Transcriptional regulation of the androgen signaling pathway by the Wilms'tumor suppressor gene WT1

The androgen-signaling pathway plays a critical role in prostate cancer dev elopment and progression. We have recently demonstrated that the Wilms' tum or suppressor gene product, WT1, binds to multiple sites in the androgen re ceptor (AR) promoter and transcriptionally represses the AR gene promoter i n vitro. We asked whether WT1 repression of the endogenous AR gene interfer es in the androgen signal transduction cascade and modifies AR target gene expression. We analyzed the effect of WT1 (-/-) overexpression on an AR tar get gene reporter construct that contains the luciferase gene, the ElB TATA box, and two copies of the androgen- respnose element (ARE), the dimeric A R binding site. Luciferase activity was determined in 293 kidney and TM4 Se rtoli cells, two nontumorigenic cell lines that express both AR and WT1. Ce lls were cotransfected by lipofectamine in the presence or absence of the s ynthefic androgen R1881. Results showed that the overexpression of WT1 down regulates ARE-reporter gene transcription in both cell lines tested. the in hibitory effect of WT1 on the AR target gene construct was dose- dependent and androgen-independent in 293 cells, whereasin TM4 cells it was androgen- dependent. Additionally, a zinc-finger mutant WT1(-/-) expression construct , R394W, failed to decrease luciferase activity, suggesting that WT1 downre gulates the ARE-reporter gene construct activity by directly repressing the endogenous AR gene promoter. Furthermore, we analyzed the expression of WT 1 and AR mRNA in several prostate cancer cell lines in order to understand the role WT1 may play in prostate cancer development and progression. Gel a nalysis of cDNA amplified by RT-PCR of AR and WT1 RNA from prostate cancer and non- prostatic cell lines showed that LNCaP and MDAPCa2b, two metastati c prostate cancer cell lines which are androgen-sensitive, expressed AR but not WT1. Du145 and Pc3, two cell lines from advanced metastatic prostate c ancer, which are characterized as androgen-independent and -insensitive, di d not express AR but expressed a high level of WT1. Two non-prostatic cell lines, T47D and 293, weakly co-expressed AR and WT1. This inverse relations hip between AR and WT1 expression in prostate cancer cell lines, together w ith WT1 repression of the AR promoter, suggest a role for WTI in the androg en signaling pathway and in prostate cancer development and progression.