression and proliferation of breast cancer cells, the explicit mechanism by which GREB1 regulates this process remains unclear. As GREB1a is the predominant isoform in cell lines and patient samples, I focused further investigation on this isoform. Our studies indicate that knockdown of GREB1 results in growth arrest and exogenous expression of GREB1 induces cellular senescence, suggesting a dynamic role for GREB1 in the modulation of proliferation. Analysis of signaling pathways known to regulate both senescence and proliferation revealed that iii GREB1 is able to modulate signaling through the PI3K/Akt/mTOR pathway. I show that GREB1 acts through the canonical PI3K pathway to regulate PIP3 levels and activation of Akt and downstream effectors. Importantly, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data demonstrate a novel mechanism by which GREB1 modulates signaling through the PI3K/Akt/mTOR pathway to regulate proliferation of breast cancer. These findings are critical to better understanding how ER-target genes drive proliferation of breast cancer and developing innovative therapies that act downstream of ER-signaling. iv Dedication This work is dedicated to my mother, Kimberley Haines, and my brother, Kel Haines, for their unconditional love and support. v Acknowledgments I am forever grateful for the numerous individuals that have supported and encouraged me throughout my graduate career. I am beyond appreciative of my graduate advisor, Dr. Craig Burd. When I decided to come to OSU I had a clear idea of the type of mentor and lab environment I was looking for: approachable, hands-on, engaging, motivating, and fun. I am thankful to say I found all of those characteristics in my mentor, Craig, and the Burd lab. Craig, your unwavering support and guidance have allowed me to become a better scientist, writer, and presenter. I have learned more in the past 5 years in your lab than I could have ever imagined. Thank you for your resourcefulness and your willingness to support my ideas. Thank you to my committee members, Drs. Sharon Amacher, Anne Strohecker, Denis Guttridge, and Philip Tsichlis for your continued support and advice. Your input and guidance helped to move my project forward and enabled me to become a better scientist. To Dr. Christin Burd, thank you for all of your advice, care, and especially your tough love. My graduate career would not have been the same without the companionship of my fellow graduate students: Clarissa Wormsbaecher, Becky Hennessey, Kyle LaPak, Brandon Murphy, and Xiangang Guan. To Ali Shapiro, vi Alina Murphy, Makanko Komara, Evi Zhang, and Tirzah Weiss, thank you for all of your help over the years. Finally, thank you to my family and friends for helping to make graduate school bearable. To my mom, Kimberley Haines, there are no words to express my immense gratitude and appreciation for everything you have done and continue to do for me that has allowed me to get to this point in my life. You are truly my inspiration and the one that has made all of this possible. Thank you. To my brother, Kel Haines, thank you for always being there to cheer me up and for encouraging my love of science. To my “work dad,” Bill Neville, thank you for your unwavering care and support, advice, and for being the best Pelotonia riding partner. To Garrett Strawser, thank you for being the one to make me laugh and smile at the end of everyday- even when nothing in lab worked and I’m hangry. You guys have been the best cheerleaders ever and I couldn’t have done it without you. Finally, thank you to my cat, Ethel, for always welcoming me home with loud meows and warm cuddles. vii Vita 2006-2010……..Grove City High School, Grove City, OH 2010-2014……..B.S. Biology, Denison University, Granville, OH 2014-2019……..Graduate Research Associate, Graduate Teaching Associate, and Pelotonia Fellow, The Ohio State University, Columbus, OH Publications Haines CN, Braunreiter KM, Mo XM, Burd CJ. (2018). “GREB1 isoforms regulate proliferation independent of ERalpha co-regulator activities in breast cancer.” Endocrine-related cancer. Fields of Study Major Field: Molecular, Cellular, and Developmental Biology Research Area: Cancer Biology viii Table of Contents Abstract .................................................................................................................ii Dedication ............................................................................................................ v Acknowledgments ................................................................................................vi Vita ..................................................................................................................... viii Table of Contents .................................................................................................ix List of Tables ...................................................................................................... xiii List of Figures ..................................................................................................... xiv Chapter 1 : Introduction ........................................................................................ 1 1.1 Breast Cancer Incidence, Risk Factors, and Diagnosis .............................. 1 1.2 Estrogen and Estrogen Receptors .............................................................. 2 1.2.1 The Estrogen Receptors ....................................................................... 3 1.2.2 Structure of the Estrogen Receptors .................................................... 4 1.2.3Estrogen Receptor Action ...................................................................... 4 1.2.4 Role of the Estrogen Receptor in Mammary Gland Development and Breast Cancer ............................................................................................... 9 1.3 Breast Cancer Treatment and Resistance ................................................ 12 1.3.1 Endocrine Therapy ............................................................................. 13 1.3.2 Other Targeted Therapies and Immunotherapy ................................. 15 1.3.3 Resistance to Endocrine Therapies .................................................... 15 1.4 Signal Transduction Pathways in Breast Cancer ...................................... 17 1.4.1 Receptor Tyrosine Kinases (RTKs) and Breast Cancer ..................... 17 1.4.2 PI3K/Akt/mTOR Pathway is Frequently Altered in Breast Cancer
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