The Prostate 74:1240^1248 (2014)

Dihydrotestosterone Enhances Castration-Resistant Prostate Cancer Cell Proliferation Through STAT5 Activation via Glucocorticoid Receptor Pathway Cheryn Song,1 Yunlim Kim,1 Gyeong Eun Min,2 and Hanjong Ahn1* 1 2

Department of Urology,Universityof Ulsan College of Medicine, Asan Medical Center, Seoul, Korea Department of Urology, Kyung Hee University College of Medicine,Gangdong Hospital, Seoul, Korea

INTRODUCTION. We aimed to evaluate STAT5 expression and cell proliferation change after dihydrotestosterone (DHT) treatment in castration-resistant prostate cancer (CRPC) cells to elucidate the mechanism in relation to different androgen receptor (AR) expression status. METHODS. Using DU145, PC3, and LNCaP cells, cell viability assay and Western blot for phosphorylated STAT5 (p-STAT5) were done after DHT treatment at various concentrations. Endogenous levels of nuclear hormone receptor mRNA and protein were identified using realtime RT-PCR and Western blot. We treated the cells with RU486 and then glucocorticoid receptor (GR)-specific small interfering RNA (siRNA), to assess change in DHT-induced STAT5 activation. Immunofluorescence staining of DU145 cells with anti-GR and anti-pSTAT5 Ab before and after DHT treatment was done and visualized. RESULTS. DHT treatment enhanced STAT5 phosphorylation and promoted proliferation of all CRPC cells. Endogenous GR was identified strongly in DU145, weakly in PC3 but not in LNCaP cells. AR was identified strongly in LNCaP but not in DU145 cells. RU486 treatment abolished DHT-induced cell proliferation and STAT5 activation in both DU145 and PC3 cells but not in LNCaP cells. Similarly, GR-specific siRNA completely suppressed STAT5 activation. On immunofluorescence, activation of STAT5 and GR translocating into the nucleus after DHT treatment was confirmed. Immunoprecipitation confirmed direct complex formation between the GR and pSTAT5. CONCLUSION. In CRPC cells, DHT activated STAT5 enhancing cell proliferation. Activation was induced regardless of presence of AR and in cells devoid of AR, DHT used GR which formed direct complex with p-STAT5. Prostate 74:1240–1248, 2014. # 2014 Wiley Periodicals, Inc.

KEY WORDS: castration-resistant; prostate neoplasm; STAT5; glucocorticoid receptor; androgen receptor; dihydrotestosterone

INTRODUCTION Prostate cancer (PC) is the most common nonskin cancer and the second leading cause of cancer-related deaths in men in the United States [1]. With the widespread use of the tumor marker prostate-specific antigen (PSA), proportion of advanced disease at diagnosis has significantly reduced; yet, together with recurrent disease after definitive therapy a substantial number of patients still remains in need of treatment for advanced PC. As most PC is initially androgendependent for cell survival, the most commonly used form of treatment for the advanced disease is androß 2014 Wiley Periodicals, Inc.

Grant sponsor: Asan Institute for Life Sciences; Grant number: 2010499. Conflict of interest: For all of the authors, there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Correspondence to: Hanjong Ahn, MD, PhD, Department of Urology, University of Ulsan College of Medicine, Asan Medical Center, 86 Asanbyungwongil, Songpa-gu, 138-736 Seoul, Korea. E-mail: [email protected] Received 26 September 2013; Accepted 28 May 2014 DOI 10.1002/pros.22841 Published online 7 July 2014 in Wiley Online Library (wileyonlinelibrary.com).

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is ideally suited for androgen signaling studies. All of the cells used in the experiment were in their three to five passages after obtaining from the ATCC. Our 10 mM stock solutions were prepared by dissolving 5-alpha-DHT (Sigma, St. Louis, MO), dexamethasone (Dex; Sigma) and the GR antagonist RU486 (Sigma) in ethanol. Working concentrations were freshly prepared daily by diluting the stock solution with 1% charcoal stripped serum containing phenol red-free RPMI 1640. Anti-phospho-STAT5 (Tyr694) (pSTAT5; Cell Signaling, Danvers, MA), anti-GR (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoJAK2 (Santa Cruz Biotechnology), and anti-b-actin (Santa Cruz Biotechnology) primary antibodies were used for Western blot analysis.

