S Jacob et al. AR as a regulator of ZEB2 21:3 473–486 Research expression in PCa Androgen receptor as a regulator of ZEB2 expression and its implications in epithelial-to-mesenchymal transition in prostate cancer Sheeba Jacob, S Nayak, Gwendolyn Fernandes1, R S Barai2, S Menon3, U K Chaudhari, S D Kholkute and Geetanjali Sachdeva Primate Biology Laboratory, National Institute for Research in Reproductive Health (NIRRH), Correspondence Indian Council of Medical Research, JM Street, Parel, Mumbai 400012, India should be addressed 1GS Medical College and KEM Hospital, Parel, Mumbai 400012, India to G Sachdeva 2The Centre for Medical Bioinformatics, NIRRH, Parel, Mumbai 400012, India Email 3Department of Pathology, Tata Memorial Hospital, Mumbai 400012, India [email protected] Abstract Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial- Key Words to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was " androgen receptor initiated to explore whether ZEB2 expression differs in prostate cancer (PCa, nZ7) and " EMT benign prostatic hyperplasia (BPH, nZ7) tissues. In PCa tissues, the levels of both " ZEB2 immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher " prostate cancer (P!0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to Endocrine-Related Cancer investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P!0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P!0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection of AR and ZEB2 cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen- dependent cells and as a negative regulator in AI PCa cells. Endocrine-Related Cancer (2014) 21, 473–486 Introduction It is well recognized that androgens play a vital role in men (Mohler 2008). Surgical ablation of androgens and anti- development, growth, and progression of prostate cancer androgen therapies are the major treatment modalities for (PCa), the most common nondermatological malignancy in locally advanced or metastatic PCa (Miyamoto et al.2004). http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/ERC-13-0514 Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 05:31:03AM via free access Research S Jacob et al. AR as a regulator of ZEB2 21:3 474 expression in PCa Although these treatment strategies are initially effective, Materials and methods the disease eventually progresses to an androgen- Antibodies independent (AI) state (Feldman & Feldman 2001). Nonetheless, AI PCa cells continue to rely on androgen Human AR MABs and secondary antibodies conjugated receptor (AR) for their growth and survival (Balk 2002). to HRP or FITC were procured from Dako (Glostrup, Hence, AR-mediated signaling and its effectors are Denmark). E-cadherin, p63, and glyceraldehyde-3-phos- considered to be attractive targets for the treatment of AI phate dehydrogenase (GAPDH) antibodies were purchased PCa, which is often associated with metastasis. from BD Biosciences (San Jose, CA, USA), Sigma–Aldrich, Sufficient evidence has shown that the epithelial- and Calbiochem (Darmstadt, Germany) respectively. to-mesenchymal transition (EMT) facilitates metastasis Human ZEB2 and a-methylacyl-CoA racemase (AMACR) of cancerous cells (Klymkowsky & Savagner 2009). EMT is antibodies were purchased from Sigma–Aldrich, mouse and characterized by the gain of mesenchymal characteristics, rabbit IgGs from Millipore (Billerica, MA, USA), Alexa Fluor such as mobility, invasiveness, and loss of epithelial 488-conjugated secondary antibodies from Life Tech- features, that is, cell–cell adhesion, cell polarity, etc. nologies, and biotinylated secondary antibodies were from Several strides have been carried out to identify the factors Vector Laboratories (Burlingame, CA, USA). contributing to EMT in PCa. Transforming growth factor beta (Zhu & Kyprianou 2010), nicotinamide adenine Tissue samples dinucleotide-dependent histone deacetylase or SIRT1 (Byles et al. 2012), platelet-derived growth factor (Kong Use of prostate tissue samples was approved by the NIRRH et al. 2009), TMPRSS2/ERG (Leshem et al. 2011), SNAI2 Ethics Committee for Clinical Studies. BPH tissues (nZ7) (SLUG) (Emadi Baygi et al. 2010), DAB2IP (Xie et al. 2010), were obtained using transurethral resection and PCa Hsp27 (HSPB1) (Shiota et al. 2013), and miRNAs (Ru et al. biopsies (nZ7), by core needle biopsy. 