AB174. VHL protein: a new therapeutic target of renal cell carcinoma

OBJECTIVE: Although VHL gene is well-known as the most important tumor suppressor gene in renal cell carcinoma (RCC), the exact mechanism of VHL gene missense mutation in the pathogenesis of RCC is unclear. We studied on the mechanism of VHL gene missense mutation in the function loss of VHL protein (pVHL), and discussed the possibility of pVHL as a new therapeutic target of RCC. METHODS: The VHL-deficient cell line 786-O was transfected with wild-type and mutant VHL vectors. Stable transfection was established through 10-day G418 selection. The expression of downstream molecular of VHL gene (HIF-2a, Glut-1) was detected to explore whether the mutant pVHL still retained its E3 ligase function. Then we used cycloheximide (CHX) method to detect the half-time of wild-type and mutant pVHL, and retested it after treating with celastrol (an extractive from a traditional Chinese medicine). Co-IP was done to study the exact mechanism of mutant pVHL degradation, and look into the effect of celastrol on mutant protein protection. RESULTS: In the stable cell line over expressed with mutant pVHL, HIF-2a and Glut-1 was obviously lower than constant cell line; the half-time of missense mutant pVHL was shorter than the wild-type one(t1/2 mut =1.5 h vs. t1/2 wt =4 h). After treated with celastrol, the half-time of mutant pVHL increased to 3 h. Co-IP indicated that celastrol increased the interaction between mutant pVHL and HSP70, and decreased the HSP90 binding. CONCLUSIONS: The mechanism of VHL gene missense mutation in tumorigenesis is not loss of protein intrinsic function, but the rapid degradation of mutant pVHL. Therapeutic modification of missense pVHL degradation may provide new strategies for treatment of RCC. Celastrol protects missense pVHL mutants by regulating the protein-protein-interaction in molecular chaperone system, and may develop into a potential new drug for RCC.