Abstract

Colorectal cancer (CRC) is the third leading cause of cancer-related death in men and women in the United States. Microsatellite instability (MSI) is found in approximately 15% of sporadic colorectal cancers and in the majority of Lynch syndrome patients. MSI has been correlated to deficiency in the mismatch repair (MMR) genes and therefore MSI-High tumors exhibit higher mutation rates then non-MSI-High tumors. We recently reported on 26 MSI-High and 558 non-MSI-High CRCs that were profiled at Caris Life Sciences (Shagisultanova et al., 2015 ASCO Annual Meeting Abstract No. e14684 “Association of increase in BRCA2 gene mutations in microsatellite instable (MSI-H) colorectal cancer (CRC) with increased c-MET expression.”). Immunohistochemistry and genomic analyses were performed in the BRCA-mutant versus BRCA wild-type MSI-High tumors. BRCA2 mutations were highly enriched (50%) in MSI-High CRC. MSI-High tumors with BRCA2 frameshift mutations had high c-MET expression. c-MET overexpression is known to be associated with aggressive metastatic CRC. We hypothesized there may be a synergistic drug interaction between drugs that are used to treat BRCA2-deficient tumors and c-MET inhibitors. We further hypothesized there may be a mechanistic link between BRCA2-deficiency, double-strand breaks following DNA damage, and c-MET overexpression. Mitomycin C (MMC) is an anti-cancer chemotherapeutic agent, which causes DNA damage by inducing double strand breaks (DSBs) through DNA cross-linking. Tumors deficient in genes encoding for proteins involved in DNA repair such as BRCA2 show hypersensitivity to MMC. Crizotinib is a small molecule inhibitor of c-MET and ALK receptor tyrosine kinases. In the present studies, we tested CRC cell lines for sensitivity to MMC plus crizotinib. CRC cell lines treated with MMC activated a DNA damage response as measured by upregulation of -H2AX. Upon BRCA2 siRNA-mediated knockdown colorectal cancer cells became more sensitive to MMC shown by cleaved-PARP as a measure of apoptosis. Crizotinib inhibited the activation of c-MET in CRC cell lines treated with Hepatocyte Growth Factor (HGF). The combination treatment of colorectal cancer cells with crizotinib and MMC led to increased apoptosis (cleaved-PARP) compared to each drug alone. Combination treatment with increasing concentrations of both drugs in a CellTiter-Glo Cell Viability assay demonstrated a synergistic effect. However, we found no evidence for c-MET up-regulation upon effective BRCA2 knockdown in the absence or presence of DNA damage. Although there is no mechanistic link between BRCA2 mutation and c-MET overexpression, c-MET is frequently overexpressed in CRC in general and BRCA2 is frequently mutated in CRC especially in MSI-High cases. The combination of crizotinib with MMC appeared synergistic regardless of whether CRC cell lines were MSI-High or non-MSI-High. Our results prompt the clinical testing of the combination of MMC and crizotinib in advanced CRC.