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Fall 12-13-2019

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

Meghana G. Vijayraghavan University of San Francisco, [email protected]

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Recommended Citation Vijayraghavan, Meghana G., "Investigating the Relationship Between Hormone Receptors DAX-1 and Within Prostate Cancer Cell Lines" (2019). Master's Theses. 1388. https://repository.usfca.edu/thes/1388

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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 that is being researched is Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on X, 1 (DAX-1). DAX-1 is known to interact with other hormone receptors, such as (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 expression of DAX-1, ERα, and ERβ. Both cell lines were found to express the 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.

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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.

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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.

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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

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List of Figures

Page number

Figure 1-1. The typical mechanism of a hormone NHR…………………….. 13

Figure 1-2. Schematic of and Beta proteins…………… 16

Figure 1-3. Schematic of DAX-1 protein………………………………………………… 19

Figure 2-1. Prostate cancer cell lines express NHR ………………………….. 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

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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

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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

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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 seven non-Hispanic black men being diagnosed with PCa over a lifetime compared to one in eight non-Hispanic white men [5]. Black men are also twice as likely to die from PCa than white men [5]. Additional risk factors associated with PCa are family genetics, mutations in the BRCA1 and BRCA2 genes, and lifestyle factors such as smoking [6-10]

Like most cancers, PCa is diagnosed and evaluated through numerous techniques. The initial test is a digital rectal exam performed by a physician followed by testing the level of prostate-specific antigen (PSA) in the blood. Healthy prostate cells secrete PSA in low numbers and over time, the cells gradually secrete an increasing amount of PSA. However, a rapid increase in an individual’s PSA levels compared to a baseline level (usually determined in the patient’s early 40s) indicates either the prostate has enlarged or prostate cells are dividing rapidly and uncontrollably indicating

9 Vijayraghavan, Meghana a cancerous [11]. If PSA levels are elevated, a biopsy of the tissue is taken and the tissue is given a Gleason score and a stage. The Gleason score determines whether the PCa looks like healthy tissue when viewed under a microscope. The higher the Gleason score, the more the cancer cells look undifferentiated [11-12] and the higher the likelihood the cancer will grow more rapidly. However, Gleason score alone cannot determine whether a patient is likely to have an aggressive form of PCa. Staging is another technique that involves evaluating the tumor based on the Tumor, Node,

Metastasis (TNM) system. The TNM system evaluates the primary tumor location and size, whether the PCa has spread to the lymph nodes, and whether the PCa has metastasized to another tissue. Based on these categories, patients are given a stage ranging from 0 to IV, with stage 0 meaning no evidence of PCa was found within the patient and stage IV meaning the PCa has metastasized beyond the prostate [13].

Treatment for PCa depends on the Gleason score combined with the cancer stage. The higher the Gleason score and the higher the stage, the more aggressive the PCa treatment for the patient.

Prostate cancers primarily rely on androgens to stimulate proliferative and survival genes via androgen receptor (AR), a type of nuclear hormone receptor [14].

Therefore, the typical course of treatment of PCa relies on androgen-deprivation therapy (ADT), also known as androgen ablation therapy. This form of treatment involves either surgical castration, known as orchiectomy, or medical castration that manipulates levels of luteinizing-hormone release-hormone (LHRH) in the hypothalamus to considerably reduce the amount of produced by the testes [15]. Randomized clinical trials have exhibited the benefits of ADT, including

10 Vijayraghavan, Meghana long-term survival, progression, and local control, in both intermediate-risk PCa and high-risk PCa [16-17]. Additionally, multiple randomized clinical trials have demonstrated the benefits of adjuvant use of radiation therapy along with ADT. The results found that the addition of radiation therapy to ADT allowed for a longer five-year survival rate compared to ADT or radiation therapy alone [18-20].

The Nuclear Hormone Receptor Family and Estrogen Receptor

While there has been significant improvement in therapies over the years, PCa that has metastasized beyond the sex organs primarily into bone and has a poor five-year survival rate of 30% [1]. These PCa cells become androgen-resistant and continue to proliferate despite low levels of testosterone in the body. These tumors are known as castration-resistant prostate cancer (CRPC) and are virtually impossible to cure with current treatments [21-22]. For this reason, scientists are looking into novel treatments for CRPC. One avenue of CRPC treatment researchers are exploring is targeting other nuclear hormone receptors (NHRs) for hormone therapies that would impact PCa proliferation and metastasis.

NHRs are a unique family of ligand-inducible transcription factors that regulate [23-24]. Typical members of this family have 6 conserved regions

(distinguished as regions A-F), each having a different function. Regions A/B comprise the variable N-terminal domain that is important in gene regulation and includes the ligand-independent transactivation domain, called activation function 1 (AF1) [25-26].

Region C contains the highly conserved DNA binding domain (DBD). The DBD contains two motifs allowing the receptor to “read” the DNA and find the appropriate five to six start of the DNA response element (RE) in the promoter of the

11 Vijayraghavan, Meghana regulated gene. DNA response elements allow transcription factors to bind DNA upstream from their gene targets, essentially allowing transcription factors to recognize their correct targets [27-28]. Region D acts as the hinge region and is a link to the conserved region E. The variable hinge region of the receptor contains a nuclear localization signal and assists with DNA recognition [24; 29-31]. Regions E and F contain the ligand binding domain (LBD) along with the variable C-terminus of the protein[24]. The LBD is composed of 12 alpha-helices that mediate ligand binding and also assist in co-receptor binding as well as homo- or heterodimerization of receptors

[32-33]. The ligand-dependent activation function 2 (AF2) is also included within this region. Depending on the nature of the ligand, NHRs can be further classified into steroid hormone receptors such as androgen receptor and estrogen receptors (ER), non-steroid hormone receptors such as those that respond to vitamin D or retinoids, and orphan receptors with no known ligands [34-37]. In total, there have been 48 NHRs identified to date, with each category of NHR having a slightly different mechanism of action.

The typical mechanism of a starts with the ligand diffusing across the plasma membrane of the cell. Once inside the cell, the ligand binds to its respective receptor located in the cytoplasm, which causes the receptor to be released from an anchoring protein. Homo- or heterodimerization of the ligand-receptor complex occurs and coactivators are recruited to these complexes. The overall ligand- receptor-coactivator complex enters the nucleus where it uses the NHR’s DBD to bind to the correct RE and activate or repress target gene transcription (Figure 1-1).

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Figure 1-1. The typical mechanism of action of a steroid hormone NHR. The ligand crosses the plasma membrane of the cell where it binds to its respective NHR. The binding of the ligand causes the anchoring HSP (heat shock protein) to release the NHR. Two activated NHRs can homodimerize and recruit coactivators to the complex. The whole complex translocates to the nucleus and binds to the DNA response element to act as a for its target gene.

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NHRs play an important role in prostate cancer, with AR acting as the main driver of PCa and therefore being the most studied NHR within this cancer. However, some studies have explored the role of ER within PCa. Estrogen receptors are NHRs that respond to as ligands. There are two distinct genes that code for ER proteins, with the two most common isoforms being ERα and ERβ1. The ESR1 gene codes for

ERα while the ESR2 gene codes for ERβ1. Both ERs are typical NHRs and contain an

N-terminal DBD and ligand-independent AF-1 domain along with the C-terminal LBD and ligand-dependent AF2 domain. The isoforms have high except in their LBDs, suggesting that different estrogens preferentially bind ERα or ERβ1

(Figure 1-2). Indeed, ERα demonstrates a higher affinity for estradiol (E2) while ERβ1 has a higher affinity for 3β-Androstanediol, an estrogen that is a metabolite of androgen conversion [38].

Historically, clinical studies have looked at the effect of the oral estrogen, diethylstilbesterol, on prostate tumors [39-41]. These efforts were deemed unfavorable when the studies noted the orally administered estrogens did not improve the overall survival of the patients yet the level of cardiovascular toxicity was highly increased [42-

43]. While these studies are quite old, more recent efforts are being made to study the effect of parenteral and dermal estrogen therapy on thromboembolic effects and bone mineral density in PCa patients [43-44].

