GPER-MEDIATED REGULATION of NUCLEAR AKT/FOXO3A SIGNALING Erin Zekas
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University of New Mexico UNM Digital Repository Biomedical Sciences ETDs Electronic Theses and Dissertations 7-1-2014 GPER-MEDIATED REGULATION OF NUCLEAR AKT/FOXO3A SIGNALING Erin Zekas Follow this and additional works at: https://digitalrepository.unm.edu/biom_etds Recommended Citation Zekas, Erin. "GPER-MEDIATED REGULATION OF NUCLEAR AKT/FOXO3A SIGNALING." (2014). https://digitalrepository.unm.edu/biom_etds/153 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Biomedical Sciences ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. Erin Zekas Candidate Biomedical Sciences Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: Eric R. Prossnitz, PhD, Chairperson Angela Wandinger-Ness, PhD Diane Lidke, PhD Helen J. Hathaway, PhD i GPER-MEDIATED REGULATION OF NUCLEAR AKT/FOXO3A SIGNALING by ERIN ZEKAS B.S. Biology, Temple University, 2005 DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biomedical Sciences The University of New Mexico Albuquerque, New Mexico July, 2014 ii © 2014, Erin Zekas iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my parents, Marlene and Joe. They have unconditionally supported me throughout this experience and throughout my entire life and for that I am more grateful than words could express. Secondly, I would like to thank my mentor, Dr. Eric Prossnitz, for pushing me to stand on my own two feet as a researcher. I would also particularly like to thank Eric for his patience. Seriously. I’ve learned so much throughout this process and have Eric to thank for helping me make it out of this in one piece. Next, I would like to acknowledge my committee. Dr. Angela Wandinger-Ness has always put things in perspective for me. Angela has also been extremely helpful with my job search and giving of her time and advice. I am truly grateful for that. Dr. Diane Lidke has been a useful asset with regards to image analysis. Diane’s expert input was always welcome and I thank her for being so helpful. Lastly, I would like to thank Dr. Helen Hathaway, who only recently joined my committee but has been a mentor to me since I started this program. Helen’s guidance and understanding have kept me sane. I would like to thank all my coworkers and fellow classmates. Dr. Niki Marjon, Dr. Sara Alcon and Dr. Allison Scaling who have always been supportive and who I still ask for help despite them all having moved on. I am also extremely grateful to Dr. Geetanjali Sharma and Tamara Howard. Gee has been so helpful in the lab and always there when I need someone to vent to. I probably wouldn’t have made it through this last year without Tamara. Every time I would go on the Leica iv and return with a new problem, Tamara would have at least five ideas about how to fix it. I am so thankful for my friends in Albuquerque as well as my friends and family back home. It’s been a long 5 years and they have always been there for me when I need them. I also need to thank my former mentor, Dr. Sriram Krishnaswamy. Krish was the first person in science to give me a chance despite having no experience. I also didn’t really have a clue what I wanted to do with my life. He gave me my own project and allowed me to fall on my face repeatedly for 2 years before things finally clicked. Krish then told me I needed to move on to either graduate school or something else. Here I am 5 years later in Albuquerque, New Mexico finishing my PhD, all because of Krish and his old friend Walt Keisel. I am so grateful to Krish for his amazing and brutally honest support throughout the past 9 years. Finally, I would like to acknowledge the Spatial Temporal Modeling Center (P50 GM085273 (Wilson, PI)) at the University of New Mexico and NIH CA163890 (Prossnitz, PI) for providing funding for this project. v GPER-MEDIATED REGULATION OF NUCLEAR AKT/FOXO3A SIGNALING By Erin Zekas B.S., Biology, Temple University, 2005 Ph.D., Biomedical Sciences, University of New Mexico, 2014 ABSTRACT 17β-estradiol (estrogen) has been demonstrated to regulate survival in breast cancer cells, which is partially mediated by its nuclear receptors ERα and ERβ and the G protein-coupled estrogen receptor (GPER). We previously established that estrogen can activate the phosphoinositide 3-kinase (PI3Kinase) prosurvival pathway via GPER stimulation resulting in PIP3 generation within the nucleus of breast cancer cells; the mechanism for this is still unclear. PIP3 generation results in Akt activation, which is known to inactivate FOXO3a, a proapoptotic transcription factor that translocates from the nucleus to the cytoplasm upon inactivation resulting in a decrease in proapoptotic gene expression and consequently an increase in cell survival. Here, utilizing a FoxO3-GFP construct, we report FOXO3a inactivation as a result of GPER stimulation by E2 and the GPER-selective agonist G-1 in the estrogen-responsive breast cancer cell line MCF7 and that ERα is not required. The p110α catalytic subunit of PI3Kinase, vi and the transactivation of the EGFR constitute the mechanism by which GPER inactivation of FOXO3a occurs. Additionally, E2 and G-1 stimulation of MCF7 cells results in a decrease in caspase activation compared to negative control. This suggests, in part, that GPER stimulation is required for survival of breast cancer cells and that GPER expression and FOXO3a localization should be utilized as prognostic markers in breast cancer treatments. Furthermore, our results indicate a need for GPER antagonists in GPER positive breast cancers in order to counteract GPER related prosurvival effects in combination with chemotherapeutic drug treatments. vii TABLE OF CONTENTS ABSTRACT...................................................................................................................... vi LIST OF FIGURES .......................................................................................................... x LIST OF ABBERVIATIONS ......................................................................................... xii CHAPTER 1 ...................................................................................................................... 1 1. Introduction ................................................................................................................. 1 1.1. Estrogen Synthesis ............................................................................................... 1 1.2. Estrogen Function ................................................................................................. 4 1.3. Estrogen Receptors and Genomic Signaling ................................................. 5 1.4. Estrogen Receptors and Nongenomic Signaling ........................................ 10 1.5. PI3Kinase ................................................................................................................ 15 1.6. FOX Proteins ......................................................................................................... 19 1.7. PI3Kinase/Akt/FOXO and Cancer ..................................................................... 22 1.8. Breast Cancer ........................................................................................................ 25 1.9. GPER-selective ligands ...................................................................................... 27 Rationale......................................................................................................................... 30 Hypothesis ..................................................................................................................... 30 Specific Aims ................................................................................................................ 30 CHAPTER 2 .................................................................................................................... 32 2. GPER activation leads to FOXO3a inactivation in MCF7 breast cancer cells.................................................................................................................................. 32 2.1. Abstract................................................................................................................... 32 2.2. Introduction ........................................................................................................... 33 viii 2.3. Materials and Methods ........................................................................................ 37 2.3. Results .................................................................................................................... 42 2.4. Discussion ............................................................................................................. 50 CHAPTER 3 .................................................................................................................... 82 3. Conclusions, significance and future directions ............................................ 82 3.1. Conclusions ........................................................................................................... 82 3.2. Significance ........................................................................................................... 88 3.3. Future directions .................................................................................................