Tumor suppressor activity of glucocorticoid receptor in the prostate

Oncogene (2007) 26, 1885–1896 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Tumor suppressor activity of glucocorticoid receptor in the prostate A Yemelyanov1, J Czwornog1, D Chebotaev1, A Karseladze2, E Kulevitch2, X Yang3 and I Budunova1 1 Department of Dermatology, Feinberg Medical School, Northwestern University, Chicago, IL, USA; 2Department of Pathology, NN Blokhin Cancer Research Center, RAMS, Moscow, Russia and 3Department of Pathology, Feinberg Medical School, Northwestern University, Chicago, IL, USA Glucocorticoids are extensively used in combination chemotherapy of advanced prostate cancer (PC). Little is known, however, about the status of the glucocorticoid receptor (GR) in PC. We evaluated over 200 prostate samples and determined that GR expression was strongly decreased or absent in 70–85% of PC. Similar to PC tumors, some PC cell lines, including LNCaP, also lack GR. To understand the role of GR, we reconstituted its expression in LNCaP cells using lentiviral approach. Treatment of LNCaP-GR cells with the glucocorticoids strongly inhibited proliferation in the monolayer cultures and blocked anchorage-independent growth. This was accompanied by upregulation of p21 and p27, downregulation of cyclin D1 expression and c-Myc phosphorylation. Importantly, the activation of GR resulted in normalized expression of PC markers hepsin, AMACR, and maspin. On the signaling level, GR decreased expression and inhibited activity of the MAP-kinases (MAPKs) including p38, JNK/SAPK, Mek1/2 and Erk1/2. We also found that activation of GR inhibited activity of numerous transcription factors (TF) including AP-1, SRF, NF-jB, p53, ATF-2, CEBPa, Ets-1, Elk-1, STAT1 and others, many of which are regulated via MAPK cascade. The structural analysis of hepsin and AMACR promoters provided the mechanistic rationale for PC marker downregulation by glucocorticoids via inhibition of specific TFs. Our data suggest that GR functions as a tumor suppressor in prostate, and inhibits multiple signaling pathways and transcriptional factors involved in proliferation and transformation. Oncogene (2007) 26, 1885–1896. doi:10.1038/sj.onc.1209991; published online 2 October 2006 Keywords: prostate carcinoma; PIN; glucocorticoid receptor; PC marker; transcription factor; MAPKs Correspondence: Dr I Budunova, Feinberg Medical School, Department of Dermatology, Northwestern University, Ward Building 9-332, 303 East Chicago Avenue, Chicago, IL 60611, USA. E-mail: Received 16 June 2006; revised 27 July 2006; accepted 28 July 2006; published online 2 October 2006 Introduction Glucocorticoid hormones regulate proliferative, inflammatory and immune responses. For years, glucocorticoids have been extensively used for the treatment of hormone refractory prostate cancer (HRPC), and the combination of paclitaxel and dexamethasone remains a standard treatment for HRPC patients in the US and other countries (reviewed by Fakih et al., 2002). Glucocorticoids have also been used as the ‘standard’ therapy arm in several randomized phase II–III clinical trials for the combination therapy of HRPC (Fakih et al., 2002; Koutsilieris et al., 2002). The cellular response to glucocorticoids is mediated through a highly specific glucocorticoid receptor (GR). In the absence of glucocorticoids, GR is sequestered in the cytoplasm by chaperone proteins. Following ligand binding, the GR dissociates from the chaperones and forms homodimers, which enter the nucleus. There are two major mechanisms of gene regulation by GR (De Bosscher et al., 2003; Necela and Cidlowski, 2004). The direct positive transcriptional regulation (transactivation) occurs via binding of the GR homodimer to palindromic promoter DNA sequences called glucocorticoid-response elements. The indirect regulation is mediated via crosstalk with other transcription factors (TFs), including activator protein 1 (AP-1), nuclear factor kappa-B (NF-kB), signal transducer and activator of transcription (STAT)-5, mothers against DPP homolog 3 (SMAD3), etc. (De Bosscher et al., 2003; Necela and Cidlowski, 2004). Most of such GR–TF interactions repress the activity of partner TFs and their target genes (transrepression). Recently, the additional mechanism of indirect gene regulation by GR has been discovered where GR blocks mitogen-activated protein kinases (MAPKs) (Kassel et al., 2001; Bruna et al., 2003). Indirect, DNA-independent mechanisms of GR gene regulation appear to be critical for the antiinflammatory effects (Schacke et al., 2002), whereas their role in the growth inhibition by glucocorticoids has never been addressed. Although the clinical effect of glucocorticoids in HRCP patients is well known, the objective responses have been found only in 20–25% of patients (Fakih et al., 2002). The limited effect of glucocorticoids in prostate carcinoma (PC) patients implies the changes in Tumor suppressor activity of GR in PC cells A Yemelyanov et al 1886 GR expression, function and/or availability of GR targets in PC cells. Indeed, we and others showed that different types of tumor cells lose their sensitivity to growth inhibition and apoptosis by glucocorticoids either because of the loss of GR expression or because of the abnormal GR function (Ray, 1996; Budunova et al., 1997; Greenstein et al., 2002). These observations suggest that intact GR signaling is crucial for the growth control of lymphoid and epithelial cells and that in some tissues GR may act as a tumor suppressor. Despite the use of glucocorticoids in the standard combinational therapies of PC patients, the information regarding GR expression in PCs is surprisingly limited and conflicting (Mohler et al., 1996; Nishimura et al., 2001). To our knowledge, GR expression in early prostate lesions such as intraepithelial neoplasia (PIN) has never been evaluated. Furthermore, GR function in the prostate cells and its role in PC have never been studied, even though the growth inhibitory effect of glucocorticoids in GR-positive human and rat prostate cells has been reported (Nishimura et al., 2001). These previous studies chiefly attribute growth inhibitory effect of glucocorticoids to the inhibition of NF-kB TF (Nishimura et al., 2001). Here, we for the first time present the comprehensive analysis of GR expression changes in the course of prostate tumorigenesis, and determine the effect of activated GR signaling on proliferation and the maintenance of transformed phenotype by PC cells. Table 1 GR expression is strongly decreased in prostate carcinomas. Tissue samples Patient cohorts Number of samples GR intensity score* +/
++ +++ BPH I II 15 52 0 0 20 4 80 96 HGPIN I II 30 5 37 0 53 40 10 60 PC (Gl. 6–7) I II 41 17 68 88 22 12 10 0 PC (Gl. 8–10) I II 30 28 70 85 20 15 10 0 Abbreviations: BPH, benign prostatic hyperplasia; Gl, Gleason score; GR, glucocorticoid receptor; HGPIN, high-grade prostatic intraepithelial neopl (...truncated)