HDAC-inhibition counteracts everolimus resistance in renal cell carcinoma in vitro by diminishing cdk2 and cyclin A

BACKGROUND: Targeted therapies have improved therapeutic options of treating renal cell carcinoma (RCC). However, drug response is temporary due to resistance development. METHODS: Functional and molecular changes in RCC Caki-1 cells, after acquired resistance to the mammalian target of rapamycin (mTOR)-inhibitor everolimus (Caki(res)), were investigated with and without additional application of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA). Cell growth was evaluated by MTT assay, cell cycle progression and apoptosis by flow cytometry. Target molecules of everolimus and VPA, apoptotic and cell cycle regulating proteins were investigated by western blotting. siRNA blockade was performed to evaluate the functional relevance of the proteins. RESULTS: Everolimus resistance was accompanied by significant increases in the percentage of G2/M-phase cells and in the IC(50). Akt and p70S6K, targets of everolimus, were activated in Caki(res) compared to drug sensitive cells. The most prominent change in Caki(res) cells was an increase in the cell cycle activating proteins cdk2 and cyclin A. Knock-down of cdk2 and cyclin A caused significant growth inhibition in the Caki(res) cells. The HDAC-inhibitor, VPA, counteracted everolimus resistance in Caki(res), evidenced by a significant decrease in tumor growth and cdk2/cyclin A. CONCLUSION: It is concluded that non-response to everolimus is characterized by increased cdk2/cyclin A, driving RCC cells into the G2/M-phase. VPA hinders everolimus non-response by diminishing cdk2/cyclin A. Therefore, treatment with HDAC-inhibitors might be an option for patients with advanced renal cell carcinoma and acquired everolimus resistance.