Estrogen Induces Androgen-repressed SOX4 Expression To Promote Progression Of Prostate Cancer Cells

BackgroundProstate cancer (PCa) is the most prevalent non-dermatological cancer and the second leading cause of cancer-related death among American men. Although androgen-deprivation treatment (ADT) remains as the mainstay treatment for advanced PCas, these therapies eventually fail and patients invariably relapse with the more aggressive castration-resistant prostate cancer (CRPC) within 18-24 months. The mechanisms underlying the development of CRPC are either androgen receptor (AR)-dependent—uch as AR mutation/amplification, intratumoral production of androgens, modified expressions of co-activators/co-repressors, and dysregulation of AR-regulated genes—or AR-independent. The reactivation of AR signaling after ADT causes altered genetic and epigenetic changes in response to different ligand binding, which consequently helps cancer cells to adapt to the androgen-depleted environment. In recent years, evidence from a large number of investigations including epidemiological, cancer cell-line, human tissue and animal studies strongly suggest that estrogen also plays an important role in the progression of PCa. The direct estrogens actions are mediated by estrogen receptor a (ERa) and estrogen receptorβ (ERP). The traditional paradigm regarding the roles of the two ERs is that ERa mediates the harmful effects of estrogen in the prostate whereas ERβ is tumor-suppressive. However, there is increasing evidence indicates that ERβ may be oncogenic in PCa. Therefore, the precise actions of ERβ in the prostate remain to be completely elucidated. The sex determing region Y-box 4 (SOX4) gene is a critical developmental transcriptional factor that is overexpressed in PCa. Although SOX4 may act as a tumor suppressor, numerous suggested that SOX4 functioned as an oncogene in multiple cancers. Indeed, SOX4 could exert distinct functions— depending on the tumor origin, the cellular context and even the stages of tumor development. Moreover, SOX4 might be a metastasis contributor in cancer progression, via regulating a number of genes important for metastasis. Most recently, we defined an important role for SOX4 in the progression of PCa by orchestrating epithelial-mesenchymal transition (EMT), and SOX4 might serve as a prognostic marker for Chinese PCa patients. While we and others have investigated the role of SOX4 overexpression in PCa, the molecular mechanism underlying its aberrant expression remains unclear.Materials and methodsImmunohistochemistry (IHC) were utilized to detect SOX4 expression and the correlation between ERβ,AR and SOX4 in clinical specimens. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Immunofluorescence staining and co-immunoprecipitation (co-IP) were performed to assess the interaction and subcellular location of ERβ and AR. Chromatin immunoprecipitation assays (ChIP) and Luciferase reporter assays were performed to explore the binding and transcriptional activities of ERβ and AR to the SOX4 promoter. Cell function was evaluated by MTS, invasion and wound healing assays.ResultsIn the current study, we showed that SOX4 expression is up-regulated in CRPC tumors compared to hormone dependent prostate cancer (HDPC) cases. Increased expression was also observed in PCa cells after ADT, which corroborates the IHC results and further indicates a potential role of SOX4 in CRPC. In vitro data indicated that SOX4 is an AR transcriptional target and down-regulated by dihydrotestosterone (DHT) via AR. DHT significantly decreased mRNA and protein expression of SOX4 in a dose-dependent manner both in LNCaP and VCaP cell lines. Maximal effects were observed at lOnM of DHT. We also found that lOnM DHT decreased SOX4 mRNA levels in LNCaP cell line via a time-dependent manner. E2 up-regulates SOX4 expression in the absence of androgen through the formation of a protein complex between ERp and AR. Knockdown of AR or ERβ blocks the E2-induced SOX4 expression, which suggested that both ERβ and AR are required for the E2-induced SOX4 expression. ChIP assays confirmed that both ERβ and AR bind to the SOX4 promoter in response to E2. These data further supported that ERP is required for the E2-induced SOX4 expression. Accordingly, either activated by DHT or E2, AR plays a fundamental role in the transcriptional regulation of SOX4 expression. In addition, AR signaling could exhibit alternative transcriptional activity in response to different ligand-binding. Functionally, silencing SOX4 significantly attenuates the proliferative effect, as well as the capacity of migration and invasion of E2 on PCa cells. Clinically, overexpression of SOX4 is significantly associated with ERβ expression in PCa. In addition, this association is still retained in CRPC patients with poor prognosis.ConclusionIn summary, we identifies SOX4 is a novel DHT-repressed AR-target gene. E2 could promote proliferation of PCa cells through the up-regulation of SOX4 under androgen-depleted environment, suggesting that SOX4 might play an important role in the transition of CRPC. Collectively, these data suggested that AR signaling controls SOX4 expression and exhibits different transcriptional activity against SOX4 in response to different ligand binding. Moreover, the E2-activated AR signaling would contribute to the further increases in SOX4 expression under androgen-depleted conditions. Like the model of selection, this cellular basis might promote those ERβ and AR positive PCa cells to grow through the up-regulation of SOX4 and gradually become resistant to castration. Addressing the regulatory program responsible for the distinct expression of SOX4 between hormone-responsive and androgen-independent setting will facilitate the detection and prevention of the emergence of CRPC.