2012) have all been identified as EMT regulators in PCa. Interestingly, the majority of these factors mediate EMT Immunohistochemistry via zinc finger E-box-binding protein (ZEB) family members, such as ZEB1 (dEF1 or AREB6) and ZEB2 Prostate tissue sections of 5 mm thickness were deparaffi- (Smad-interacting protein 1 (SIP1)) (Gregory et al. 2011). nized and rehydrated in descending grades of methanol. Endocrine-Related Cancer The role of the androgen axis in PCa pathogenesis has After quenching endogenous peroxidase activity, sections been described previously (Nieto et al. 2007). However, were microwaved for 10 min in antigen unmasking data available on the role of androgens in EMT regulation solution (Vector Laboratories). For detection of nuclear are contradictory. While some investigators have reported proteins, sections were permeabilized in 0.1% Triton androgen-induced activation of EMT and its effectors (Zhu X-100 in PBS (pH 7.4) for 10 min. After blocking, sections & Kyprianou 2010, Anose & Sanders 2011), others have were incubated with either the respective primary demonstrated EMT activation in response to androgen antibody or rabbit/mouse IgGs and then with biotinylated deprivation (Sun et al. 2012). A recent study by Izumi et al. secondary antibodies. Reaction products were detected (2013) has implicated AR as an inhibitor of EMT activation using avidin–biotin–HRP complex and diaminobenzidine. in PCa cells. These conflicting data warrant more studies Immunostained sections were counterstained with directed toward deciphering the role of androgenic stimuli hematoxylin. in EMT and PCa metastasis. Staining intensities of the immunoprecipitates In this study, ZEB2 expression was examined in were determined using an image analysis software – i) primary PCa and benign prostatic hyperplasia (BPH) Aperio Image Scope, version v11.2.0.780 (Aperio, Vista, tissues, ii) an androgen-dependent (AR-positive) PCa cell CA, USA). A minimum of ten areas from each section were line (LNCaP), before and after the attenuation of AR randomly selected for measuring integrated optical expression, and iii) AI PC3 and DU145 cells (expressing density. low levels of endogenous AR), before and after the forced expression of AR. We also investigated whether the Cell lines and their maintenance modulation in ZEB2 expression is accompanied by any changes in downstream functions (e.g. invasion and Androgen-dependent prostate carcinoma LNCaP-FGC migration). cell line (CRL-1740) was procured from the American http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/ERC-13-0514 Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 05:31:03AM via free access Research S Jacob et al. AR as a regulator of ZEB2 21:3 475 expression in PCa ZEB2 AR AMACR p63 BPH AD G J PCa BE HK –ve C FIL M ZEB2N AR 1.5×107 1.0×107 1.2×107 8.0×106 Endocrine-Related Cancer 9.0×106 ** 6.0×106 6.0×106 4.0×106 *** Integrated intensity 3.0×106 Integrated intensity 2.0×106 0 0 PCa BPH PCa BPH Figure 1 Immunolocalization of ZEB2 (A and B), AR (D and E), AMACR (G and H), replaced with IgGs. Magnification 20!. (M and N) semiquantitative and p63 (J and K) proteins to the prostate tissues of patients with analysis to compare the intensities of immunoreactive proteins ((M) benign prostatic hyperplasia (BPH) (A, D, G and J) and prostate cancer (PC) ZEB2 and (N) AR) in BPH and PCa tissues. **P value !0.001 and (B, E, H and K). (C, F, I and L) sections where primary antibodies were ***P value !0.0001. Type Culture Collection (Manassas, VA, USA), whereas 5a-Dihydrotestosterone stimulation experiments AI PCa cell lines, PC3 (CRL-1435) and DU145 (ATCC- LNCaP cells (1!105/well), seeded in RPMI-1640 supple- HTB-81), were obtained from National Centre for Cell mented with 10% charcoal-stripped FBS, were stimulated Sciences (Pune, India). The cell lines were grown in with different concentrations of 5a-dihydrotestosterone phenol red-free RPMI-1640 media (Sigma–Aldrich), (DHT). After 72 h, cell pellets were resuspended in protein supplemented with 10% fetal bovine serum (FBS), 100 lysis buffer (9 M urea, 4% CHAPS