Regulator of cullins-1 expression knockdown suppresses the malignant progression of muscle-invasive transitional cell carcinoma by regulating mTOR/DEPTOR pathway

Abstract

Background: Regulator of cullins-1 (ROC1) is a key subunit in the cullin-RING ligase (CRL) protein complex. Our previous study indicated that ROC1 was essential for bladder cancer cell survival and that ROC1 knockdown inhibited CRL activity, triggering G2 phase arrest and senescence. However, the role of ROC1 in the malignant progression of bladder cancer remained unknown.

Methods: ROC1 expression in cancer cells was knocked down by siRNA silencing. The effects of ROC1 silencing were evaluated by in vitro assays for cell migration and by an in vivo mouse metastasis model. Epithelial-mesenchymal transition (EMT) induction was evaluated by immunofluorescence staining and western blotting of EMT-associated proteins. ROC1 expression in human tumours was further evaluated by immunohistochemical analysis.

Results: ROC1 knockdown suppresses bladder cancer cell migration by inhibiting EMT. ROC1 knockdown inhibited EMT by inhibiting mammalian target of rapamycin (mTOR) activity via the accumulation of the mTOR-inhibitory protein DEPTOR, a CRL substrate. DEPTOR knockdown partially rescued ROC1 knockdown-inhibited EMT and the ROC1-induced inhibition of cancer cell migration. Furthermore, in vivo studies using a nude mouse metastasis model confirmed the in vitro data. Finally, tissue microarray analysis of clinical bladder cancer specimens indicated a positive correlation between ROC1 expression and EMT.

Conclusions: ROC1 has an important role in the malignant progression of bladder cancer via the mTOR/DEPTOR pathway. ROC1 may serve as a novel therapeutic target for the treatment of muscle-invasive transitional cell carcinoma.

Figure 1

Inhibition of MI-TCC cell migration after ROC1 knockdown. (A, C and E) 253J cells; (B, D and F) EJ cells. Cells were transiently transfected with siROC1 or siCONT for 24 h and then subjected to cell viability CCK8 (A, B), wound-healing (C, D) or transwell migration assay (E, F). Representative results of three independent experiments are shown as means± s.e.m. *P<0.05.

Figure 2

ROC1 knockdown induction of MI-TCC cell MET. ROC1 knockdown-induced MET in 253J (A) and EJ (B) cells. Cells were transfected with siROC1 or siCONT for 96 h, stained for E-cadherin (red) and vimentin (green), and then examined under a confocal microscope. Nuclear DNA was stained with DAPI (blue). Scale bar=10 μm. The expression of EMT-associated markers in 253J (C) and EJ (D) cells were examined using western blots; the quantification relative to GAPDH was accomplished with densitometric analysis by Image J software. Representative results of three independent experiments are shown.

Figure 3

Effects of ROC1 knockdown on the mTOR pathway. mTOR pathway-associated protein expression in 253J (A) and EJ (B) cells was examined via western blot. EJ cells were transfected with siRNA for 96 h and then subjected to western blotting analysis (C) and transwell migration assays (D). Representative results of three independent experiments are shown. Columns, means of three independent experiments; bars, s.e.m. *P<0.05. Treated cells were subject to western blot analysis with GAPDH as a loading control, and the quantification relative to GAPDH was conducted by densitometric analysis using Image J software.

Figure 4

The effects of rapamycin-induced inhibition of the mTOR pathway on EMT. (A, C) 253J cells. (B, D) EJ cells. Cells were treated with different concentrations of rapamycin (0, 50 and 100 nmol l⁻¹) for 72 h and analysed via western blotting (A, B) or transwell migration assays (C, D). Representative results of three independent experiments are shown. Columns, means of three independent experiments; bars, s.e.m. *P<0.05. Treated cells were subject to western blot analysis with GAPDH as a loading control, and the quantification relative to GAPDH was conducted by densitometric analysis using Image J software.

Figure 5

Effects of ROC1 knockdown on metastasis in vivo. (A) Representative photographs of systematic metastasis detection in nude mice 12 weeks after the injection of shROC1 or shCONT cells. (B) IHC of lung metastasis nodules with the indicated antibodies in both groups. Columns, the mean luciferase activity of 10 mice; bars, s.e.m. ***P<0.01. Scale bar=50 μm.

Figure 6

ROC1 expression in human bladder cancer tissues. (A) Immunohistochemical staining of ROC1, DEPTOR and E-cadherin in human bladder cancer. Representative immunohistochemistry images of non-muscle-invasive or muscle-invasive cancer are shown. (B) Correlation of the immunostaining intensities of ROC1, DEPTOR and E-cadherin with cancer invasiveness. Immunostaining intensity was divided into two groups (high and low), and the association of intensity with cancer invasiveness (non-muscle-invasive cancer or muscle-invasive cancer) was analysed by a χ²-test for each protein. The P-value for each association is shown. Scale bar=50 μm.