gen-deprivation therapy (ADT). However, sensitivity or dependence on androgen changes after a median treatment of 24 months, and almost all invariably progresses to androgen-independent or castrationresistant state. The definition for castration-resistant prostate cancer (CRPC) is rising PSA, radiographic cancer progression, and/or worsening of clinical symptoms with castrate levels of serum testosterone [2,3]. Several mechanisms of PC cells developing castration resistance have been proposed which include increased androgen uptake or increased expression of key enzymes involved in androgen synthesis [4,5], but changes in androgen receptor (AR) are considered most critical and frequent. These include AR amplification [6], mutation leading to AR splice variants [7], or increased sensitivity [8,9]. Signal transducer and activator of transcription 5 (STAT5) is a member of STAT family of transcription factors and its role in PC cell survival, differentiation, progression, and metastasis is increasingly gaining attention. STAT5 expression in human PC tissues correlates with histologically poor differentiation predicting early recurrence after radical prostatectomy [10,11]. Tissue expression of STAT5 in advanced PC also correlates with progression to androgenindependent state, its expression increasing after ADT and further after becoming CRPC [12]. Functional and direct interaction between the STAT5 and AR has been demonstrated suggesting a putative role in the process of PC becoming castration-resistant. However, structural as well as functional integrity of the AR’s in different types of CRPC cells are highly variable. The objectives of the current study were to evaluate STAT5 expression and cell survival changes after dihydrotestosterone (DHT) treatment in CRPC cells to elucidate the mechanism in relation to different expression status of AR. We show that in CRPC cells without functioning AR, DHT enhances cell proliferation by activating STAT5 pathway, which in turn promoted CRPC cell survival, by directly interacting with glucocorticoid receptor (GR) abundantly present in the cells.

Cell viability was assessed using the cell proliferation reagent AlamarBlue (AbD Serotec, Kidlington, Oxford, England) and Trypan blue staining. AlamarBlue is an indicator dye, which incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to the chemical reduction of growth medium, resulting from cell growth. In brief, 2,500–3,000 cells were seeded in each well in the 96-well plate and were incubated at 37°C overnight before drug treatment. After 3 days, the cell viability was determined. AlamarBlue solution was added to each well at a 1:10 dilution, and cells were incubated at 37°C in 5% CO2 for 4 hr. Cell viability was assessed by absorbance at 570 and 600 nm using an automatic ELISA plate reader. In addition, for trypan blue staining, 20 mM of the cell suspension was mixed with 20 uM of trypan blue solution and transferred to a hemocytometer twin chamber. The number of viable cells (trypan blue positive) after treatments was counted, and the percentage of viable cells was calculated by the number of the trypan blue stained cells of the treatment group divided by that of the untreated control group.

MATERIALS AND METHODS

Western Blot Analysis

Cell lines, reagents, and antibodies. Three human PC cell lines, DU145 PC3, and LNCaP (American Type Culture Collection; Manassas, VA) were routinely maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 mg/ml of streptomycin in a 5% CO2 atmosphere at 37°C. For androgen induced proximity experiments, the cells were transferred and cultured in 1% charcoal-stripped serum (Hyclone, Logan, UT) containing phenol-red free media for 24 hr. The charcoal-stripped serum has reduced hormone levels and

Human PC cells were incubated in RPMI 1640 without phenol red containing 1% charcoal-stripped FBS for 18–24 hr. Cells were then treated with 0.1, 1, and 10 nM concentration of DHT for 24 hr followed by treatment with or without RU486 (1 mM) for 24 hr. After treatment, cells were lysed and their protein concentrations measured using the Bradford protein assay (Bio-Rad, Hercules, CA). The proteins were subjected to SDS–PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with primary anti-

Measurement of Cell Viability

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bodies, and subsequently incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Millipore Corp., Bedford, MA).

VECTASHIELD with DAPI (Vector Laboratories, Burlingame, CA). Confocal images were obtained using a confocal laser microscopy system (Leica, St-Gallen, Switzerland).

