Androgen receptor modifications in prostate cancer cells upon long‐termandrogen ablation and antiandrogen treatment

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Abstract
To study the mechanisms whereby androgen‐dependent tumors relapse in patients undergoing androgen blockade, we developed a novel progression model for prostate cancer. The PC346C cell line, established from a transurethral resection of a primary tumor, expresses wild‐type (wt) androgen receptor (AR) and secretes prostate‐specific antigen (PSA). Optimal proliferation of PC346C requires androgens and is inhibited by the antiandrogen hydroxyflutamide. Orthotopic injection in the dorsal‐lateral prostate of castrated athymic nude mice did not produce tumors, whereas fast tumor growth occurred in sham‐operated males. Three androgen‐independent sublines were derived from PC346C upon long‐term in vitro androgen deprivation: PC346DCC, PC346Flu1 and PC346Flu2. PC346DCC exhibited androgen‐insensitive growth, which was not inhibited by flutamide. AR and PSA were detected at very low levels, coinciding with background AR activity in a reporter assay, which suggests that these cells have bypassed the AR pathway. PC346Flu1 and PC346Flu2 were derived by culture in steroid‐stripped medium supplemented with hydroxyflutamide. PC346Flu1 strongly upregulated AR expression and showed 10‐fold higher AR activation than the parental PC346C. PC346Flu1 proliferation was inhibited in vitro by R1881 at 0.1 nM concentration, consistent with a slower tumor growth rate in intact males than in castrated mice. PC346Flu2 carries the well‐known T877A AR mutation, causing the receptor to become activated by diverse nonandrogenic ligands including hydroxyflutamide. Array‐based comparative genomic hybridization revealed little change between the various PC346 lines. The common alterations include gain of chromosomes 1, 7 and 8q and loss of 13q, which are frequently found in prostate cancer. In conclusion, by in vitro hormone manipulations of a unique androgen‐dependent cell line expressing wtAR, we successfully reproduced common AR modifications observed in hormone‐refractory prostate cancer: downregulation, overexpression and mutation.