Calmodulin protects androgen receptor from calpain-mediated breakdown in prostate cancer cells

Abstract

Although inactivation of the androgen receptor (AR) by androgen-ablation or anti-androgen treatment has been frontline therapy for disseminated prostate cancer for over 60 years, it is not curative because castration-resistant prostate cancer cells retain AR activity. Therefore, curative strategy should include targeted elimination of AR protein. Since AR binds to calmodulin (CaM), and since CaM-binding proteins are targets of calpain-mediated proteolysis, we studied the role of CaM and calpain in AR breakdown in prostate cancer cells. Whereas the treatment of prostate cancer cells individually with anti-CaM drug or calcimycin, which increases intracellular Ca ++ and activates calpain, led to minimal AR breakdown, combined treatment led to a precipitous decrease in AR protein levels. This decrease in AR protein occurred without noticeable changes in AR mRNA levels, suggesting an increase in AR protein turnover rather than inhibition of AR mRNA expression. Thus, CaM inactivation seems to sensitize AR to calpain-mediated breakdown in prostate cancer cells. Consistent with this possibility, purified recombinant human AR (rhAR) underwent proteolysis in the presence of purified calpain, and the addition of purified CaM to the incubation blocked rhAR proteolysis. Together, these observations demonstrate that AR is a calpain target and AR-bound CaM plays an important role in protecting AR from calpainmediated breakdown in prostate cancer cells. These observations raise an intriguing possibility that anti-CaM drugs in combination with calpain-activating agents may offer a curative strategy for the treatment of prostate cancer, which relies on AR for growth and survival. KeywordsAndrogen receptor; calmodulin; calpain; hormone-refractory prostate cancer Prostate cancer is the most frequently diagnosed non-skin cancer and second leading cause of cancer deaths in American men (Jemal et al., 2009). Androgen, by activating the androgen receptor (AR), plays a pivotal role in prostate cancer cell proliferation and viability (Magi-Galluzzi et al., 1998). Hence, androgen ablation has been frontline therapy for the treatment of advanced prostate cancer. However, androgen-deprivation therapy (ADT) is only palliative and most patients receiving ADT eventually succumb to castrationresistant prostate cancer (CRPC) that is also resistant to chemotherapy (Feldman and Feldman, 2001). Furthermore, whether normal or mutated, AR continues to play an important role in progression of CRPC (Feldman and Feldman, 2001;van der Kwast et al., 1991) and is required for proliferation and survival of both androgen-sensitive as well as androgen-independent AR-positive prostate cancer cells (Haag et al., 2005;Snoek et Yuan et al., 2006;Zegarra-Moro et al., 2002). Therefore, developing a curative strategy for the treatment of hormone-refractory disease requires identifying effective means to eliminate AR protein in prostate cancer cells .Overexpression of AR is reported to be involved in the progression to CRPC (Chen et al., 2004). Besides AR gen...

Hydrazinobenzoylcurcumin inhibits androgen receptor activity and growth of castration-resistant prostate cancer in mice

“…This study identified HBC as one such agent. We have shown previously that CaM binds to the NTD of AR and plays an important role in AR transcriptional activity and AR protein stability [10-12]. In the present study, we showed for first time that CaM is overexpressed in PCa (Fig.…”

“…A number of proteins interact with NTD and play an important role in regulation of AR activity [9]. Calmodulin (CaM) is one such co-regulator that binds to the AR NTD and regulates AR activity; CaM-antagonists, including CaM-siRNA inhibit AR activity and suppress AR protein stability in PCa cells [10-12]. Thus, targeting CaM may offer a viable strategy to inhibit AR activity through a mechanism that does not involve intact AR LBD.…”

“…Since CaM regulates AR activity [10, 12], and HBC binds exclusively to CaM [20], we investigated the effect of HBC on AR activity and on the androgen-regulated transcriptome in PCa cells. In addition, we evaluated the efficacy of HBC in suppressing the growth of tumors derived from metastatic CRPC cells in mice.…”

Androgen receptor and its splice variants in prostate cancer

“…The first mechanism forwarded for the production of the naturally occurring AR isoforms lacking LBD in CWR22 cell lines was calpain-mediated proteolysis [30, 31] although not without contradictory data [32]. However, it is now evident that splicing events generates some variants.…”

Comparison of the protein expression of calpain-1, calpain-2, calpastatin and calmodulin between gastric cancer and normal gastric mucosa

“…In addition, calmodulin (CaM) is another endogenous regulator of proteolytic activity of calpains (10). By binding to the PEST sequence [a sequence that is rich in proline (P), glutamic acid (E), serine (S) and threonine (T)], which is the target sequence of calpain, CaM protects the protein to be cleaved by calpain (11)(12)(13)(14).…”