Fig. 7: CDK1 promotes tumor progression through destabilizing pVHL and inversely correlates with pVHL expression in TNBC.

A MDA-MB-231 cells stably expressing control, CDK1 or VHL shRNAs were generated and western blotting was performed with indicated antibodies. B Total RNA was isolated from MDA-MB-231 cells. Relative expression of VEGF, GLUT1 and MMP2 in cells stably expressing control, CDK1 or VHL shRNAs were determined by quantitative PCR. Results represent the mean ± SD of three independent experiments. **p < 0.01, shCDK1 vs Ctrl. ^##p < 0.01, shCDK1shVHL vs shCDK1. C MDA-MB-231 cells were treated with RO-3306 and western blotting was performed with indicated antibodies. (D) Cell proliferation assay was performed in MDA-MB-231 cells. Results represent the mean ± SD of three independent experiments. ***p < 0.001, RO-3306 vs Vehicle, RO-3306 + shVHL vs RO-3306. E Transwell assays were performed to measure effects of RO-3306 on migratory (left) and invasive (right) abilities of BT-549 cells. Results represent the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001, RO-3306 vs Vehicle, RO-3306 + shVHL vs RO-3306. F MDA-MB-231 cells were treated with RO-3306. Cell survival was determined. Results represent the mean ± SD of four independent experiments. **p < 0.01, ***p < 0.001, RO-3306 vs Vehicle. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, RO-3306 + shVHL vs RO-3306. G, H Mice with subcutaneously established tumors from MDA-MB-231 cells stably expressing indicated shRNAs were treated with Vehicle, RO-3306 (4 mg/kg), cisplatin (2 mg/kg) or combination. Mice were sacrificed to dissect xenograft tumors and measure tumor weights (H). Results represent the mean ± SD of six independent experiments. **p < 0.01, Vehicle + Cisplatin vs Vehicle, RO-3306 + Cisplatin vs Vehicle + Cisplatin, RO-3306 + shVHL + Cisplatin vs RO-3306 + Cisplatin. I Cell proliferation assay was performed in MDA-MB-231 cells. Results represent the mean ± SD of three independent experiments. ***p < 0.001, FLAG-VHL WT + RO-3306 vs FLAG-VHL WT Vehicle + , FLAG-VHL SA + RO-3306 vs FLAG-VHL SA + Vehicle, FLAG-VHL SD + RO-3306 vs FLAG-VHL SD + Vehicle, FLAG-VHL SA + Vehicle vs FLAG-VHL WT + Vehicle, FLAG-VHL SD + Vehicle vs FLAG-VHL WT + Vehicle. J Mice with subcutaneously established tumors from MDA-MB-231 cells stably expressing indicated plasmid were treated with Vehicle or RO-3306 (4 mg/kg). K Representative images of IHC staining of CDK1, PIN1 and pVHL in TNBC patient samples (n = 71). Scale bars, 100 μm. L, M Correlation analysis of CDK1 and pVHL expression levels as well as PIN1 and pVHL expression. N The working model to illustrate that CDK1 and PIN1 destabilize pVHL and promote tumor progression.