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Although many androgen receptor (AR) mutations have been detected in patients with prostate cancer (CaP), their role in cancer initiation remains unknown. We have previously shown that different CaP-derived AR mutations have different transactivational activities, ranging from loss of function for a few alleles to gain of function for the majority of alleles. We hypothesized that gain-of-function (GOF) AR mutations would contribute to the development of CaP. To test this hypothesis, wild-type AR (ARWT) and GOF K580R AR mutant (ARK580R) alleles were stably transfected into the immortalized human prostate epithelial pRNS-1-1 cell line that lacks the expression of the AR and fails to grow in nude mice. The untransfected and vector-transfected cell lines were used as controls. The expression level of the transfected receptors was confirmed using RT-PCR, Western blot analysis and immunohistochemistry (IHC). In response to stimulation by the synthetic androgen R1881, both the ARWT and ARK580R protein were able to translocate from cytoplasm to nuclei, and to activate a PSA promoter, inducing a 5.5- and 15-fold increase in luciferase activity, respectively. We tested the tumorigenicity of AR-transfected prostate epithelial cells first in soft agar and then in nude mice. In soft agar, the ARWT-transfected and the control cell lines formed 0.3-0.7% colonies, while the GOF AR subline produced 3.2% colonies, resulting in a 5-10-fold increase in colony formation. The ability of ARK580R cells to grow in soft agar can be significantly inhibited using specific AR siRNA, indicating that the anchorage-independent growth of ARK580R cells is mediated through the GOF AR. In male nude mice, the ARWT-transfected and the control cell lines were unable to grow up to six months, while tumor formed in 3 of 10 mice 3-4 months after subcutaneous injection of ARK580R-transfected pRNS-1-1 cells. Notably, regrown cells from an ARK580R xenograft formed tumor in 5 of 6 male mice 6-8 weeks after injection. This finding presents the first evidence that introduction of a GOF AR mutation into a transformed, benign prostatic epithelial cell line induces the formation of prostate cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]