Induction of clusterin by AKT--role in cytoprotection against docetaxel in prostate tumor cells

Abstract

Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3-dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells.

Figure 1

AKT activation accompanies docetaxel resistance in DU145 and PC3 human prostate tumor cells. Paired parental and DR DU145 and PC3 prostate tumor cells were lysed and analyzed by Western blotting with specific antibodies against CLU. Activated AKT was evaluated using antibodies directed against the phosphorylation site at Thr³⁰⁸ or Ser⁴⁷³. Equal loading was monitored by β-actin levels. Immunoblot analysis of pAKT and sCLU indicated that active AKT and sCLU were increased in DU145-DR and PC3-DR cells compared with their matched parental cells.

Figure 2

API-2 blockade of AKT function reduces sCLU expression and induces chemosensitivity to docetaxel in drug-resistant tumor cells. A, DU145-DR and PC3-DR tumor cells were pretreated 2 h with the indicated doses of API-2, a potent AKT inhibitor, and exposed to docetaxel-containing medium for 48 h. Cell lysates were then prepared and analyzed by Western blotting with antibodies against pAKT. The lysates were also probed with antibodies specific for total AKT and sCLU. Treatment with API-2 caused a dose-dependent inhibition of active AKT and sCLU protein expression. B, CWR22Rv1 tumor cells were pretreated 2 h with the indicated doses of API-2 and cultured for 48 h in complete medium. Cell lysates were made and Western blot analysis was done to detect pAKT, total AKT, sCLU, and β-actin. Treatment with API-2 caused a dose-dependent inhibition of active AKT and sCLU protein expression. C, DU145-DR and PC3-DR tumor cells, similarly treated with API-2 for 2 h, were analyzed for cell apoptosis by Annexin V/PI staining after a further 48-h culture in docetaxel medium. Both cell lines succumbed to docetaxel-induced cell death after API-2 treatment to deplete AKT function.

Figure 3

Expression of DN-AKT disrupts sCLU expression. A, DU145-DR tumor cells were transfected with DN-AKT and cultured for 48 h in docetaxel-containing medium. Control transfection was with the empty vector PCDNA3. Transfection efficiency was monitored by Western blot analysis for total AKT. pAKT assessment indicated that the overexpressed DN-AKT is not phosphorylated, as expected, due to mutation at the critical threonine and serine sites, thus showing equal levels of pAKT in control-transfected and DN-AKT–transfected cells. Downstream AKT function was assessed by analysis of GSK-3β phosphorylation, which is a known AKT target. DN-AKT effectively suppressed GSK-3β phosphorylation and correspondingly suppressed sCLU expression. B, the above experiment was repeated on PC3-DR cells and produced confirmation of the loss of AKT to result in loss of pGSK-3β and sCLU. C, analysis of sCLU gene expression was done by RT-PCR on DU145DR and PC3-DR cells that were either treated with DMSO or API-2, or transfected with control PCDNA3 vector or DN-AKT. Both API-2 treatment and DN-AKT transfection markedly suppressed CLU mRNA expression in DU145-DR and PC3-DR tumor cells compared with their respective controls. Equal loading was monitored by GAPDH mRNA levels.

Figure 4

AKT inhibition by API-2 treatment or DN-AKT transfection induces chemosensitivity to docetaxel via a caspase-3–dependent pathway. A, DU145-DR and PC3-DR tumor cells, pretreated with API-2 or DN-AKT transfection (including DMSO or PCDNA3 controls), were treated with zVAD.fmk, a general caspase inhibitor, and zVAD.amc, a caspase-1–specific inhibitor. These cells were then exposed to docetaxel for 48 h before analysis of active caspase-3. High levels of active caspase-3 were detected in API-treated and DN-AKT–expressing tumor cells. This activity was inhibited by zVAD.fmk but not by zVAD.amc. B, visual assessment of cell survival by methylene blue staining of the same cells showed few cells surviving in DU145DR and PC3-DR tumor cells treated with API-2 or transfected with DN-AKT, whereas the control DMSO–treated and PCDNA3-expressing cells showed a robust growth.

Figure 5

Overexpression of sCLU overcomes API-2-induced chemosensitivity to docetaxel. A, DU145-DR tumor cells were transfected with either control PCDNA3 or myc-tagged sCLU. These cells were then pretreated with API-2 for 2 h and incubated in docetaxel medium for 48 h. Western blot analysis with anti-myc was done to confirm efficient transfection, and further analysis of sCLU, pAKT, and total AKT was pursued. It is confirmed that API-2 can suppress AKT function in both PCDNA3- and sCLU-expressing tumor cells. B, these same cells were also analyzed for cell growth and survival by methylene blue staining. API-2 effectively induced docetaxel sensitivity and cell death in DU145-DR tumor cells but could not do so in the sCLU-overexpressing DU145-DR tumor cells.

Figure 6

Inhibition of AKT function leads to STAT1 inactivation, whereas CA-AKT leads to STAT1 activation and CLU induction. A, DU145-DR tumor cells, pretreated with API-2 or DMSO in one set or transfected with DN-AKT or PCDNA3 control vector in a second set, were further cultured 48 h in docetaxel medium. Cells were then lysed and analyzed by Western blotting with anti-pSTAT1 or total STAT1. Equal loading was also monitored with anti–β-actin. B, parental DU145 tumor cells were transfected with CA-AKT or with the empty vector PCDNA3, followed by 48-h culture in medium. Transfection efficiency was monitored by Western blot analysis with anti-AKT, and functional activation was assessed by analysis of AKT phosphorylation. CA-AKT led to STAT1 activation and upregulation of CLU expression.