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Investigating the Relationship Between Hormone Receptors DAX-1 and Estrogen Receptor Within Prostate Cancer Cell Lines

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Investigating the Relationship Between Hormone Receptors DAX-1 and Estrogen Receptor Within Prostate Cancer Cell Lines The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Fall 12-13-2019 Investigating the Relationship Between Hormone Receptors DAX-1 and Estrogen Receptor Within Prostate Cancer Cell Lines Meghana G. Vijayraghavan University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biology Commons, and the Cancer Biology Commons Recommended Citation Vijayraghavan, Meghana G., "Investigating the Relationship Between Hormone Receptors DAX-1 and Estrogen Receptor Within Prostate Cancer Cell Lines" (2019). Master's Theses. 1388. https://repository.usfca.edu/thes/1388 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. 8/14/2021 Vijayraghavan, Meghana Abstract Prostate cancer (PCa) is estimated to be the second leading cause of cancer- related deaths among men in the United States in 2019. While most prostate tumors rely on androgens for growth signaling, there are subsets of tumors that become androgen-resistant. For these tumors, known as castration-resistant PCa, the conventional treatment of androgen-deprivation therapy fails and the cancer cells continue to proliferate despite low levels of androgens in the body. Consequently, research has focused on targeting different hormone receptors for novel therapeutics. The most common hormone receptors being researched are ERα and ERβ. However, their roles within PCa are complicated and have not been fully elucidated. Another lesser-known hormone receptor that is being researched is Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1). DAX-1 is known to interact with other hormone receptors, such as androgen receptor (AR) and estrogen receptor (ER), and suppress the proliferative effects of their signaling. Previous research has shown DAX-1 binds to AR in PCa cell lines, but little work has been done on the association of ER and DAX-1 in these cell lines. Therefore, the aim of this project was to explore DAX-1 and ER binding in the context of PCa cell lines. We utilized two different PCa cell lines, an androgen-dependent line and an androgen-resistant line, and analyzed gene and protein expression of DAX-1, ERα, and ERβ. Both cell lines were found to express the proteins of interest at varying degrees. We also used immunofluorescence microscopy to detect the protein localization and found DAX-1 and ERα as well as DAX-1 and ERβ co-localize in PCa cell lines, regardless of the androgen response of the cell line. This novel finding suggests that the hormone receptors bind in vivo in PCa cell lines. GST pull-down assays along with co- immunoprecipitation assays were then performed to assess bona fide interaction of DAX-1 and ERα. Although the results from these assays were inconclusive, our previous results of hormone receptor co-localization support the idea that DAX-1 does bind ERs in vivo. These results strongly suggest DAX-1 utilizes both ER and AR as binding partners in PCa cells and mediates its anti-proliferative effects in prostate cancer through these interactions. 2 Vijayraghavan, Meghana Acknowledgements First and foremost, this thesis work could not have been completed without the unwavering support of my advisor, Dr. Christina Tzagarakis-Foster. Dr. T, thank you for allowing me to join your lab and taking the time to mentor me over the past few years. You have shown me what it means to be a strong woman in science, one who can have it all and remain graceful and balanced throughout. I wouldn’t have crossed the finish line without you, so thank you for always believing in me and always encouraging me. Dr. Mary Jane Niles and Dr. John Paul, thank you both for being on my committee and providing the patience, feedback, and support needed for this project to blossom. I will always appreciate the open-door policy you both had for me (even when I would crash lab meetings and undergraduate office hours). I am thankful for the faculty and staff at USF, especially Dr. Naupaka Zimmerman, Dr. Janet Yang, Jeff Oda, and Matt Helms, for checking in on me and brainstorming with me when facing stubborn science. Those quick chats in the hallway meant the world to me. Jeff, thank you for being the equipment “wizard” and somehow managing to conjure up lab supplies out of thin air when I most needed it. To the fellow students, both graduate and undergraduate, in the Tzagarakis-Foster lab, thank you for the support you showed me