DNA-PKcs interference sensitizes colorectal cancer cells to a mTOR kinase inhibitor WAY-600

Colorectal cancer (CRC) is one leading contributor of cancer-related mortalities. Mammalian target of rapamycin (mTOR), existing in two complexes (mTORC1/2), is a valuable target for possible CRC interference. In the current study, we showed that WAY-600, a potent mTOR inhibitor, only exerted moderate activity against primary and HT-29 CRC cells. We proposed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) could be the major resistance factor of WAY-600 in CRC cells. DNA-PKcs inhibitors, including NU7026 and NU7441, dramatically enhanced WAY-600-induced cytotoxic and pro-apoptotic effect against the CRC cells. Further, WAY-600-exerted cytotoxicity was significantly increased in DNA-PKcs-silenced (by targeted siRNA/shRNA) CRC cells, but was attenuated with DNA-PKcs overexpression. Our evidence suggested that DNA-PKcs Thr-2609 phosphorylation might be critical for WAY-600's resistance. Mutation of this site through introducing a dominant negative DNA-PKcs (T2609A) dramatically potentiated WAY-600's sensitivity in HT-29 cells. Meanwhile, overexpression of protein phosphatase 5 (PP5) dephosphorylated DNA-PKcs at Thr-2609, and significantly increased WAY-600's sensitivity in HT-29 cells. In vivo, WAY-600-induced anti-HT-29 xenograft growth activity was significantly potentiated with NU7026 co-administration. These results suggest that DNA-PKcs could be the major resistance factor of WAY-600 in CRC cells.