Real-Time Reverse Transcription-PCR Analysis

RNA Interference Experiments

Total RNA was extracted from samples using TRIzol reagent (Invitrogen). Total RNA (2 mg) from each sample was subjected to reverse transcription using the SuperScript II First-Strand cDNA Synthesis Kit (Invitrogen) and the cDNAs were subjected to real-time PCR analysis for hormone receptors. Real-time PCR reactions were carried out in an ABI 7000 sequence detector system (Applied Biosystems, Foster City, CA). Primers were designed as follows: AR forward, 50 CAGTGGATGGGCTGAAAAAT-30 , reverse, 50 -AAGC GTCTTGAGCAGGATGT-30 ; GR forward, 50 -GCGATG GTCTCAGAAACCAAAC-30 , reverse, 50 -GCAGAGGATAACTTCCTCTGTAATCTC-30 ; estrongen receptor (ER) forward, 50 -GACAGGGAGCTGGTTCACAT-30 , reverse, 50 -AGGATCTCTAGCCAGGCACA-30 ; progesterone receptor (PR) forward, 50 -GTCAGTGGGCAGATGCTGTA-30 , reverse, 50 -TGCCACATGGTAAGGC ATAA-30 ; and ACTB forward, 50 -CACCACCATGTACC CTGGCA-30 ; reverse, 50 -ACATCTGCTGGAAGGTGG AC-30 . The cycler was programmed with the following conditions: (i) initial denaturation at 94°C for 2 min, followed by 35 cycles of; (ii) 94°C for 40 sec; (iii) annealing of the primer template at 58°C for 40 sec; and (iv) extension at 72°C for 40 sec. The PCR amplification efficiency and linearity for targeted and control genes were tested.

PC3 and DU145 cells were plated at 60% confluency 24 hr before transfecting with small interfering RNA (siRNA). Cells were transiently transfected with either a GR-specific siRNA or nonspecific control siRNA using HiPerfect transfection reagent (Qiagen). Following transfection, cells were harvested at 72 hr and cell extracts were screened for GR expression by Western blot analysis. Cells showing the most efficient GR knockdown were used for further experiments such as GR and pSTAT5 localization by immunofluorescence as previously described. Immunoprecipitation DU145 cells were lysed in high-salt buffer. Equal amounts of the lysates were immunoprecipitated with 10 mg of the GR antibodies with 20 ml of protein A/G agarose with constant rotation overnight and washed with 10 mM HEPES (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40 twice with 200 ml each. The precipitated proteins were eluted with 30 ml of SDS–PAGE sample buffer and boiled for 5 min. The eluted proteins were electrophoresed by 8% SDS– PAGE, transferred to PVDF membranes, and probed with anti-pSTAT5 antibodies. Statistical Analysis

Immunofluorescence Staining and Confocal Imaging PC3 and DU145 cells were plated on coverslips in each well of a 12-well plate containing 1% charcoalstripped FBS for 18–24 hr. Cells were then treated with 0.1, 1, and 10 nM concentration of DHT for 24 hr followed by treatment with or without RU486 (1 mM) for 24 hr. In addition, cells were treated with 0.1 nM dexamethasone. Cells were then fixed with 4% formalin for 20 min. Then, coverslips were rinsed with PBS, treated with 0.2% bovine serum albumin in PBS for 45 min and with 0.5% Triton X-100 in PBS for 10 min, and incubated with anti-pSTAT5 (1:100; Cell Signaling) and anti-GR antibody (1:500; Santa Cruz Biotechnology) at 4°C overnight in PBS with 0.5% Triton X-100. After washing with PBS, the cells were incubated with Alexa Fluor 488 goat anti-mouse (1:400; Molecular Probes, Eugene, OR) and Alexa Fluor 555 donkey antirabbit (1:400; Molecular Probes) antibodies to detect the primary antibodies. The samples were mounted in The Prostate

Data were obtained from at least three independent experiments and are presented as mean  SD. Statistical evaluation of the results was done by one-way ANOVA (Statistical Package for Social Sciences, Amonk, NY, USA, ver 18.0) and a P-value < 0.05 was considered statistically significant. RESULTS Following DHT treatment, all cells showed dosedependent increase in cell proliferation until DHT 1 nM concentration with LNCaP cells demonstrating the strongest proliferative activity (Fig. 1A, all P < 0.05 compared to respective controls). Similar to the proliferative activity, STAT5 phosphorylation also increased with increasing DHT concentration, in all the cells examined (Fig. 1B). Dose-dependent increase in STAT5 activation was more distinct in the PC3 and DU145 cells, and it continued until DHT 10 nM concentration. We also observed that in PC3 and DU145 cells,

