Quantitative proteomic and phosphoproteomic studies reveal novel 5-fluorouracil resistant targets in hepatocellular carcinoma.

The development of chemoresistance remains the major obstacles to successful chemotherapy of hepatocellular carcinoma. The molecular mechanisms of drug resistance are complex. Identifying the key markers is crucial for development of therapeutic strategies to overcome resistance. In this study, we employed a cell-line model consisting of the 5-fluorouracil resistant Bel/5-Fu cell line and its parental Bel cell line. Using stable isotope dimethyl labeling combined with high-resolution mass spectrometry, in total, 8272 unique proteins and 22,095 phosphorylation sites with high localization confidence were identified. Our data indicated that the GnRH signaling pathway was involved in acquiring drug resistance, which has not been well elucidated. The western blotting results confirmed that the expression levels of PLCβ3 and PLCβ3 pS1105 in Bel/5-Fu cells were increased as compared to Bel cells. Furthermore, the protein levels of SRC and PKCδ, which could phosphorylate PLCβ3 at ser1105, were higher in Bel/5-Fu cells than in Bel cells. The knockdown of SRC, PKCδ and PLCβ3 increased the susceptibility of Bel/5-Fu cells to 5-Fu. Besides, the increased transcription levels of PLCβ3, PKCδ and SRC were significantly associated with decreased overall survival. Together, our deep proteomic and phosphoproteomic data reveal novel therapeutic targets for attenuating 5-Fu resistance in anti-cancer therapy. SIGNIFICANCE: It was reported that many hepatocellular carcinoma patients are resistance to 5-Fu. Although some studies related to drug resistance have been reported, the underlying mechanisms were not well elucidated. Unlike many single molecular studies, we focused on the global proteome and phosphoproteome analysis of Bel and Bel5-/Fu cell line using stable isotope dimethyl labeling to identify the previously unrecognized signaling pathway for causing 5-Fu resistance. Our results showed that the phosphorylation levels of PLCβ3 pS1105 and the protein levels of PLCβ3, PKCδ and SRC, which are major components of GnRH signaling pathway were higher in Bel/5-Fu cells than in Bel cells. Furthermore, knockdown of PLCβ3, PKCδ and SRC increased the susceptibility of Bel/5-Fu cells to 5-Fu. Overall, this is the first comprehensive proteomic and phosphoproteomic studies on 5-Fu resistant cell line Bel/5-Fu to identify the potential targets of attenuating chemoresistance in hepatocellular carcinoma.