Androgen receptor induces EPHA3 expression by interacting with transcription factor SP1

Abstract

Erythropoietin‑producing hepatocellular carcinoma cell surface type‑A receptor 3 (EPHA3) has been found to promote the proliferation and survival of prostate cancer (PCa) cell lines and prostate tumor development in nude mice. However, the regulation of EPHA3 in PCa remains largely unknown. This study is aimed to investigate the association between EPHA3 expression and androgen receptor (AR) signaling and the potential mechanism. We determined mRNA and protein levels of EPHA3 and AR signaling‑related genes in the PCa cell line 22Rv1 by reverse transcription‑polymerase chain reaction (RT‑PCR) and western blotting, respectively. The EPHA3 mRNA and protein levels were both found to be elevated by dihydrotestosterone (DHT) hormone in a dose‑ and time‑dependent manner, as AR and prostate‑specific antigen (PSA) expression were increased. Similarly, EPHA3 protein levels were also increased in the PCa cell line LNCaP stimulated with DHT or mibolerone (Mib). Overexpression of pEGFP‑AR in 22Rv1 cells significantly increased the EphA3 level, while AR knockdown with small interfering RNA (siRNA) for AR (siAR) markedly decreased the expression of EPHA3. The key EPHA3 promoter region associated with AR regulation was evaluated by co‑transfection of various pGL3‑basic‑luciferase reporter plasmids, containing EPHA3 core promoter fragments differing in length, with the AR plasmid or siAR into 22Rv1 cells. AR overexpression in 22Rvl cells raised the EphA3 promoter transcription activity of pGL3‑EPHA3‑Luc (EPHA3‑Luc)‑789, and vice versa. Similarly, luciferase activity of EPHA3‑Luc‑317 was also clearly affected. However, truncated EPHA3‑Luc‑237 without the transcription factor specific protein 1 (SP1) binding sites or EPHA3‑Luc‑789ΔSP1 with modified SP1 binding sites clearly decreased EPHA3 promoter activity regardless of whether AR was overexpressed or blocked. Treatment of 22Rv1 cells with 10 and 100 nM of the SP1 inhibitor mithramycin A for 24 and 48 h significantly reduced EPHA3 mRNA and protein levels. Additionally, selective inhibition of SP1 with siRNA SP1 (siSP1) at various concentration from 25 to 75 nM, reduced the EPHA3 protein level in PCa LNCaP cells, accordingly. Co‑immunoprecipitation (co‑IP) and chromatin IP (ChIP) assays were performed to determine whether AR forms a transcription factor complex with Sp1 that binds the EPHA3 core promoter region to sense androgen induction. The result suggests that the interaction of AR and SP1 contributes to regulate EPHA3 expression, and the SP1 binding sites (‑295~‑261) in the EPHA3 core promoter region is crucial to the regulation of EPHA3 expression in response to androgen hormone stimuli.

The prognosis value of EphA3 and the androgen receptor in prostate cancer treated with radical prostatectomy

“…27 In addition, Diao et al found that EphA3 expression was induced by the interaction of the AR with SP1 transcription factor. 23 These findings suggest that there is some correlation between EphA3 and PCa progression.…”

“…Regarding PCa, EphA3 is a significantly upregulated gene in androgen‐independent cells, and this phenomenon might be due to a mechanism of gene amplification or DNA methylation . In addition, Diao et al found that EphA3 expression was induced by the interaction of the AR with SP1 transcription factor . These findings suggest that there is some correlation between EphA3 and PCa progression.…”

“…Diao et al found that the EphA3 mRNA and protein levels were both elevated by the hormone DHT in a dose‐ and time‐dependent manner accompanied by increased AR expression. The overexpression of pEGFP‐AR in 22Rv1 cells significantly increased the EphA3 level, whereas AR knockdown with small interfering RNA (siRNA) specific for the AR (siAR) markedly decreased the expression of EphA3 . Another study showed that prostate androgen induces the prostate leucine zipper gene‐promoted expression of EphA3 .…”

“…22 The mRNA expression level of EphA3 was higher in the androgen-dependent PCa cell lines 22Rv1 and LNCaP with elevated AR expression levels than in the androgen-independent PCa cell lines DU145 and PC-3 without AR expression. 23 Immunoprecipitation (IP) analysis of human tumor cell lines indicated the lack of EphA3 in DU145 cells, and androgen-dependent PCa cell lines (LNCaP and 22Rv1 cells) were EphA3 + . 23 However, Brian et al demonstrated that the transcript level for EphA3 was elevated in LNCaP and PC-3 cells but reduced in DU145 cells.…”

“…23 Immunoprecipitation (IP) analysis of human tumor cell lines indicated the lack of EphA3 in DU145 cells, and androgen-dependent PCa cell lines (LNCaP and 22Rv1 cells) were EphA3 + . 23 However, Brian et al demonstrated that the transcript level for EphA3 was elevated in LNCaP and PC-3 cells but reduced in DU145 cells. 24 We speculated that there might be some correlation between EphA3 and AR in PCa, which needs to be confirmed.…”