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Fig. 1. Survival and STAT5 activation after DHT treatment in CRPC cells.Treatment of DHT for 72 hr enhanced cell proliferation until 1nM concentration (A). Activation of STAT5 also increased with DHT treatment. STAT5 activation following DHT treatment was distinctively dose-dependent and continued until 10 nM concentration (B). Results are expressed as mean  SD of three independent experiments.
,#,&P < 0.05 by one-way ANOVA, compared with the controlgroup.

increase in phospho-JAK2 accompanied increase in pSTAT5 expression. To examine the presence and functioning status of AR in these cells, expression levels of endogenous AR mRNA were measured. Endogenous AR mRNA was expressed strongly in LNCaP cells, very weakly in PC3 but was absent in DU145 cells (Fig. 2A). To explain the DHT-induced cell proliferation in the cells without AR, we examined other nuclear hormone receptor levels because steroid hormone receptors share a region of homologous configuration and the crossbinding between the ligands and the nonmatching receptors have been previously observed: GR, ER, and PR. We confirmed varying levels of hormone receptor mRNAs in each cell line cells (Fig. 2A). Most distinctively, levels of GR mRNA demonstrated a conspicuous contrast to the levels of AR mRNA. It was expressed about fivefold stronger in DU145 compared to PC3, but was absent in LNCaP cells. Protein levels for endogenous AR and GR demonstrated a similar pattern (Fig. 2B). Treatment with increasing doses of DHT reduced AR mRNA expression in LNCaP cells but not in the other two cells (Fig. 2C). On the other hand, treatment with DHT reduced GR mRNA expression, immediately in the PC3 cells and dose-dependently in the DU145 cells (Fig. 2D). Protein levels for endogenous AR and GR demonstrated a similar

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pattern. Changes in ER mRNA expression after DHT treatment were variable and diverse across the cell lines (Fig. 2E). To test whether DHT actions were mediated by the GR in PC3 and DU145 cells, we incubated the cells with GR-antagonist, RU486 before treating with DHT. Pretreatment with RU486 nearly completely abolished DHT-induced cell proliferation enhancement in PC3 and DU145 cells (Fig. 3A). Paralleling the decrease in cell proliferation, STAT5 activation with treatment of DHT was nearly completely suppressed after RU486 pretreatment in both PC3 and DU145 cells (Figs. 3B and C). We checked SGK1 and phospho-Foxo3a levels, which are immediate products of GR target genes by Western blot to see if DHT induced these effects by GR target gene activation. We observed minimal change in these proteins. Similarly on immunofluorescence staining, treatment with increasing doses of DHT increased nuclear GR (green) and pSTAT5 (red) compared to control, whereas pretreatment with RU486 suppressed these activations (Figs. 4A and B). Cytoplasmic GR’s were markedly depleted after treatment with RU486, and although DHT treatment increased activity of the remaining GRs in the nucleus, overall activation process was significantly suppressed. STAT5 activation and nuclear translocation activity paralleled the GR activation level. To specifically block GR activation, GR knockout DU145 cells were constructed using siRNA (Fig. 5A). Similar to RU486 treatment, blocking GR with siRNA suppressed DHT-induced STAT5 activation, but the interaction was more specific and explicit than RU486 (Fig. 5B). Immunofluorescence staining confirmed these findings (Figs. 5C and D). Treatment with 1 nM dexamethasone in the CRPC cells increased nuclear GR and slightly pSTAT5 as well but the activation was completely blocked with siRNA. Lastly, DHT-treated cell lysates were immunoprecipitated first with antiGR antibodies and then detected with anti-phosphorylated STAT5 (p-STAT5) antibodies (Fig. 6). After DHT treatment, pSTAT5 band was observed, confirming that these interactions were the result of a direct complex formation between GR and pSTAT5 following DHT stimulation. DISCUSSION In the current study, we have demonstrated that DHT directly activated STAT5 in CRPC cells enhancing cell proliferation. Activation of STAT5 by DHT was induced regardless of presence of AR. In cells devoid of ARs, DHT utilized GRs which formed direct complex with activated STAT5 to translocate into the nucleus to continue downstream action initiating cell proliferation. The Prostate

