Characterization of the Truncated Androgen Receptor Generated by Calpain-Dependent Proteolysis in Prostate Cancer

摘要

Androgen ablation therapy is effective in treating androgen-dependent prostate tumors; however, tumors that can proliferate in castrate levels of androgen eventually arise. We previously reported that in CWR22Rv1 (Rv1) cells, the protease calpain 2 can cleave the androgen receptor (AR) into a constitutively active approximately 80 KDa low molecular weight (LMW) form. In this study, we further dissect the mechanisms that produce the AR LMW forms using Rv1 cells and the related CWR22-R1 (R1) cells. The 39 a.a. insertional mutation in Rv1 cells sensitizes this AR (E3DM-AR) calpain 2 proteolysis. R1 cells encode the same AR molecule as the parental CWR22 xenograft. Using anti-calpain 2 siRNA and calpeptin, we find that calpain 2 plays a role in the generation of the LMW- AR in R1 cells. Furthermore, LMW-AR expression is regulated by the activation of calpain 2 by Extracellular Signal-Regulated Kinases 1/2 (ERK). Inhibition of ERK phosphorylation or siRNA-mediate decrease of ERK expression reduces LMW-AR levels in R1 cells. Conversely, activation of the MAPK pathway and increased ERK phosphorylation results in increased levels of LMW-AR. Finally, analyses of human tumor samples found that LMW-AR levels are higher in tumors that have an increased calpain/calpastatin ratio and/or increased levels of phospho-ERK (pERK), suggesting that a higher calpain/calpastatin ratio collaborates with activation of the MAP kinase pathway to promote the generation of the LMW-AR. Furthermore, cellular localization analysis of AR shows that the LMW-AR is the predominant form (approximatel 90%) present in the nucleus in Rv1 cells cultured in the absence of androgen, allowing us to study the chromosomal binding sites of LMW- AR using chromatin immunoprecipitation (ChIP) combined with DNA microarray analysis (Chip-on-Chip).