Laboratory-based studies have explored the roles of ERα and ERβ in the context of PCa. ERα is thought to behave as oncogene as activation of ERα via E2 binding mediates proliferation signals and prostatic carcinogenesis. Adding to the evidence that

ERα is oncogenic, mice ERα knockout models do not develop PCa after treatment with

14 Vijayraghavan, Meghana estrogen or testosterone, indicating ERα is necessary for PCa progression [45]. In contrast ERβ, which tends to be down-regulated as PCa progresses, seems to act in a non-oncogenic manner as ERβ agonists have slowed the proliferation of prostatic epithelium cells [46]. ERβ activation lead to apoptosis in the CRPC cell lines DU145 and

PC3 [47]. These findings indicate ERα and ERβ have larger roles in PCa than previously thought. However, studies are needed to further explore the role of estrogen receptors and other NHRs in the progression of PCa tumors, specifically in the progression of CRPC, in order to identify novel therapeutic targets.

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Figure 1-2. Schematic of Estrogen Receptor Alpha and Beta proteins. The two ER proteins are coded by different genes, ESR1 and ESR2. ESR1 codes for ERα and is located on chromosome 6 while ERβ is coded by ESR2 which is located on chromosome 14. ERα and ERβ are typical NHRs with an AF1 region and DBD region in the N-terminus and LBD and AF2 regions in the C-terminus. The proteins share a high level of homology in their DBD domains but only a 55% sequence homology in their LBD domains. This suggests that each ER preferentially binds different estrogen ligands.

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DAX-1: an unusual NHR

One such NHR is the orphan receptor DAX-1, encoded by the NR0B1 gene.

DAX-1 is known as an orphan receptor since it has no known ligand. The protein plays an important role in mammalian steroidogenesis, , and sex development

[48-52]. Originally, the NR0B1 gene was found to be associated with two disorders.

Adrenal hypoplasia congenita (AHC) in which the adrenal glands fail to develop properly and hypogonadotropic hypogonadism (HH) in which DAX-1 is over-expressed resulting in male-to-female sex reversal. Because of its association with these two disorders, the protein’s name stands for Dosage sensitive sex reversal, Adrenal hypoplasia congenita critical region on chromosome X, gene 1 [53]. The gene is expressed in tissues highly influenced by hormones such as the developing , pituitary gland, hypothalamus, and [54]. The DAX-1 protein is unusual as it lacks several conserved functional domains typical of other NHRs. Specifically, instead of having an

N-terminal domain containing an AF-1, DBD, and hinge region, DAX-1 contains three full-length alanine/glycine-rich repeat domains and one half-length domain. These repeats harbor short leucine motifs known as LXXLL motifs that are commonly seen in coactivators and corepressors [53; 55-56] (Figure 1-3). Because of these leucine-rich motifs, DAX-1 has been studied in the context of other nuclear hormone receptors. It has been shown to act as a transcriptional corepressor of other nuclear hormone receptors including steroidogenic factor 1, estrogen receptor, androgen receptor, , and liver receptor homologue 1 [57-63]. It is proposed that DAX-1 binds to the coactivator-binding surface in the LBD of other NHRs using its LXXLL motifs, specifically the leucine amino acids, found in its N-terminus. By

17 Vijayraghavan, Meghana binding to the coactivator-binding surface, DAX-1 is able to competitively inhibit NHR from binding coactivators thus blocking subsequent transcription [60].

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Figure 1-3. Schematic of DAX-1 protein. DAX-1, encoded by the NR0B1 gene, is an unusual, orphan NHR. The protein differs from typical NHRs by not housing a DBD region, AF1 region, or hinge region within its N-terminus. Instead the protein exhibits 3.5 leucine-rich LXXLL motifs which help the protein bind to typical NHRs in their LBDs. Though DAX-1 maintains LBD and AF2 regions in its C-terminus, no known ligand has been elucidated for this NHR, making it an orphan.

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In light of DAX-1’s role in transcriptional regulation, studies have shifted to focus on its possible role in various tumors. Adrenocortical were the first type of tumors to be analyzed for DAX-1 expression [64]. One study proposed that higher DAX-

1 expression in adrenocortical tumors limits steroidogenic factor 1 transcriptional activity thereby resulting in reduced cell proliferation and tumor formation [65]. Additional studies have correlated DAX-1 to clinical parameters in different types of human tumors suggesting the altered expression of DAX-1 could act as a clinical marker for certain cancers. It is worth noting the differing antibodies and IHC techniques used in these studies make for variable interpretation of the results. In adenocarcinomas, high

DAX-1 expression was associated with lymph node metastasis [66]. Conversely, in breast cancer and endometrial carcinomas, DAX-1 immunoreactivity was inversely correlated with nodal status and histological grade, respectively [67-68]. Similarly, in prostate cancer DAX-1 immunoreactivity correlated inversely with tumor Gleason scores. However, the study found no correlation between the status of DAX-1 and the status of the NHRs tested including ERβ and AR in patient samples [69]. This is an interesting discrepancy since previous work has demonstrated the ability of DAX-1 to act as a corepressor of ERs in vitro and in vivo using COS-7 cells that are fibroblast-like cells derived from primate kidney tissue [60]. COS-7 cells are mainly used since they are mammalian cells, widely available, and easy to transfect. However, in the context of human prostate cancer cells, both hormone-naïve and CRPC cells, there are no published studies to date that explore the relationship between DAX-1 and ERs. If an association is found between the NHRs, the ER-DAX-1 heterodimer could be further studied as a possible target for novel prostate cancer therapeutics.

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We hypothesize that DAX-1 binds ER within prostate cancer cell lines and this binding serves to act as a transcriptional repressor of proliferative and metastatic genes.

The first aim of this study was to determine whether DAX-1 and both isoforms of ER were endogenous to both hormone-naïve and CRPC cell lines. PCR, immunoblotting, and immunofluorescence microscopy suggested that all three NHRs were present in both types of PCa cell lines, with DAX-1 being mostly localized to the nuclei of the cells and ER being diffused among the cytoplasm and the nuclei of the cells. The next aim of this study was to determine if DAX-1 binds to ER within the PCa cell lines. GST pull- down assays along with co-immunoprecpitation assays were used to evaluate the protein-protein interaction both in vitro and in vivo. The results from this portion of the study were inconclusive and future work needs to be carried out to confirm this interaction exists and if so, to determine the strength and context of the interaction.

Elucidating the mechanisms of DAX-1 and ER within PCa cell lines will allow new target

NHRs to be established as novel targets for castration-resistant prostate cancer treatment.

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Chapter 2

DAX-1 and ER expression in prostate cancer cell lines

Introduction

As described in Chapter 1, androgen deprivation therapy (ADT) is the main form of treatment for PCa. However, ADT eventually fails leaving few other options for cancer treatment. The need for other forms of hormonal and NHR-mediated treatments are glaring when ADT fails.

While the status of ERα and ERβ within PCa has been reported in many studies, the results are conflicting due to the variation of PCa models used (mice models, cell lines, etc.) and the variety of techniques employed. ERα is mainly expressed in stromal cells of healthy prostate tissue, though it has been found in basal-epithelial cells occasionally. ERβ is primarily localized to the basal-epithelial cells within the prostate

[70-72]. Crosstalk between the two compartments, the stroma and the epithelium, plays an important role in the progression of prostate carcinogenesis as reviewed by Niu and

Xia [73]. Therefore, the differential expression of ERs within these compartments indicate ERα and ERβ have different roles in healthy prostate tissue and likely have differing roles within PCa tissues. Within PCa cell models and tissue samples, ERα is considered to be an oncogene that is highly expressed in epithelial tissues, as opposed to its stromal expression in healthy prostate tissue. ERα mediates proliferation, inflammation, and prostate carcinogenesis. ERα knockout mice that were treated with testosterone and estrogen did not develop prostate carcinomas indicating that within

PCa mice models, ERα is a necessary component to induce prostate carcinogenesis

[45]. Additionally, ERα, but not ERβ, is highly up-regulated in the prostate of transgenic

22 Vijayraghavan, Meghana mice models which closely mimics the pathogenesis of human PCa compared to wild- type models, suggesting once again, ERα is the predominant ER mediating prostate carcinogenesis in mice models [74]. Within human samples, ERα is repeatedly detected in tissues from patients with higher Gleason scores and poor survival rates [75-76].

