calpastatin and Prostatic-Neoplasms

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 80,000 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-amino acid insertional mutation in the Rv1-AR (E3DM-AR) sensitizes this AR to calpain 2 proteolysis. R1 cells encode the same AR molecule as the parental CWR22 xenograft. Using calpain 2 small interfering RNA 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 ERK 1 and 2. Inhibition of ERK phosphorylation or small interfering RNA-mediated decrease of ERK expression reduces LMW-AR levels in R1 cells. Conversely, activation of the MAPK pathway results in increased ERK phosphorylation and 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). This suggests that a higher calpain/calpastatin ratio collaborates with activated ERK to promote the generation of the LMW-AR.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Transformed; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Weight; Phosphorylation; Prostatic Neoplasms; Receptors, Androgen; Recurrence; RNA, Small Interfering

p107 protein, a member of the retinoblastoma family protein, suppresses growth promotion in cancer cells. We have already reported evidence that calpain, a calcium dependent protease is involved in the cleavage of p107 protein. We show here that p107 protein can also be a substrate for ubiquitination. A negative growth regulator, the HMG-CoA reductase inhibitor lovastatin was found to induce loss of p107 protein which was reversible by a specific protease inhibitor lactacystin as well as calpain inhibitor. Following treatment with lovastatin higher molecular weight ubiquitinated forms of p107 were detected by anti-p107 immunoprecipitation and anti-ubiquitin Western blotting. These forms further increased when lactacystin was added to culture medium. These results indicate that ubiquitin-proteasome pathway plays a potential role in the degradation as well as calpain. The data presented here suggest a model in which calpain and ubiquitin-proteasome system possibly play a cooperative role in targeting the protein under certain conditions.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Western; Calcium-Binding Proteins; Calpain; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Humans; Lovastatin; Male; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Sarcoma, Ewing; Transfection; Tumor Cells, Cultured; Ubiquitins