Identification of genes involved in RAS oncogenic activity in human colorectal cancer cell line HCT116

The study aimed to identify genes essential for the maintenance of the transformed phenotype of the colorectal cancer cell line HCT116, which is dependent on the continued expression of the activated KRAS oncogene (KRASG13D). We generated HCT116 cell lines stably expressing an inducible shRNA-expressing retroviral vector targeting KRAS (Ngo et al., Nature, 2006). In cells engineered to express the bacterial tetracycline repressor, the shRNA is expressed specifically upon doxycycline addition. With this system, we showed that inducible down-regulation of KRAS is triggering cell death in the HCT116 cells, suggesting oncogene addiction in this cell line. In this study, we compared the gene expression profile of HCT116 cells where KRAS has been downregulated for different lengths of time, aiming at identifying RAS-target genes. We also compared the gene expression profile of the parental HCT116 cell line with two derived isogenic cell lines, Hke3 and Hkh-2, where the activated KRAS gene has been deleted by homologous recombination. HCT116 cell line stably expressing inducible shRAS vectors (Ngo et al., Nature, 2006) were treated with 25 ng/ml docycycline or vehicle control for 3 days or 6 days. Hke3, Hkh-2 and parental HCT116 were cultured in basal condition. All conditions were done in triplicate.