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Fig. 2. Endogenous hormone receptor mRNA and protein expression and changes after DHT treatment. Basal expression levels of AR, GR, ER, and PGRwere compared across the CRPC cells (A). Protein levels of AR and GR in CRPC cells (B).Changes in AR mRNA expression after DHT treatment for 72 hr in LNCaP cells (C).Changes in GR mRNA expression after DHT treatment for 72 hr in PC3 and DU145 cells (D).Changesin ERmRNA expression after DHT treatmentfor 72 hr (E ).

STAT5 is the principal signaling protein mediating the effects of mitogenic cytokines, peptide hormones, and growth factors in both normal and malignant prostate cells [10]. Once the cytokine or hormone binds to its transmembrane receptor, Janus-activated kinase (JAK) on the cytoplasmic side becomes activated which then phosphorylates the STAT monomers to form a dimer. The p-STAT5 dimer translocates into the The Prostate

nucleus and binds to promoter regions to activate transcription of specific genes. While the JAK-STAT signaling pathway was recognized to be critical for the viability of the PC cells, it was previously considered to be unrelated to AR pathway, until research by Tan et al. [12] revealed that activated STAT5 and liganded AR physically interacted with each other to enhance reciprocal nuclear translocation.

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Fig. 3. Survival and STAT5 activation after blocking GR before DHT treatment in PC3 and DU145 cells. Incubation with RU486 1 mM for 24 hr before treating with DHT nearly completely abolished DHT-induced cell proliferation (A). DHT-induced STAT5 phosphorylation was similarly suppressed in both PC3 (B) and DU145 cells (C). SGK1and phopho-Foxo3a proteins showed minimal change after DHT treatment (D).Results are expressed asmean  SD of threeindependentexperiments.
,#P < 0.05 byone-way ANOVA.

In the previous CRPC experiments, it was suggested that autocrine prolactin or cytokines and growth factors activated STAT5, which in turn interacted with AR bound to DHT. In our experiment, we observed that DHT-STAT5 phosphorylation reaction was accompanied by increase in JAK2 phosphorylation (Fig. 1B). One of the primary actions liganded GR initiates is activating many different kinds of kinases. This needs further exploration, but we are assuming that liganded GR phosphorylated JAK2 among many other receptor kinases, which in turn activated STAT5 in the cytoplasm. Activated STAT5 would then form complex with liganded GR to translocate and initiate further reaction. More interestingly, in DU145 cells, which are devoid of AR or in PC3 cells with little AR, DHT bound to GR to initiate the proliferative signals. One of the defining characteristics of PC cells becoming castration-resistant is the acquisition of ability to grow and proliferate despite castrate level of androgenic stimulation. One of the critical molecular mechanisms explaining CRPC proliferating and progressing in this condition was AR mutation leading to AR supersensitivity and promiscuous stimulation by other cytokines, growth factors, and trace amounts of DHT [6–9]. In addition to the previously described routes, we have demonstrated that in CRPC cells

devoid of AR, DHT exerted its effect through GR. This result can explain how clinically CRPC progresses in the presence of antiandrogens blocking AR. Comparing the proliferation activities, growth stimulation through GR was less effective than through AR (Fig. 1A) but sufficient to increase population. Glucocorticoid hormone is a major regulator of various transcriptional pathways in homeostasis, metabolism, cognition, and inflammation. It has proapoptotic property eliciting anti-inflammatory and immunosuppressive actions [13]. However, specific transcriptional action induced by the hormone depends on the cell or tissue where the hormone bound with its receptor [14–16]. Once the hormone binds with the receptor in the cytoplasm, it translocates into the nucleus where the complex binds to the glucocorticoid response element on the DNA. Transcriptional activity depends on the genomic loci where the complex bound to, and the binding site is cell and tissue specifically predetermined before hormone. Therefore, despite widespread expression of the GR, hormonal activity is directed and highly regulated by the cell or the tissue where the hormone bound with its receptor [16]. In our experiment, we observed that in CRPC cells devoid of AR, DHT bound with GR and liganded GR functioned like liganded AR activating The Prostate

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Fig. 4. Immunofluorescence staining of GR and pSTAT5 with DHT treatment with and without RU486.Dose-dependent activation and nuclear translocation of both GR (green) and pSTAT5 (red) were observed in both PC3 (A) and DU145 cells (B). Incubation with RU486 1 mM for 24 hr depleted cytoplasmic GR significantly which suppressed DHT-induced activation.