In contrast to the widely accepted oncogenic effects of ERα, ERβ has a more controversial role within PCa. Generally, it is thought that ERβ acts in opposition to ERα and has anti-proliferative, pro-apoptotic effects within PCa. In ERβ knockout mice, the absence of ERβ compared to wild-type mice resulted in an increase in proliferation of epithelial cells, a decrease in apoptotic cells, and an increase in undifferentiated cells

[77-78]. When stimulated with 3β-androstanediol, a ligand that only activates the ERβ receptor, wild-type mice had reduced AR expression levels in comparison to the ERβ knockout mice. The reduction of AR levels suggests ERβ potentially regulates AR expression. Since AR is known to be a driver of PCa progression, it would be worthwhile to look into ERβ-specific treatments for hormonal therapies. In human cancer models, ERβ is down-regulated as the PCa progresses suggesting that the NHR is needed to maintain a lower grade phenotype [79]. ERβ activation and up-regulation in

CPRC cell lines inhibited cell proliferation and cell invasiveness while triggering apoptosis [47; 80-81]. However, the anti-proliferative effects of ERβ have been disputed within PCa cell line models. When stimulated with estradiol (E2) and an ERβ-selective agonist, diarylpropionitrile, both a normal human prostate cell line and a CRPC cell line exhibited an increase in DNA synthesis, an increase in the cyclin D2 protein that mediates the cell cycle, and an increase in proliferation [82]. These findings are in direct contrast to the proposed role of ERβ within PCa progression, highlighting the fact that

23 Vijayraghavan, Meghana more studies need to be performed to elucidate the location and the roles of ERs within

PCa models.

Another NHR of particular interest to our lab is DAX-1. As mentioned in Chapter

1, expression of DAX-1 in cancerous steroidogenic tissues has been correlated to better grades or statuses of those cancers. Though these studies are only correlative, they point to an unknown role that DAX-1 plays in these cancers. DAX-1 has been detected in both cancerous and non-cancerous prostatic tissues and is reportedly strongly expressed in normal prostate epithelial cells but is absent in the CRPC cell line, PC3

[62; 83]. However, DAX-1 exhibited a variable detection pattern in patient PCa tissues with expression inversely correlating to Gleason score [69]. The variable pattern was unexpected given that DAX-1 expression seems to decrease as PCa progresses.

Further studies are required in order to clarify the ill-defined expression pattern of DAX-

1 within cancerous prostatic tissues.

Therefore, the objective of this portion of the thesis work was to investigate the expression and localization of endogenous DAX-1 protein in a hormone-naïve and a

CRPC cell line. ERα and ERβ expression and localization was also assessed in these cell lines and co-localization of the ERs with DAX-1 was explored. Furthermore, DAX-1 was overexpressed using adenovirus-based delivery methods in order to assess whether DAX-1 co-localizes with ERα and ERβ.

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Materials and Methods

Cell Culture

LNCaP and PC3 cell lines were obtained from ATCC (American Type Culture

Collection; Manassas, VA). The cell lines were grown in complete media consisting of

Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific;

Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher

Scientific), 1% Penicillin-Streptomycin antibiotic solution (Invitrogen; Carlsbad, CA), 1%

HEPES buffer (Mediatech Inc; Manassas, VA), 1% sodium pyruvate (Thermo Fisher

Scientific), 1% glucose (Mediatech Inc), and 0.2% sodium bicarbonate (Mediatech Inc.).

Cells were incubated at 37°C with 5% CO2 in a humidified chamber. Cells were sub- cultured at least twice a week using 0.25% Trypsin-EDTA (Thermo Fisher Scientific) as per their respective ATCC guidelines, with a 1:3-1:6 split ratio.

LNCaP cells were originally isolated from a 50 year old Caucasian male in 1977.

These cells are hormone naïve. PC-3 cells were derived from a bone metastasis of a grade IV adenocarcinoma in a 62 year-old Caucasian male in 1979. The cells are representative of advance PCa. They do not respond to androgens nor do they express

DAX-1. PC-3 cells have high metastatic potential compared to other prostate cancer cells.

Adenovirus transduction

Recombinant human DAX-1 adenovirus was obtained from Creative Biogene

Biotechnology (Shirley, NY) at a concentration of 2.62 x 108 vp/mL. DAX-1 adenovirus inoculum was prepared using 1 μL of virus per 1 mL of complete media. LacZ adenovirus inoculum was prepared by adding CMV LacZ adenovirus (Applied Biological

25 Vijayraghavan, Meghana

Materials Inc.; Richmond, BC, Canada) at the same concentration as the DAX-1 viral inoculum.

LNCaP cells, or alternatively, PC3 cells were seeded into 6-well dishes in duplicates at varying densities depending on the assay. Cells were incubated in complete media overnight (12-16 hours) in a humidified chamber at 37°C with 5% CO2.

The next day, the cells were checked for confluence of at least 40% prior to infection.

Viral inoculum was prepared according to the protocol above. Old media was gently aspirated and the viral inoculum was added for a total of 2 mL/well. Cells were seeded in duplicates and were either untreated, treated with LacZ adenovirus, or treated with

DAX-1 adenovirus. The cells were incubated with the virus in a humidified chamber at

37°C with 5% CO2 for 48-72 hours, depending on the assay being performed. After incubation, cells were harvested and either RNA or protein lysate were collected.

RNA extraction

Following a 48 hours infection with no treatment, LacZ adenovirus, or DAX-1 adenovirus, cells were harvested and RNA isolated according to the manufacturer’s instructions using the Qiagen RNeasy Midi Prep Kit (Qiagen; Hilden, Germany). The collected RNA was quantified using the ND-1000 Nanodrop (Thermo Fisher Scientific).

RNA was immediately used for cDNA synthesis and the remaining RNA was stored at -

20°C. cDNA synthesis

A calculated amount of RNA (1000 ng) was reversed transcribed into cDNA using the Quantabio qScript cDNA Supermix kit (Quantabio; Beverly, MA) according to

26 Vijayraghavan, Meghana the manufacturer’s instructions. cDNA was either immediately used for PCR reactions or stored at -20°C until use.

Reverse-transcription polymerase chain reaction

Synthesized cDNA was amplified via polymerase chain reaction (PCR) and used to detect expression of the NR0B1 (DAX-1), ESR1 (ERα), and ESR2 (ERβ) genes.

GAPDH was used as a housekeeping gene and loading control. GAPDH

(glyceraldehyde 3-phosphate dehydrogenase) is a gene that encodes the GAPDH enzyme that is critical for glycolysis to occur. This gene remains constant in the cells and is unlikely to change when DAX-1 is up-regulated. The forward and reverse primers for each gene are detailed below (Table 2-1). Each PCR reaction was assembled for a final volume of 20 μL using 10 μL GoTaq Green Master Mix (Promega; Madison, WI), 1

μL of 10 μM forward and reverse primers, 1 μL cDNA, and 8 μL of nuclease free dH2O.

The PCR reactions were performed using the Bio-Rad C1000 Touch Thermal cycler

(Bio-Rad; Hercules, CA) following the protocol described in Table 2-2. PCR products were resolved in a 2% agarose gel and imaged using the Bio-Rad ChemiDoc XRS+ machine with Image Lab software (Bio-Rad).

27 Vijayraghavan, Meghana

Table 2-1. Forward and Reverse primer sequences for genes of interest

Gene of Forward primer (5’ to 3’) Reverse primer (5’ to 3’) Expected Interest size (base pairs) NR0BI GACTCCAGTGGGGAACTCAG ATGATGGGCCTGAAGAACAG 154 bp ESR1 GACAGGGAGCTGGTTCACAT AGGATCTCTAGCCAGGCACA 110 bp ESR2 TGAAAAGGAAGGTTAGTGGGAACC TGGTCAGGGACATCATCATGG 528 bp GAPDH CCATCACCATCTTCCAGGAGCG AGAGATGATGACCCTTTTGGC 149 bp

Table 2-2. Thermal cycler conditions for standard PCR.

Step Temperature Time Initial denaturation 95°C 5 minutes 35 cycles: Denaturation 95°C 30 seconds Annealing 56°C 30 seconds Elongation 72°C 40 seconds Final extension 72°C 5 minutes Hold 4°C ∞

28 Vijayraghavan, Meghana

Protein extraction

LNCaP or PC3 cells were seeded in 6-well dishes and treated with adenovirus as outlined above. At 72 hours post infection, cells were washed once with 1X ice cold

PBS. The cells were then harvested by adding a solution of 300 μL M-PER Mammalian

Protein Extraction Reagent (Thermo Fisher Scientific) and 3 μL of HALT Protease

Inhibitor Cocktail (Thermo Fisher Scientific) to each well and gently rocking the plate for

5 minutes at room temperature. After incubation, the cell lysate was scraped out of each well, transferred to a sterile 1.5 mL microcentrifuge tubes, and centrifuged at 14,000 x g for 7 minutes at 4°C to pellet cell debris. The supernatant was collected and transferred to new, labeled microcentrifuge tubes. Lysate was stored at -20°C until further use.

