Characterization of the Truncated Androgen Receptor Generated by Calpain-Dependent Proteolysis in Prostate Cancer
Abstract
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 ~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 (~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).
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 31, 2009
- Accession Number
- ADA513068
Entities
People
- Honglin Chen
Organizations
- University of California