Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells

Abstract

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)-pre-treated castration-resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc-sensitive and Doc-resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc-sensitive and Doc-resistant CRPCs.

Keywords: AZD2014; antitumour; castration-resistant prostate cancer; docetaxel; mTORC1 and 2.

Figure 1

The biochemical activity of AZD2014 in blocking mTORC1/C2 signalling in prostate cancer cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were either treated with dimethyl sulfoxide (DMSO) (CTR) or increasing final concentrations of AZD2014 (10 nM, 50 nM, 100 nM, 500 nM, 1000 nM) or rapamycin (10 nM, 50 nM, 100 nM, 500 nM, 1000 nM) and further cultured for 48 hours before lysis and immunoblotting. A‐D, Expression of the mTORC1 and mTORC2 signalling proteins was tested by Western blotting. E‐H, IC50 values were generated by GraphPad Prism version 7.0 from CCK8 assay data. The experiments in this figure were repeated three times, which yielded similar results

Figure 2

AZD2014 inhibited proliferation and induces apoptosis in CPRC cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were either treated with DMSO (CTR) or treated with the indicated concentrations of AZD2014 or rapamycin. The following experiments were performed after 48 hours. A‐D, Cell growth curves of all prostate cancer cell lines were determined by CCK8 assay every 24 hours. E, G, I, K, Cell apoptosis was detected by Annexin V fluorescence‐activated cell sorting (FACS), and the diagram shows representative apoptosis rates in prostate cancer cell lines treated with DMSO, AZD2014 or rapamycin. F,H,J,L, Statistical analysis of the apoptosis rates of prostate cancer cell lines. M, Western blot analysis of the relative expression of Bax and cleaved PARP. GAPDH served as a loading control. Data are presented as the mean ± SD from at least three independent experiments. (*P < .05, **P < .01)

Figure 3

AZD2014 induced cell cycle arrest at G0/G1 phase in CRPC cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were treated with either DMSO (CTR) or AZD2014 or rapamycin at the indicated concentration and further cultured for several days. A‐H, After 48 hours, flow cytometry was used to determine the proportion of all prostate cancer cells in distinct cell cycle phases. I, After 48 hours, Western blot analysis of the relative expression of CDK4 and cyclin D1 was carried out. β‐Tubulin served as a loading control. The results are plotted as the mean ± SD of three independent experiments. (*P < .05,**P < .01; #P < .05, ##P < .01).

Figure 4

AZD2014 activated autophagy in CRPC cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were treated with either DMSO (CTR) or AZD2014 or rapamycin at the indicated concentration. The expression of related proteins was tested by Western blotting. A, Cells were treated with AZD2014 or rapamycin at concentrations of 200, 250, 400 and 600 nM for 48 hours in the presence or absence of 3‐MA. The cells were then stained with MDC to detect the formation of autophagosomes and immediately observed with a confocal microscope. Bars, 50 μm. B, The expression of Beclin‐1 and LC3A/B (LC3A/B‐1 and LC3A/B‐2) in prostate cancer cells treated as described above was assessed by immunoblot analysis

Figure 5

The effects of AZD2014 on migration and invasion in CRPC cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were treated with either DMSO (CTR) or AZD2014 or rapamycin at the indicated concentration. A, C, The invasive abilities of prostate cancer cell lines were determined with Transwell assays (representative photographs are magnified 200×). B, D, Representative quantification of migrated and invaded cells. E, The migratory abilities of prostate cancer cells were determined with wound‐healing assays (representative photographs are magnified 200×). F, Migrated cells were quantified. The inhibition of cell invasion by rapamycin and AZD2014 was observed in prostate cancer cells. The inhibition of cell migration by rapamycin or AZD2014 was observed in prostate cancer cells. Data are shown as the mean ± SD. (*P < .05, **P < .01)

Figure 6

The effects of AZD2014 on EMT in CRPC cells. C4‐2, CWR22RV1, C4‐2 DOCR and CWR22RV1 DOCR cells were treated with either DMSO (CTR) or AZD2014 or rapamycin at the indicated concentration. The following experiments were performed after 48 hours. A, Immunofluorescence staining of prostate cancer cells treated as described above for E‐cadherin and vimentin. Bars, 50 μm. B, Prostate cancer cells were treated as described above, lysed and immunoblotted. The expression of EMT markers was detected via Western blotting. Rapamycin and AZD2014 increased the expression of E‐cadherin and decreased the expression of N‐cadherin and vimentin in prostate cancer cells. Data are shown as the mean ± SD. (*P < .05, **P < .01)