Western Blot

SDS-PAGE was performed using the XCell SureLock Mini Cell (Invitrogen) according to the NuPAGE Novex Bis-Tris Protocol (Invitrogen). Protein concentration in lysate was normalized using the Bio-Rad Protein Assay Kit II (Bio-Rad). Samples were prepared for a total volume of 25 μL using 10 μg of protein, 6.25 μL of 4X NuPAGE sample buffer, 2.5 μL of 10X NuPAGE reducing agent (Thermo Fisher Scientific), and

DI H2O. Samples were heated at 70°C for 10 minutes and immediately electrophoresed in a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen) at 200 V for 45 minutes. Proteins were transferred to a PVDF membrane using the XCell II Blot Module run at 125 mA for 90 minutes. Membranes were blocked on a plate shaker in a 5% Blotto/1X TBS-T (tris-

29 Vijayraghavan, Meghana buffered saline + Tween-20) solution at room temperature for 60 minutes followed by an overnight incubation on a plate shaker at 4°C in the appropriate primary antibody solution diluted in 5% Blotto (Table 2-3). The next day, membranes were washed three times with TBS-T at 5 minutes per wash on a shaker at room temperature. Appropriate

HRP-conjugated secondary antibody dilutions were prepared in 1X TBS-T. Membranes were incubated with the secondary antibody dilutions for 60 minutes at room temperature with gentle shaking (Table 2-4). After incubation, membranes were washed three times at 5 minutes per wash in TBS-T followed by development using the Clarity

Western ECL Substrate (Bio-Rad) according to manufacturer’s protocol. The immunoblots were visualized using the Bio-Rad ChemiDoc XRS+ machine with Image

Lab software (Bio-Rad).

30 Vijayraghavan, Meghana

Table 2-3. Primary antibodies used in Western Blots

Protein Target Animal of origin Brand Dilution DAX-1 Mouse monoclonal Active Motif 1:1000 (Cat # 39983) ERα Rabbit monoclonal MilliporeSigma 1:1000 (Cat # 04-820) ERβ Rabbit monoclonal MilliporeSigma 1:1000 (Cat # 04-842) GAPDH Rabbit monoclonal Cell Signaling 1:5000 Technology (Cat # 2118S)

Table 2-4. Secondary antibodies used in Western Blots

Probing against Animal of origin Brand Dilution HRP goat anti-mouse Goat BD Pharmigen 1:1000 IgG (Cat # 554002) HRP goat anti-rabbit Goat GeneTex 1:1000 (for ERα and IgG (Cat # GTX213110-01) ERβ) 1:2000 (for GAPDH) HRP goat anti-biotin Goat Cell Signaling 1:5000 (for biotinylated Technology protein ladder) (Cat # 7075)

31 Vijayraghavan, Meghana

Immunofluorescence Microscopy

Cells were plated in a 6-well dish with sterilized No. 1.5 22x22mm Corning cover glasses (Sigma Aldrich) that were previously incubated with FBS overnight. Treatments occurred in duplicate on each plate. Following 72-hour treatments with the appropriate adenovirus, cells were washed 3x with 1X PBS for 5 minutes per wash on a shaker. The cells were fixed in a 4% paraformaldehyde solution at 37°C for 20 minutes at room temperature. Following three 1X PBS washes, cells were permeabilized using 0.1%

Triton X-100 in 1X PBS for 15 minutes at room temperature. Following three washes of

10 minutes each, cells were further fixed and permeablized for 30 minutes using an ice- cold 1:1 methanol:acetone solution. Following this final fixation and permeabilization step, cells were washed three times with 1X PBS and blocked for 1 hour at 37°C using a solution of 2% bovine serum albumin in 1X PBS. Primary antibodies probing for either

DAX-1 and ERa or DAX-1 and ERβ were diluted in antibody dilution buffer (1% BSA,

0.5% Tween-20, 1X PBS) (Table 2-5). Primary antibody dilutions were added to one set of untreated, LacZ adenovirus treated, and DAX-1 adenovirus treated cells. The other set, dubbed “secondary antibody only controls”, received antibody dilution buffer only.

The cells were incubated for 1 hour at 37°C in a humidified chamber. After primary antibody incubation, three PBS washes occurred. Secondary antibodies with conjugated fluorophores were diluted in antibody dilution buffer and added to all cover slips (Table

2-5). The cover slips were incubated in the dark for 30 minutes at 37°C in a humidified chamber. Following this incubation step, cells were washed three times with PBS with precaution taken to minimize the exposure to light. The cells were mounted onto 1 mm microscope slides using ProLong Diamond Antifade Mountant (Thermo Fisher

32 Vijayraghavan, Meghana

Scientific) and allowed to dry for a 24 hours in the dark before being sealed with nail polish. Slides were imaged using Zeiss A1 AxioObserver fluorescence microscope (Carl

Zeiss Microscopy, LLC, Thornwood, NY) with ZEN Black software. Images were processed using ZEN Blue software and ImageJ software (National Institute of Health;

Bethesda, MD).

33 Vijayraghavan, Meghana

Table 2-5. Protein targets and corresponding primary and secondary antibodies used in immunofluorescence microscopy experiments

Protein Target Primary antibody Secondary antibody DAX-1 Active Motif ThermoFisher (Cat # 39983) (Cat # A-11004) mouse anti-DAX-1 Alexa Fluor 568 goat anti-mouse IgG Dilution 1:250 Dilution 1:250 ERα Abcam ThermoFisher (Cat # 75635) (Cat # A-11034) rabbit anti-ERα Alexa Fluor 488 goat anti-rabbit IgG Dilution 1:100 Dilution 1:100 ERβ Abcam ThermoFisher (Cat # 3576) (Cat # A-11034) rabbit anti-ERβ Alexa Fluor 488 goat anti-rabbit IgG Dilution 1:100 Dilution 1:100

34 Vijayraghavan, Meghana

Results

Prostate tumor cells express NHR genes

Before the co-localization of DAX-1 and both ERs could be determined, it was important to first evaluate gene expression followed by protein expression of these

NHRs in prostate tumor cell lines, LNCaP and PC3. LNCaP cells are still hormone- naïve meaning they respond to the androgen-deprivation and stop proliferating in the absence of androgens. PC3 cells are aggressive CRPC cells that continue to proliferate despite the absence of androgens [84]. LNCaP cells and PC3 cells have differential gene and protein expression, thus it was necessary to investigate the presence of mRNA and protein expression of DAX-1, ERα, and ERβ within these cell lines. The expression of the GAPDH gene and GAPDH protein were used as positive controls for both mRNA and protein assays since it is stably expressed in mammalian cells. ERα and ERβ were also evaluated in the presence of over-expressed DAX-1 in order to assess whether increased levels of DAX-1 regulate gene or protein expression of ERs.

To evaluate mRNA expression, cells were treated with virus accordingly and then harvested for mRNA. The mRNA was reversed transcribed into cDNA and cDNA was evaluated via RT-PCR for the presence of the NR0B1, ESR1, ESR2, and GAPDH genes. The NR0B1 gene codes for DAX-1 while ESR1 and ESR2 code for ERα and

ERβ, respectively. PCR results indicated LNCaP cells constitutively express the NR0B1 gene at detectable levels with no differences between mock-treated and untreated cells.

Results also indicated that cells were permissible to infection with the DAX-1 adenovirus since NR0B1 had stronger expression when the LNCaP cells were exposed to the virus, meaning the DAX-1 adenovirus was successful in increasing levels of NR0B1

35 Vijayraghavan, Meghana expression in LNCaP cells. LNCaP cells were observed to express the ESR1 and ESR2 genes at low levels. Up-regulation of NR0B1 did not change the expression of the genes compared to untreated and mock-treated cells. GAPDH remained consistent across all treatments (Figure 2-1).

PCR results indicate PC3 cells normally express NR0B1 in low quantities, though infection with DAX-1 adenovirus indeed caused an up-regulation of NR0B1 production.

Furthermore, results suggested that no ERα was present in these cells due to a lack of signal of the ESR1 gene. However, ESR2 was detected in all cells despite treatment differences. ESR2 was not up-regulated when cells were infected with either DAX-1 adenovirus or LacZ adenovirus. GAPDH remained consistent across all treatments

(Figure 2-1). Overall, results indicate prostate cancer cell lines do express genes for

ERs and for DAX-1, supporting the model that DAX-1 binds ERs in PCa models.

