Radiosensitization of HRAS-Mutant Bladder Cancer

Abstract

The ability of activated HRAS to promote radiation resistance and predict for sensitivity to potential radiosensitizers was tested in a bladder cancer model system. Mutations in HRAS, KRAS, and NRAS were analyzed using cBioPortal in a bladder cancer cohort (TCGA). We employed a panel of urothelial carcinoma cell lines – T24 (HRASG12V), J82 and 5637 (RASWT). Cell growth and survival was tested with colony formation and proliferation assays. Radiation survival was assessed by clonogenic survival assay. Immunoblots were used to confirm mutant HRAS expression, and target activation/knockdown in overexpression and knockdown studies. Radiosensitization of mutant-HRAS bladder cancer was tested using a flank cell line xenograft model in nude mice. Approximately 10% of bladder cancers feature alterations in one of three RAS genes; mutations in HRAS comprise greater than 50% of these. An HRAS activating (G12V) mutation was detected in T24. Consistent with other cancer lines featuring activated RAS, T24 is relatively insensitive to radiation. Direct inhibition of the MEK/ERK or PI3K/MTOR pathway by selumetinib or BEZ-235, respectively, decreased target protein activation and resulted in significant growth inhibition compared to control (p<0.05). Treatment with either selumetinib or BEZ-235 radiosensitized HRASG12V expressing cells (SER 1.3-1.8), but had more modest effects on cells with wildtype HRAS. Compared to control, T24 exhibited higher levels of phosphorylated DNA-PK and γH2AX one hour after combination treatment with radiation and selumetinib or BEZ-235. siRNA knockdown of HRAS radiosensitized T24, but not J82 or 5637 cells relative to non-targeting control. The combination of selumetinib and radiation therapy significantly delayed the growth of T24 tumors in vivo. Over 10% of bladder cancers feature alterations in the RAS family of genes. These mutations often lead to resistance to conventional therapies, including radiation. Inhibition of downstream MEK/ERK and PI3K/MTOR pathways can radiosensitize tumors with activating mutations in HRAS. Identification of additional mutation/drug combinations that result in radiosensitization may be valuable to advance the design of personalized radiation therapy.