ERK5 is a promising therapeutic target for Clear Cell Renal Cell Carcinoma
INTRODUCTION
Clear-cell renal cell carcinoma (ccRCC) is characterized by a mutation in the von Hippel-Lindau tumor suppressor gene (VHL). A recent study revealed that extracellular signal-regulated kinase 5 (ERK5) is degraded through the ubiquitin-proteasome system, in a process mediated by VHL and HIF. Several studies have demonstrated that microRNA 143 (miR143), which displays decreased expression in RCC cells, regulates ERK5 expression. Our objective was to examine the effect of ERK5 in ccRCC and to investigate the potential of ERK5 as a therapeutic target.
METHODS
ERK5 expression in tumor cells of RCC patients was investigated using immunohistochemistry (IHC). Effect of proteasome inhibition on ERK5 in RCC cell lines was examined by western blot analysis. The expression of miR143 in human RCC cell lines was analyzed by quantitative PCR. MTS assays, flow cytometry, and western blotting were used to assess the effect of XMD8-92-mediated ERK5 inhibition in human RCC cell lines. We introduced ERK5 siRNA into A498 cells to analyze the apoptotic effect. Furthermore, RCC xenografts in mice were treated with XMD8-92 to investigate the effect of ERK5 inhibition.
RESULTS
A total of 168 surgical specimens taken from patients with localized RCC were analyzed using IHC—82 (48.8 percent) were ERK5 positive and 20 (11.9 percent) were ERK5 strongly positive. Proteasome inhibition appeared to enhance ERK5 expression in wildtype cell lines, but not in VHL-mutant cell lines. An inverse correlation between ERK5 expression and miR143 quantification was observed in RCC specimens (n=48, Fisher’s exact test p=0.0625). Furthermore, ERK5 inhibition resulted in the increase of the sub-G1 population as observed by flow cytometry, and cleaved RARP expression indicated an increase in apoptotic cells as seen by western blotting. ERK5 inhibition also reduced cell viability and ERK5 knockdown downregulated RARP and Bcl-2 expression. Also, XMD8-92 showed anti-tumor activity in tumor xenograft mice and it was observed Ki67 and CD34 expression downregulated following the treatment.
CONCLUSION
We observed that ERK5 expression in RCC was suppressed by miR143 expression. ERK5 degradation appears to involve the VHL pathway. The inhibition of ERK5 by XMD8-92 suggests induction of apoptosis. The anti-tumor effect of XMD8-92 shown in nude mouse xenograft tumor models may be due to the inhibition of angiogenesis. Our results suggest that ERK5 is a promising therapeutic target for the treatment of ccRCC.
Funding: JSPN KAKENHI Grant-in-Aid for Scientific Research(C)