36 Vijayraghavan, Meghana

Figure 2-1. Prostate cancer cell lines express NHR genes. RNA harvested from untreated, LacZ adenovirus treated, or DAX-1 adenovirus treated cells was reverse-transcribed to cDNA and analyzed for NR0B1, ESR1, and ESR2 expression. Results indicate hormone-naïve LNCaP cells expressed gene transcripts for the NHRs and higher levels of NR0B1 did not affect expression of ESR1 or ESR2. CRPC PC3 cells expressed NR0B1 at low levels though gene expression was increased following treatment with DAX-1 adenovirus. PC3 cells did not express ESR1 but consistently expressed ESR2, regardless of treatment. GAPDH was used as a positive control and was consistently expressed across all cell types and treatments.

37 Vijayraghavan, Meghana

DAX-1 co-localizes with ERα and ERβ within PCa cell lines

Following the detection of the NR0B1, ESR1, and ESR2 genes, respective protein expression was determined in LNCaP and PC3 cells. The goal of these experiments was to verify protein expression matched mRNA expression suggested in the PCR experiments. Cells were untreated, LacZ adenovirus treated, or treated with

DAX-1 adenovirus for 72 hours after which lysates were collected. Proteins were separated via SDS-PAGE and immunoblotted using antibodies specifically against

DAX-1, ERα, and ERβ. GAPDH was used as a positive loading control. Surprisingly, the western blot results partially contradicted the PCR results. The western blots suggested that in LNCaP cells, there is very little to almost no expression of DAX-1 unless the cells are treated with DAX-1 adenovirus. This result contrasted the expected result (based on the PCR assays) that DAX-1 protein would be more detectable in the untreated and mock-treated samples. In agreement with the PCR results, LNCaP cells expressed similar levels of ERα and ERβ, regardless of the overexpression of DAX-1. GAPDH was consistent across all cell treatments (Figure 2-2). Protein detection in PC3 lysates was minimal, barring GAPDH protein detection. A band around 50 kDa corresponding to

DAX-1 was faintly observed only in the cells treated with DAX-1 adenovirus. There was no detectable DAX-1 protein in the untreated or LacZ adenovirus treated samples which was to be expected based on the minimal expression of NR0B1 from the PCR results.

No ERα signal was observed in any PC3 samples which corresponded to the PCR results of ESR1. ERβ was faintly detected in across all samples, although higher expression was expected (Lau 2000). GAPDH was equally expressed across all cell treatments (Figure 2-2). While these results were an important first step in determining

38 Vijayraghavan, Meghana

DAX-1, ERα, and ERb expression in PCa cell lines, they were inconsistent with the earlier PCR results suggesting the need for resolution. Published studies have noted discrepancies between PCR and western blots and suggest differing rates of mRNA translation in cell lines or lack of protein stability in certain cell lines are to blame [85-

86]. Perhaps ERβ is regulated at the protein level in PCa cell lines which is why this study utilized immunofluorescence microscopy to clarify expression in these PCa cell lines.

39 Vijayraghavan, Meghana

Figure 2-2. Prostate cancer cell lines express NHR proteins. Cells were untreated, LacZ adenovirus treated, or DAX-1 adenovirus treated for 72 hours after which lysates were collected. Protein detection occurred by immunoblotting using antibodies specifically against DAX-1, ERα, and ERβ. GAPDH acted as a loading control.

40 Vijayraghavan, Meghana

Immunofluorescence microscopy (IF microscopy) was performed as an alternative way of detecting proteins as well as a means of determining the localization of specific NHRs within PCa cell lines. LNCaP or PC3 cells were grown on coverslips and were uninfected or treated with either LacZ adenovirus or DAX-1 adenovirus for 48-

72 hours. After treatment, cells were fixed, permeabilized, and probed for DAX-1 and either ERα or ERβ. DAX-1 was stained using Alexa Fluor 568 (red fluorescence) while

ER was stained using Alexa Fluor 488 (green fluorescence). DAPI (blue fluorescence) was used to stain the nuclei of cells. In contrast to the gene expression and protein expression results, the IF microscopy results suggest DAX-1 and both ERs are expressed within both PCa cell lines.

In untreated LNCaP cells, DAX-1 had low basal expression in the nuclei which was to be expected given the results from the earlier western blot experiments. As expected, when treated with DAX-1 adenovirus, the expression of DAX-1 was up- regulated. DAX-1 was seen to be primarily localized to the nuclei of the cells and had even dispersal throughout the nuclei. LacZ adenovirus treated LNCaP cells did not differ from untreated cells indicating the infection of adenovirus did not interfere with DAX-1 expression or localization (Figure 2-3). LNCaP cells also exhibited strong expression of both ERa and ERβ protein. Both ERs demonstrated a localization preference to the cytoplasm of the cells, though both ERs were seen in the nuclei of the cells as well

(Figure 2-3). ERβ had less of a presence in the nuclei compared to ERα. Additionally,

ERβ had a distinct punctate dispersal pattern in the cytoplasm compared to ERα suggesting that ERβ was sequestered specific locations in the cell more so than its counterpart (Figure 2-3B). When captured images were merged (along with the DAPI

41 Vijayraghavan, Meghana images for nuclei reference), DAX-1 and both ERs indeed co-localized partially in the cytoplasm but mostly in the nuclei of the LNCaP cells. This is the first documentation of this observation.

The localization pattern of DAX-1 in LNCaP cells was observed to be similar in

PC3 cells (Figure 2-4). Again, DAX-1 primarily localized to the nuclei of PC3 cells, though there was residual expression in the cytoplasm of the cells. When DAX-1 was overexpressed via adenovirus, it was apparent DAX-1 preferentially localized to the nuclear compartment of the cells. In contrast to the LNCaP cells, PC3 cells exhibited fairly equal ERα expression across the cells (Figure 2-4A). While in LNCaP cells, ERα showed a clear affinity for cytoplasmic localization, in PC3 cells ERα only exhibited a slight affinity for the cytoplasm over the nucleus. ERβ expression in PC3 cells was similar to expression in LNCaP cells. Once again, ERβ had less of a presence in the nuclei compared to ERα. It also had more punctate dispersal in the cytoplasm compared to ERα. In contrast to ERβ in LNCaP cells, an increased amount of ERβ was present in the nuclei of PC3 cells (Figure 2-4B). When images were merged, DAX-1 and both ERs again co-localized partially in the cytoplasm but mostly in the nuclei of the

PC3 cells. The results from these IF microscopy experiments are the first evidence of

ERα, ERβ, and DAX-1 co-localization in prostate cancer cell lines.

42 Vijayraghavan, Meghana

43 Vijayraghavan, Meghana

Figure 2-3. DAX-1 and ERs co-localize in the nuclei of LNCaP cells. Immunofluorescence microscopy was performed on LNCaP cells to detect the localization of DAX-1 and ERα (A), and DAX-1 and ERβ. DAPI was used to stain the nuclei of cells. A) ERα was localized primarily to cytoplasm of the cells, though it was detected in the nuclei of cells. DAX-1 was localized to the nuclei of cells. DAX-1 and ERα were co-localized in the nuclei. B) ERβ was localized primarily to cytoplasm of the cells but was also detected in the nuclei. DAX-1 was localized to the nuclei of cells. DAX-1 and ERβ were co-localized in the nuclei. Scale bars = 20 μm

44 Vijayraghavan, Meghana

45 Vijayraghavan, Meghana

Figure 2-4. DAX-1 and ERs co-localize in the nuclei of PC3 cells. Immunofluorescence microscopy was performed on PC3 cells to detect the localization of DAX-1 and ERα (A), and DAX-1 and ERβ (B). DAPI was used to stain the nuclei of cells. A) ERα was dispersed almost evenly throughout the cells, though it did show slight preference for the cytoplasm of cells. DAX-1 was localized to the nuclei of cells. DAX-1 and ERα were co-localized in the nuclei. B) ERβ was localized primarily to cytoplasm of the cells but was also detected in the nuclei. DAX- 1 was localized to the nuclei of cells. DAX-1 and ERβ were co-localized in the nuclei. Scale bars = 20 μm

46 Vijayraghavan, Meghana

Discussion

This study explored the expression and localization of the NHRs, DAX-1, ERα, and ERβ within human PCa cell lines. Two commonly used PCa cell lines were chosen based differential response to ADT. The first cell line, LNCaP, is known to express AR in the cytoplasm of cells [87]. The cell line demonstrates a decrease in proliferation when androgens are removed making it a good model for hormone-naïve PCa. The second cell line, PC3, has also been shown to express AR [88], but does not stop proliferating despite a lack of androgens in the environment. This cell line is widely observed to be an example of CRPC, which is nearly impossible to treat. Because the two PCa cell lines behave differently in response to hormone treatments, it is important to characterize the statuses of NHRs and later, the mechanisms of NHRs since there are likely differences between hormone-naïve and castration-resistant PCa tumors which could be exploited for drug therapeutics.

