Phosphorylation Of TOPK By ERK2 Is Involved In The Pathogenesis Of TKI Resistance In RCC

Objective:Our study found that TOPK is highly expressed in renal cell carcinoma and is associated with poor prognosis.In this study,by screening and verifying the new site S32 of ERK2 phosphorylation of TOPK,we explored the role and mechanism of TOPK phosphorylation in TKI resistance in RCC.Methods:1.The expression of RCC dataset TOPK in TCGA database was analyzed.2.Bioinformatics predict the potential phosphorylation sites of ERK2 phosphorylation of TOPK.3.We analyzed the phosphorylation of TOPK and truncated TOPK1-31del by in vitro kinase assay,and detected p-TOPK(S32)by intracellular kinase assay.4.ERK2 was knocked down in 786-0 and ACHN cells,and the level of p-TOPK(S32)was detected.5.The wild-type TOPK(TOPK-WT)and mutant TOPK(TOPK-S32A)were overexpressed in JB6 C141 and Caki-1 cell lines,and the differences in tumor cell proliferation and transformation were evaluated by growth curve and Softagar assay.6.The expression levels of p-TOPK(S32)in 786-0,786-0SR and ACHN cell lines were compared under sorafenib incubation.7.Sorafenib was combined with TOPK inhibitor to incubate 786-O-SR and ACHN cells,and the cell proliferation level was analyzed by MTT method and flow cytometry.8.Nude mice were inoculated with 786-O-SR,and the tumors in different drug groups were observed and the expression of p-H3(S10)was analyzed by immunohistochemistry.9.The expression of HGF,p-TOPK(S32),p-ERK1/2 and p-c-MET in 786-O-SR was analyzed by Western-Blot.Results:TOPK is highly expressed in RCC.ERK2 phosphorylate TOPK at the S32 site.Phosphorylation of TOPK S32 promote cell proliferation and transformation.p-TOPK(S32)was not inhibited by sorafenib in sorafenib-resistant RCC cells.Sorafenib combined with TOPK inhibitor can enhance the drug sensitivity of sorafenib-resistant cell lines.In the process of sorafenib resistance in RCC,elevated HGF is involved in activating the ERK2/TOPK pathway and inducing the resistance of renal cancer cells to sorafenib.Conclusion:TOPK is highly expressed in RCC and may serve as a potential prognostic indicator in RCC.ERK2 can phosphorylate TOPK at S32 and promote tumor cell proliferation.TOPK inhibitors combined with sorafenib can enhance the drug sensitivity of sorafenib-resistant RCC cells,and TOPK inhibitors have important potential clinical application value.HGF/c-MET is involved in activating the ERK2/TOPK pathway to induce sorafenib resistance in RCC cells.