over the past two years. I appreciate all of you for listening to my lab presentations and for always giving an encouraging word when I needed it. 3 Vijayraghavan, Meghana To the biology graduate students at USF, thank you all for the many laughs and positive words over the years. I’m glad we all found the best garlic fries in SF. Lastly, I am so lucky to have the love and support of my family. Karun and Deepthi, thank you both for being amazing role models to look up to and for helping me figure out my path through graduate school. Mom and Dad, thank you for being here for me every step of the way. I absolutely could not have done this without either of you. Thank you both for never stifling my dreams and for always encouraging my curiosity. 4 Vijayraghavan, Meghana Table of Contents Page number Abstract………………………..………………………..…………………..……….………….2 List of Figures………………………..………………………….………..…………………... 6 List of Tables………………………..………….……………….………..………………....... 7 List of Abbreviations……..………..………….……………….………..………………....... 8 Chapter 1: Introduction to prostate cancer and nuclear hormone receptors……... 9 Chapter 2: DAX-1 and ER expression in prostate cancer cell lines Introduction……………………………………………………………………….. 22 Materials and Methods………………………….……………………………….. 25 Results…………………………………………………………………………….. 35 Discussion…………………...……………………………………………………. 47 Chapter 3: Validation of DAX-1 and ER protein interaction Introduction………………………………………….…………………………….. 51 Materials and Methods………………………….……………………………….. 53 Results…………………………………………………………………………….. 58 Discussion…………………...……………………………………………………. 63 Chapter 4: Concluding remarks…………………………….…………..………………... 65 References……………..…………………………………………..………………………… 68 5 Vijayraghavan, Meghana List of Figures Page number Figure 1-1. The typical mechanism of a steroid hormone NHR…………………….. 13 Figure 1-2. Schematic of Estrogen Receptor Alpha and Beta proteins…………… 16 Figure 1-3. Schematic of DAX-1 protein………………………………………………… 19 Figure 2-1. Prostate cancer cell lines express NHR genes………………………….. 37 Figure 2-2. Prostate cancer cell lines express NHR proteins…….…...................... 40 Figure 2-3. DAX-1 and ERs co-localize in the nuclei of LNCaP cells………...……. 43 Figure 2-4. DAX-1 and ERs co-localize in the nuclei of PC3 cells…………...…….. 45 Figure 3-1. GST pull-down assays using LNCaP lysate……………………………... 60 Figure 3-2. Co-immunoprecipitation assays using anti-DAX-1 coupled beads and LNCaP lysate…………………………………………………….…………….. 62 6 Vijayraghavan, Meghana List of Tables Page number Table 2-1. Forward and Reverse primer sequences for genes of interest……….. 28 Table 2-2. Thermal cycler conditions for standard PCR…………………………….. 28 Table 2-3. Primary antibodies used in Western Blots………………………………... 31 Table 2-4. Secondary antibodies used in Western Blots…………………....………. 31 4. Protein targets and corresponding primary and secondary antibodies used in immunofluorescence microscopy experiments………………… 31 Table 3-1. Primary antibodies used for western blot analysis of co-IP reactions. 57 Table 3-2. Secondary antibodies used for western blot analysis of co-IP reactions…………………………………….…………………………………… 57 7 Vijayraghavan, Meghana List of Abbreviations ADT: androgen deprivation therapy AF1: activation function 1 AF2: activation function 2 AR: androgen receptor Co-IP: co-immunoprecipitation CRPC: castration-resistant prostate cancer DAX-1: Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 DBD: DNA binding domain E2: estradiol ER: estrogen receptor GST: glutathione-S-transferase HSP: heat shock protein IF: immunofluorescence LBD: ligand binding domain LHRH: luteinizing-hormone release-hormone NHR: nuclear hormone receptor PCa: prostate cancer PSA: prostate-specific antigen RE: response element PCR: reverse-transcription polymerase chain reaction TNM system: tumor, node, metastasis system 8 Vijayraghavan, Meghana Chapter 1 Introduction to Prostate Cancer and Nuclear Hormone Receptors Prostate cancer Prostate cancer (PCa) is estimated to be the most diagnosed cancer in men in the United States in 2019. Approximately, one in five new male cancer diagnoses will be prostate cancer, totaling 174,650 new cases. PCa is also estimated to be the second most deadly cancer in men in the United States accounting for 10% of total male cancer deaths [1]. The median age of patients diagnosed with PCa is 66 years old [2]. Men have an increased risk in developing high-risk PCa as a result of age [3-4]. Race also plays a large factor in predicting which men will be diagnosed with PCa. Those of African-American descent have the highest chance of being diagnosed with PCa, with one in
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