This study focused the on the localization of ERs since there is controversy around ER presence and mechanisms within PCa tumors. Additionally, we focused on the localization of DAX-1, an orphan NHR that is known to bind to ERs and AR and whose up-regulation is correlated with a more favorable prognosis of PCa [69]. PCR was initially used to detect mRNA transcripts corresponding to each NHR in LNCaP and

PC3 cells. Results indicated LNCaP cells expressed mRNA transcripts pertaining to the

DAX-1, ERα, and ERβ genes. Despite clear DAX-1 up-regulation via adenovirus, the levels of ERα and ERβ mRNA transcripts qualitatively remained similar to levels of mRNA in untreated cells suggesting that under these conditions, DAX-1 does not induce nor suppress transcription of estrogen receptors. This is unexpected because

47 Vijayraghavan, Meghana unpublished findings from our lab suggest DAX-1 down-regulates AR expression via an

AR negative feedback loop. The study suggests activated AR increases transcription of the NR0B1 gene and once the mRNA transcripts are translated, the DAX-1 protein acts as co-repressor to AR. Our results with ER do not indicate DAX-1 and ER are involved in a feedback loop.

PCR results in PC3 cells support the observations seen in the LNCaP cells. The untreated and mock-treated PC3 cells expressed low levels of DAX-1 mRNA while the

DAX-1 adenovirus treated cells had higher levels of DAX-1 mRNA. Despite an up- regulation in DAX-1, PC3 cells did not qualitatively exhibit an up-regulation in ERβ mRNA. However, these results would be better supported with quantitative data via quantitative PCR (qPCR) reactions. Curiously, and in contrast to previous studies, no

ERα mRNA was detected within PC3 cells [70, 89]. Possible explanations for this discrepancy include the use of different PCR primers or differences in PCR conditions that do not detect low expression levels.

Because of the discrepancy in our PCR results compared to previous studies, protein detection methods of immunoblotting and IF were used to confirm the results and to add to the general understanding in terms of expression and localization patterns of DAX-1, ERα, and ERβ within the chosen PCa cell lines. Immunoblotting results suggested LNCaP cells expressed all three NHRs while PC3 cells did not express ERα, and only slightly expressed DAX-1 and ERβ. Interestingly, the IF microscopy results in

PC3 cells contradicted our immunoblotting results. The results demonstrated the presence of DAX-1, ERα and ERβ proteins within the cytoplasm and the nuclei of the cells. These results are relevant in terms of the known literature because there are

48 Vijayraghavan, Meghana conflicting results of ERa protein expression within PC3 cells. One study detected ERα protein via immunoblotting while another study used the same technique but did not find evidence of ERα expression [89-90]. It is worth noting numerous studies have suggested ERα is up-regulated in prostate cancer tissues as the cancer becomes progressively worse [74-76]. Therefore, the detection of ERα expression in PC3 cells, a cell line derived from an aggressive form of PCa, aligns with these previously published studies.

It is less clear what the role of ERβ is in PCa. Results demonstrate ERβ expression in the cytoplasm and partially in the nuclei of both LNCaP and PC3 cells.

While there are published studies that have demonstrated the presence of ERβ within these cells [89-90], other studies have demonstrated the loss of ERβ in prostatic tissue as PCa progresses [47; 91-92]. Therefore, it is interesting to note sustained ERβ expression in the CRPC cell line, PC3. Recent studies have suggested perhaps ERβ is actually sustained in CRPC tissues and isoforms of ERβ have an impact on cell physiology and behavior [93-95]. In light of these studies, our results support the observations that ERβ expression does not disappear as PCa progresses and in fact, is sustained even in CRPC cell lines.

While ER expression and localization have been extensively profiled in PCa cell lines, few studies have focused on DAX-1 expression and localization in PCa cells. This study found that DAX-1 was mainly localized to the nuclei of hormone-naïve and CRPC cells, though the protein can also be detected in low levels in the cytoplasm. These results echo the results of other studies that found DAX-1 localized mainly to the nuclei of other types of cancer cells and tissues [96-97]. More importantly, this study

49 Vijayraghavan, Meghana suggested the novel finding that DAX-1 is co-localized with both ERα and ERβ regardless of the severity of PCa. The implication of this finding is that because DAX-1 is expressed in the same place at the same time as ERs, the orphan NHR can potentially bind ER in vivo and mediate the negative effects of ER signaling with PCa cell lines. These findings indicate there is the potential to develop alternative therapies for PCa that function to increase the expression of DAX-1 as a means of inhibiting cell proliferation and tumor growth.

50 Vijayraghavan, Meghana

Chapter 3

Validation of DAX-1 and ER protein interaction

Introduction

In order to evaluate the effects of DAX-1 coupled with ER in PCa, it was necessary to elucidate the mechanisms behind these NHR interactions. It has previously been determined that the ability of DAX-1 to directly bind a variety of transcription factors is through the LXXLL motifs present in its N-terminus of DAX-1. Of particular interest are those transcription factors in the same family as ER. DAX-1 has demonstrated binding affinity for: ER within COS-7 cells [60], AR within LNCaP and

COS-7 cells [61-62], progesterone receptor within T47D breast cancer cells [62], within COS-1 cells and A549 lung cancer cells [98], and estrogen receptor-related receptor gamma within MCF-7 breast cancer cells [99]. In these reported studies, DAX-1 was shown to act as a co-repressor by binding to the

AF2 sites in the various nuclear receptors. Typically, DAX-1 was overexpressed via transient transfection of a DAX-1 plasmid in order to increase the amount of DAX-1 present in these assays. Different techniques, including GST (glutathione-S- transferase) pull-down assays and co-immunoprecipitation assays (Co-IPs), were employed to determine whether a physical interaction of DAX-1 and the nuclear receptor of interest existed. Though all of the aforementioned studies are over a decade old, the techniques used are still relevant and still the gold standard to detect protein- protein interactions within mammalian cell models.

For this study, GST pull-down assays and co-IPs were used in order to evaluate

DAX-1 and ERα interaction within LNCaP cells. To date, data by Zhang et. al

51 Vijayraghavan, Meghana demonstrating the interaction between DAX-1 and ERα in addition to DAX-1 and ERβ within COS-7 cells remains the only published work to have looked at the DAX-1/ER interaction. Therefore, this current study will contribute to our understanding of NHR interactions within cancer cells.

52 Vijayraghavan, Meghana

Materials and Methods

GST Pull-down Assays

Competent BL21 (DE3) E.coli cells (New England Biolabs; Ipswich, MA) were thawed on ice in 50 μL aliquots and transformed with 100 ng of either GST-EGFP control vector (henceforth referred to as GST-only) or custom-made GST-DAX-1 vector expressing the T7 promoter (Genecopoeia; Rockville, MD) according to the NEB protocol. Transformed BL21 cells was plated at 25 μL on separate LB + Ampicillin (100 ug/mL ampicillin) plates and grown overnight at 37°C. The next day, individual colonies were picked and added to individual starter cultures of 5 mL of LB-Ampicillin broth

(1:1000 ampicillin) and grown overnight at 37°C with shaking. The following day, 500 mL of LB-Ampicillin broth was inoculated with the 5 mL starter culture and grown at

37°C in a shaker until an optical density of at least 0.500 was reached. The cultures were then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for a final concentration of 0.2 mM IPTG. Induced cultures were grown for an additional 4 hours at

30°C with shaking. Cultures were then aliquoted into 50 mL conical vials and centrifuged at twice at 3500 x g for 20 minutes at 4°C to pellet the cells. The pellets were then stored at -20°C for at least 60 minutes prior to lysis.

In order to lyse bacterial cells and purify either GST-only protein or GST-DAX-1 protein, pellets were thawed for 10 minutes on ice, centrifuged at 7500 x g for 10 minutes at 4°C to get rid of any residual supernatant and to concentrate the pellet, resuspended in 2 mL of 1X PBS-Lysis (PBS-L) buffer (1X PBS, 1X HALT protease inhibitor, 1% Triton X-100, 1 mM EDTA, 1 mg/mL lysozyme), and then sonicated to further disrupt the membranes. Lysate was then centrifuged at 10,000 x g for 10

53 Vijayraghavan, Meghana minutes at 4°C. Supernatant was transferred 1.5 mL ftubes and either used immediately or stored at -20°C.

