Starvation microenvironment activates Yes-associated protein 1 to regulate autophagy and promote proliferation of bladder cancer cells

Abstract

Objective To explore the changes of malignant proliferation of bladder cancer cells under starvation microenvironment and its possible mechanism. Methods Bladder cancer T24 cells were divided into 2 groups: the normal group was cultured normally, while the starvation group was treated with serum-free HBSS for 6 h to simulate starvation microenvironment for cancer cells. The changes of autophagy and proliferation ability of cells were observed after treating with autophagy inhibitor CQ or transfection of siRNA of Yes-associated protein 1 (YAP1) gene, respectively. Finally, a risk prediction model was established based on the expression of YAP1 and autophagy-related gene LC3B, and the correlation between their levels and clinical prognosis was subsequently analyzed. Results CCK-8 and clone formation assays showed that the proliferation ability of bladder cancer cells was significantly increased after starvation induction (P < 0.01), which was caused by promoting cell cycle from G1 phase to S phase, while there was no significant difference in apoptosis between the 2 groups. The degradation of autophagy-related marker P62 protein and LC3-Ⅱ/Ⅰ ratio were detected by Western blotting and immunofluorescence assay, and the Results indicated that starvation induced up-regulation of autophagy in bladder cancer cells (P < 0.05). However, the proliferation of cancer cells was no longer affected by starvation after the autophagy inhibitor CQ blocked the autophagy tide. Overexpression and declined phosphorylation of YAP1 protein were observed after starvation induction (P < 0.01), and this result was not affected by inhibition of autophagy. In addition, the autophagy level and the proliferation of cells were significantly inhibited by siRNA interference in YAP1 expression (P < 0.05). Bioinformatics analysis showed that the level of YAP1 was higher in the cancer tissues of patients with higher malignant grade of bladder cancer, and the patients with higher expression of YAP1 and LC3B genes had worse prognosis. Conclusion The overexpression of YAP1 protein induced by starvation promotes the malignant proliferation of bladder cancer cells by enhancing the level of autophagy, and YAP1 and autophagy play a synergistic role in the development of bladder cancer.

Keywords

• bladder cancer
• autophagy
• yes-associated protein 1
• cell proliferation
• tumor microenvironment