STAT5, forming a complex and translocating into the nucleus to initiate DHT-induced cell proliferation. Because both AR and GR belong to a superfamily of nuclear receptors sharing structural and functional properties, a direct cross-binding of the DHT to GR in CRPC cells could be hypothesized. Alternately, GR in CRPC may contain regions of “unstructured” proteins that endows flexibility in its receptor function as the frequency of the intrinsically disordered proteins has been reported to be higher in cancer cells [17,18]. Similar to our experiment, functional interaction between STAT5 and the GR through direct binding [19,20] or through JAK activation has also been previously suggested [21]. In our experiment, increase in GR after DHT treatment was accompanied by increase in JAK2 phosphorylation suggesting DHT-bound GR induced The Prostate

Fig. 5. Suppression of STAT5 activationby siRNAGR. After constructing GR knockout DU145 cells with siRNA (A), DHT 1nM was treated to test STAT5 activation. STAT5 activation with DHT was clearly suppressed in the knockout cells (B). On immunofluorescence staining DHT 1 and 0.1nM dexamethasone increased nuclear GR (green) and pSTAT5 (red) activity in both PC3 and DU145 cells. After siRNA GR treatment, the activity was completely blocked (C).

phosphorylation of JAK2, which in turn activated STAT5. Complex formation between activated GR and pSTAT5 was demonstrated by fluorescent immunocytochemistry and immunoprecipitation, which is a novel finding. Applied to a clinical situation during combined ADT, our results indicate that GR could provide a bypass for DHT to invalidate the actions of antiandrogens blocking the AR or a route for action in cells without AR. Our result provides evidence for the necessity to continue ADT in PC that has progressed to castration resistant state, because in addition to mutated AR or increased DHT utilization, GR bypass may provide sustained growth promotion with DHT. On

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2. Chi KN, Bjartell A, Dearnaley D, Saad F, Schroder FH, Sternberg C, Tombal B, Visakorpi T. Castration-resistant prostate cancer: From new pathophysiology to new treatment targets. Eur Urol 2009;56:594–605. 3. Oh WK, Kantoff PW. Management of hormone refractory prostate cancer: Current standards and future prospects. J Urol 1998;160:1220–1229. 4. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers CL. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–39.

Fig. 6. Immunoprecipitation of DU145 cells with anti-GR and anti-pSTAT5 antibodies. Cell lysates after treating with DHT 1nM were precipitated with anti-GR Ab and western blotted with antipSTAT5 Ab.

the other hand, GR could be an additional target for therapy when PC progresses to castration resistant state. Dexamethasone is a potent GR agonist, prolonged administration of which causes GR expression reduction. Including dexamethasone in a cytotoxic chemotherapeutic regimen for CRPC brings variable declines in serum PSA in 20–79% of patients compared to a regimen with cytotoxic agent alone [22,23]. While the additional clinical response with dexamethasone has been consistently observed, the exact mechanism had remained elusive. We have demonstrated that in CRPC cells there is a direct stimulatory pathway between DHT, GR, and pSTAT5 and that concomitant with GR and STAT5 activation, DHT induced GR mRNA expression reduction. Notably, dexamethasone treatment slightly induced increase in nuclear GR, but no definite STAT5 activation could be observed indicating that DHT-induced STAT5 activation through GR is a ligand-specific or ligand-dependent process. These findings may provide a lead to further understanding. CONCLUSION In CRPC cells, DHT activated STAT5 enhancing survival and proliferation. Activation of STAT5 by DHT was induced regardless of presence of AR. In cells devoid of AR, DHT directly interacted with GR, which formed complex with activated STAT5 to translocate into the nucleus and continue downstream action initiating cell proliferation. REFERENCES 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29.

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