To immobilize the bait protein, a 50% slurry of Pierce Glutathione Agarose beads

(Thermo Fisher Scientific) was prepared in a solution of 1X PBS with 1X HALT protease inhibitor. A solution of 50 μL 50% slurry and either 300 μL GST-only or GST-DAX-1 was incubated overnight at 4°C in a rotator to bind the protein to the agarose beads. After the pre-bind step, the beads were centrifuged at 5000 x g for 2 minutes at 4°C and the supernatant was decanted. The beads were then resuspended in ~50 μg of untreated

LNCaP lysate. If E2 treatment was appropriate, E2 was added for a final concentration of

1 μM. Suspensions were incubated for 4 hours at 4°C in a rotator. After binding, the beads were centrifuge at 5000 x g for 2 minutes at 4°C. Supernatant was discarded at the beads were washed 5 times with 500 uL 1X PBS. In between washes, beads were centrifuged at 12000 x g for 15 minutes at room temperature and supernatant decanted via pipetting. After the final wash, the beads were prepared for protein separation via

SDS-PAGE. Each sample was resuspended in 32.5 μL 1X PBS, 12.5 μL of 4X NuPAGE sample buffer, and 5.0 μL of 10X NuPAGE reducing agent (Thermo Fisher Scientific).

Samples were then heated at 70°C for 10 minutes to elute the proteins off the beads then centrifuged at 10,000 x g for 2 minutes at room temperature. The supernatant was loaded and electrophoresed and transferred to a PVDF as described in the Western

Blot methods in chapter 2. DAX-1 and ERα were probed for using the primary and secondary antibodies at the dilutions detailed in Chapter 2 (Table 2-3, Table 2-4).

54 Vijayraghavan, Meghana

Co-Immunoprecipitation assays

Co-immunoprecipitation assays (co-IPs) were performed using the Dynabeads

Co-Immunoprecipitation kit (Invitrogen) according to the manufacturer’s protocol. Briefly,

DAX-1 antibody (Active Motif) was covalently coupled to the Dynabeads M-270 Epoxy beads at a ratio of 6 ug antibody/mg beads. The bead-antibody complex was assembled so as to use 1.5 mg/ co-IP reaction. Antibody coupling procedure followed the manufacturer’s protocol. Coated beads were used immediately or were stored at

4°C for no more than one week following antibody coupling.

Two confluent 15 cm dishes of LNCaP cells were seeded and left untreated or treated with LacZ adenovirus for 72 hours. After treatment, cells were washed twice with

1X PBS and harvested using 0.25% Trypsin-EDTA. Cells were transferred to 15 mL conical vials, centrifuged at 300 x g for 5 minutes, washed once with 1X PBS, and centrifuged again at 300 x g. Supernatant was decanted and cell pellets were gently suspended in Dynabeads Extraction Buffer A supplemented with 100 mM NaCl, and 1X

HALT protease inhibitor according to the manufacturer’s recommendation. Lysates were placed on ice for 15 minutes then centrifuged at 2600 x g for 5 minutes at room temperature to separate large cell debris. The supernatants were transferred to 1.5 mL microcentrifuge tubes and immediately used in co-IP reactions. In each co-IP reaction,

1.5 mg of antibody-coupled beads was added to fresh lysate and incubated for 45 minutes on a rotator at 4°C. After incubation, beads were concentrated in the tube using a magnet. The supernatant was transferred to a 1.5 mL microcentrifuge tube labeled

“Flow-through.” Beads were washed three times with 200 uL extraction buffer. After every wash, the beads were collected using a magnet and the supernatant was

55 Vijayraghavan, Meghana transferred to a 1.5 mL microcentrifuge tube labeled “3X washes”. The co-IP reactions were then washed one final time using the Dynabeads “last wash buffer” supplemented with 0.02% Tween-20 and beads were collected and supernatant was discarded. The beads were suspended in 60 μL of the Dynabeads EB buffer and incubated on a rotator for 15 minutes at room temperature. The beads were collected using a magnet and the supernatants were transferred to clean 1.5 mL microcentrifuge tubes labeled “eluted protein.” The tubes containing the beads were labeled “beads only” and contents were directly prepared for analysis via SDS-PAGE. Co-IPs were analyzed via western blot using conditions outlined in chapter 2. Samples were prepared using 16.25 μL protein sample, 6.25 μL 4X NuPAGE sample buffer, and 2.5 μL 10X NuPAGE reducing agent.

Immunoblots were probed using antibodies against ERα, AR, or DAX-1 (Table 3-1,

Table 3-2).

56 Vijayraghavan, Meghana

Table 3-1. Primary antibodies used for western blot analysis of co-IP reactions

Protein Target Animal of origin Brand Dilution DAX-1 Mouse monoclonal Active Motif 1:1000 (Cat # 39983) ERα Rabbit monoclonal MilliporeSigma 1:1000 (Cat # 04-820) AR Rabbit polyclonal GeneTex 1:1000 (Cat # GTX100056)

Table 3-2. Secondary antibodies used for western blot analysis of co-IP reactions

Probing against Animal of origin Brand Dilution HRP goat anti-mouse Goat BD Pharmigen 1:1000 IgG (Cat # 554002) HRP goat anti-rabbit Goat GeneTex 1:1000 (for ERα and IgG (Cat # GTX213110-01) AR)

57 Vijayraghavan, Meghana

Results

Determination of DAX-1 and ER interaction

This study used two methods, GST pull-down assays and co-IPs, to investigate whether a direct protein-protein interaction between DAX-1 and ERα exists within

LNCaP cells. Before all interactions in the desired cell lines were explored, protocols were optimized using one cell line (LNCaPs) and primarily focused on the DAX-1/ERα interaction. For the GST pull-down assays, purified GST-only protein or GST-DAX-1 protein was bound to glutathione-agarose beads and incubated with LNCaP lysate that was either untreated or treated with 1 μM E2. The complex was then isolated, separated via SDS-PAGE, transferred to a PVDF membrane and probed for DAX-1 and ERα.

When probing for DAX-1, bands approximately 52 kDa were detected in every lane except for the GST-only lane (Figure 3-1). When comparing GST-only lanes with

LNCaP lysate to GST-DAX-1 lanes with LNCaP lysate, bands were detected that migrated at a lower molecular weight than the bands detected in the GST-DAX-1 lanes.

It was also observed that there were two bands in the GST-DAX-1 + lysate lanes compared to the GST-only + lysate lanes. The lower bands were approximately the same molecular weight as the bands in the GST-only lanes (Figure 3-1, lower arrow).

We speculate that the bands seen at the lower size in both the GST-only and the GST-

DAX-1 lanes were residual DAX-1 protein from the lysate indicating more stringent washes were needed after the beads and complexes were pulled down. This conclusion is supported by the lack of band in the GST-only lane, indicating that there was no unspecific binding of the antibody that would have given rise to the bands seen in the

GST-only lanes.

58 Vijayraghavan, Meghana

When probing for ERα, bands around 66 kDa were detected in every lane exposed to LNCaP lysate (Figure 3-1). Interestingly, the lanes with GST-DAX-1 had faint bands of ERα when compared to the GST-only lanes, regardless of ERα activation via E2. Even more curious, GST-DAX-1 incubated with LNCaP lysate produced a fainter band compared to GST-DAX-1 with lysate and E2 treatment. While these results seem to indicate there is a difference in ERα to DAX-1 in the presence of hormone, further optimization of the GST binding experiments is needed to provide conclusive evidence of this interaction.

59 Vijayraghavan, Meghana

Figure 3-1. GST pull-down assays using LNCaP lysate. GST-only and GST-DAX-1 proteins were purified and bound to glutathione agarose beads. The beads were exposed to either LNCaP lysate alone or LNCaP lysate and 1uM E2 then pulled out of solution, washed, and proteins dissociated off the beads via heat. The dissociated proteins were separated via SDS- PAGE and probed for DAX-1 and ERα. Results indicate GST-DAX-1 was purified properly as bands against DAX-1 protein were expressed around the predicted molecular weight of 78 kDa (GST = 26 kDa, DAX-1 = 52 kDa). Bands around 58 kDa were thought to be residual DAX-1 protein expressed in the LNCaP lysate that had not been thoroughly washed away from the beads.

