Genetic alterations and sensitivity to anti-cancer drugs in colorectal cancer cells

Abstract

Colorectal cancer (CRC), the third most common cancer in the world, develops through complicated genetic mutations that occur in a stepwise progression. Therefore, examining cells for genetic alterations has been considered important in furthering research into the treatment of CRC. In support of such research, we first investigated the effects of mutation of KRAS oncogene, which is the most common genetic alteration in CRC. To determine the effects of KRAS substitutions in different CRC cell lines, we initially generated plasmids for 7 KRAS mutation subtypes that occur frequently. We then compared downstream signaling and KRAS activity in transfected HEK293 cells and CRC cell lines harboring KRAS mutations. In HEK293 cells, the G12D and Q61L mutant substitutions increased RAS downstream signaling, and KRAS activity was markedly increased in both G12S and G13D. In contrast, among 18 CRC cells, SW480 harboring KRAS G12V exhibited the greatest level of signaling activation and KRAS activity was noticeably high in HCT15 CRC cells harboring G13D. Therefore, it seems that mutation at codon 12 and 13 activates K-RAS signaling and activity in CRC. To investigate further the relationship between genetic alterations and drug sensitivity, we initially confirmed the presence of genetic alterations of major 5 genes (NRAS, KRAS, BRAF, PI3KCA and PTEN) in 18 CRC cell lines by using Sanger sequencing. Then, we selected hot-spot mutation of 5 genes based on Cancer Cell Line Encyclopedia data (CCLE). Drug sensitivities of the 18 cell lines to 6 anti-cancer drugs (5-FU, oxaliplatin, SN-38, AZD6244, regorafenib and PLX4720) were evaluated. Due to non-computable IC50 values, the drug sensitivities of the 18 cell lines were arranged by survival at clinically relevant concentrations. Mann Whitney U test results indicated a significant association between PIK3CA mutation and oxaliplatin sensitivity. In addition, we confirmed that cell lines with a BRAF mutation showed sensitivity to AZD6244 and PLX4720. In conclusion, the presence of different effects of KRAS substitutions on CRC and the presence of different relationships between genetic alterations and drug sensitivity are indicative of variability in CRC cell lines.