BRAF基因敲除HCT116细胞

The oncogenic BRAF(V600E) mutation activates the ERK1/2 pathway and is detected in 10% of human colorectal cancers (CRCs) where it is associated with poor prognosis. Inhibitors of BRAF have shown only modest efficacy in patients with CRC due to intrinsic drug resistance. We studied the CDK2/CDK9 inhibitor, fadraciclib, alone and in combination with the BRAF inhibitor encorafenib in isogenic human RKO CRC cells with two, one, or no BRAF alleles (RKO, A19, T29) and in BRAF wild-type HCT-116 cells, including Bax knockout HCT-116 cells. Treatment with fadraciclib was shown to suppress MCL-1 and phospho-MCL-1 (Ser64), induce a Bax-dependent apoptosis, and inhibit colony formation in a BRAF gene dose-dependent manner. Fadraciclib decreased phosphorylation of RNA polymerase II, indicating suppression of RNA transcription. The tumor growth inhibitory effect of fadraciclib plus encorafenib was synergistic. Fadraciclib decreased Rb phosphorylation, inhibited cell cycle progression, and promoted DNA damage as evidenced by cleavage of PARP, increased pH2AX (ser139), and activation of p53. In RKO versus A19 or T29 cells, drug treatment was associated with greater suppression of p-Rb and inhibition of apoptosis and the cell cycle. In a zebrafish xenograft model, fadraciclib plus encorafenib significantly reduced tumor size, concurrent with increased caspase-3 activation. In human CRCs, BRAF mutation was associated with overexpression of CDK2, and CDK9 overexpression was associated with worse patient survival. In conclusion, fadraciclib depletes MCL-1 to potentiate apoptosis and, combined with encorafenib, synergistically suppresses tumor cell growth in a BRAF gene dose-dependent manner. These data suggest a novel therapeutic strategy in CRCs with BRAF.

In colorectal cancer, and are major mutation points in the RAS-MAPK signaling pathway. These gene mutations are known to be important causes of resistance to anti-EGFR antibody therapies. Recently, it has been reported that Aurora kinase A (AURKA), one of the mitotic kinases, interacts with the EGFR-RAS-MAPK signaling pathway. In this study, we examined whether the combination of MK-5108 (AURKA inhibitor) and trametinib (MEK inhibitor) enhanced the antitumor effect for colon cancer cell lines. The combination of MK-5108 and trametinib showed synergistic enhancements of antitumor effect in three colon cancer cell lines harboring or mutation. Cell cycle analysis showed induction of G2/M and G1 arrests by MK-5108 and trametinib, respectively, and the potential enhancement of G1 arrest with the two drug combination. The addition of MK-5108 to trametinib enhanced the suppression of -ERK and other G1/S progression-related proteins expression. In HCT116 cells, harboring wild-type , the combination therapy induced more potent cell proliferation suppression and apoptosis induction than in knockout cells. These were related to potential enhancement of p53 expression and caspase activation. In conclusion, the combination of MK-5108 and trametinib may synergistically inhibit tumor cell division with or without mutation, and with either or mutation. Furthermore, the combination therapy could be more effective in wild-type cells.