60 Vijayraghavan, Meghana

In addition to the GST pull-down assays, co-immunoprecipitation experiments aimed at examining the interaction of DAX-1 with ERα were performed. The anti-DAX-1 antibody was covalently coupled to magnetic beads and incubated with freshly lysed

LNCaP lysate that was either untreated or treated with LacZ adenovirus. The goal of these experiments was to examine endogenous protein-protein interactions, therefore there was no exogenous DAX-1 expression via adenovirus or upregulation of ER via E2 treatment. Lysate was collected from four points in the assay: the flow-through after the beads had been exposed to LNCaP lysate, the wash buffer used to rinse the exposed beads, the beads themselves which were re-suspended in SDS-PAGE sample buffer, and the eluted protein complexes. Through these experiments, it was discovered that

ERα was only expressed in the flow-through lysate (Figure 3-2). Since previous experiments suggested DAX-1 physically interacted with AR in LNCaP cells, the immunoblot was probed with an AR antibody. Results show faint bands (marked by arrows) in the eluted protein lanes of both untreated and LacZ adenovirus treated

LNCaP cells, indicating the positive control was detected and the assay itself worked

(Figure 3-2).

61 Vijayraghavan, Meghana

Figure 3-2. Co-immunoprecipitation assays using anti-DAX-1 coupled beads and LNCaP lysate. DAX-1 antibody was covalently coupled to magnetic beads. The beads were exposed to either untreated LNCaP lysate or LacZ adenovirus treated LNCaP lysate. At various points in the assay, samples were collected and analyzed via SDS-PAGE and immunoblot. ERα was only detected in the flow-through after the covalently coupled anti-DAX-1 beads were exposed to lysate. No ERα was detected in any other lane. AR, a known binding partner of DAX-1, was detected in both the flow-through and in the purified, eluted protein sample (indicated by the arrows at roughly 110 kDa) demonstrating DAX-1 had the ability to bind another NHR under these assay conditions.

62 Vijayraghavan, Meghana

Discussion

This portion of thesis work aimed to clarify the protein-protein interaction of DAX-

1 and ER within PCa cell lines. Previous research by Zhang et al. indicated DAX-1 utilizes the repeating LXXLL motifs to bind to the AF2 domain of ERα and ERβ. This complex was determined to exist using three methods: GST pull-down assays, co-IPs, and yeast two-hybrid assays. The Zhang study then went on to characterize the functional effects of DAX-1 binding to ER and found that DAX-1 repressed transcriptional activity of ERα and ERβ in a dose-dependent manner [60]. This thesis work aimed to determine whether this interaction and functional effect was present within PCa cell lines. Specifically, these studies focused on detecting an interaction between DAX-1 and ERα in LNCaP cells.

Initially, GST pull-down assays were performed and results indicated that GST- only and GST-DAX-1 proteins were successfully purified. However, the results from these assays were not conclusive due to the presence of DAX-1 in samples, most likely due to low-stringency washes, suggesting that it is necessary to increase the number of stringent washes when performing the assay to minimize residual DAX-1 binding. When probing for ERα in the GST pull-down assays, higher ERα expression in the GST-only lanes compared to the GST-DAX-1 lanes was observed. This indicates that more stringent washes were needed in order to determine whether the GST-only beads were actually capturing the ERα or whether expression was just an artifact of non-specific binding. Interestingly, the results from the GST-DAX-1 construct indicate direct binding to ERα which increased upon exposure to E2. These results support previous results

(unpublished) from our research lab that demonstrate DAX-1 binds directly to ERα in

63 Vijayraghavan, Meghana vitro. Further optimization of the GST pull-down experiments needs to be conducted before any conclusions can be drawn.

As an alternative to the GST pull-down assays, co-IPs were used to evaluate the

DAX-1/ER interaction within LNCaP lysate. The results from these experiments indicate

DAX-1 does not bind with ERα. However, further optimization needs to be performed.

When AR was used as a positive control for DAX-1 interaction, only a faint band was detected, but not what was expected given that other studies have found a moderate amount of AR interacting with DAX-1 [62]. In our case, the lysis buffer used to extract proteins from the LNCaP cells may have disrupted the protein-protein interactions, which hindered our ability to detect the interaction. Therefore, further optimization is required. One possible approach could be to overexpress DAX-1 and ERs either through transient transfection with expression plasmids for DAX-1, ERa and ERb through the use of adenovirus. While this approach doesn’t address endogenous protein-protein binding, it is still a viable way of detecting whether DAX-1 has the ability to bind ERs within PCa cell lines.

Should future studies find that DAX-1 and ERs interact within PCa cell lines, functional assays determining the effect of DAX-1 on activated ER activity will have to be performed. Though the functions of ERs within PCa are still highly debated, it is expected that overexpressing DAX-1 will repress the transcriptional activity of the ERs causing PCa cells to halt in proliferation and metastasis. Therefore, this work was a necessary first step in determining whether DAX-1, ERα, and ERβ are viable therapeutic options for prostate cancer patients that remain without any other hormone- dependent options for treatment.

64 Vijayraghavan, Meghana

Chapter 4

Concluding remarks

The rise and progression of prostate cancer involves a complex network of biological processes and protein interactions that are mainly driven via the nuclear hormone receptor, androgen receptor. Thus, the most common targeted treatment for prostate cancers involves depleting the body of androgens so androgen receptor is never activated. However, this treatment, known as androgen-deprivation therapy, eventually fails and prostate cancer cells continue to proliferate and metastasize.

Presently, there is no other viable option for hormonal treatment due to the lack of research involving other nuclear hormone receptors in the context of prostate cancer.

Therefore, this thesis work aimed to elucidate the presence and location of three nuclear hormone receptors, DAX-1, ERα, and ERβ, within prostate cancer cells lines.

Studies have demonstrated DAX-1 is present in prostatic tissues, but the level of

DAX-1 protein in tissues is variable depending on the patient. DAX-1 is known to act as a co-repressor of other nuclear hormone receptors, but its relationships to ERs within prostate cancer cells has never been explored. ERα is classified as an oncogenic protein that mediates proliferation, inflammation, and carcinogenesis of prostate cells.

ERβ has a more controversial role within PCa with some studies demonstrating its anti- oncogenic properties while other studies dispute earlier findings and suggest ERβ also mediates an increase in proliferation of castration-resistant prostate cancer cells.

Because of the potential effects that these three NHRs have on prostatic carcinogenesis, the research conducted throughout this thesis aimed to clarify the

65 Vijayraghavan, Meghana physical interactions between nuclear hormone receptors, ERα, ERβ and DAX-1, all of which have the potential to be therapeutic targets for new treatments of prostate cancer.

The first portion of this thesis work, as detailed in Chapter 2, identified gene and protein expression of DAX-1, ERα, and ERβ in two different PCa cell lines, an androgen-dependent line known as LNCaP and an androgen-resistant line known as

PC3. Results of PCR experiments indicated that LNCaP cells expressed gene transcripts for all the NHRs. PC3 cells expressed the DAX-1 gene at low levels and also expressed the ERβ gene. This cell line had no detectable gene expression for ERα.

When DAX-1 was over-expressed in both cell lines via adenovirus, the gene expression levels of ERα and ERβ did not change, suggesting that DAX-1 plays no role in regulating gene expression of both ERs. Once gene expression was evaluated, protein expression was analyzed via immunoblotting. Surprisingly, DAX-1 protein expression in

LNCaP cells partially contradicted expected results indicated by PCR experiments as

DAX-1 was only detected in samples that had been treated with adenovirus. This suggests that in LNCaP cells, DAX-1 may be regulated at the DNA or RNA level and not be translated into protein. However, in agreement with the PCR results, LNCaP cells did express both ERs and ER expression was not dependent on DAX-1 expression.

Detection of NHR proteins in PC3 lysates was minimal, with one faint band detected when DAX-1 was overexpressed via adenovirus. ERβ was faintly detected across all conditions, but no ERα was detected. To further investigate the pattern of NHR expression in PCa cells, a second protein-detection technique was employed. Using immunofluorescence microscopy, it was determined that all three NHRs were expressed in both LNCaP and PC3 cells. These finding indicate that DAX-1 is able to

66 Vijayraghavan, Meghana co-localize with ERα and ERβ within each cell line, regardless of cell line androgen sensitivity.

The findings detailed in Chapter 2 were used to guide the work detailed in

Chapter 3. In summary, the interaction of the various NHRs were explored via GST pull- down assays and co-immunoprecipitation assays. Although two independent approaches were utilized, the results remain inconclusive. Future work will be focused on refining the co-IP assays and examining the functional effects of DAX-1 and ER binding. It is still strongly theorized that DAX-1 binds both ERα and ERβ within prostate cancer cell lines and this interaction may be utilized to mediate anti-proliferative effects.

67 Vijayraghavan, Meghana

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