Introduction. Mutational activation of the KRAS proto-oncogene is an initiating event during the development of 45% of human colorectal carcinomas (CRC). The aim of this study was to assess whether endogenous mutant KRAS is essential during the late metastatic stages of tumor growth when cells have acquired multiple additional genetic changes. Experimental procedures. RAS status of C26 murine CRC cells was assessed by RAS activity assays, RT-PCR and cDNA sequencing. The mutant KRAS mRNA was targeted by RNA interference using a lentivirus-based expression system. Knockdown of mutant KRAS was tested by Western blotting. Cell morphology, cell proliferation and cell cycle distribution were all tested with standard assays. Liver metastases were induced by intrasplenic injection of tumor cells in BALB/c mice. The formation of liver metastases was analyzed by bioluminescence imaging. Subcutaneous tumor growth was analyzed by caliper measurements. CD8+ CTL were depleted by intraperitoneal injections of a monoclonal anti-CD8-antibody. MHC-I expression was assessed by FACS analysis. Results. C26 cells harbor constitutively active KRAS due to an activating mutation in codon 12. We isolated stable cell lines in which the mutant KRAS allele was stably suppressed by RNA interference (C26-KRKD). C26-KRKD cells showed reversion of morphological transformation, exhibited a reduced proliferation rate, showed restored contact inhibition and an increased percentage of cells in G1. While mice challenged with control cells developed massive liver metastases within 12 days, the potential to form liver metastasis was completely abrogated in KRAS-suppressed cells. C26-KRKD cells did grow out as benign subcutaneous tumors in 40% of the mice but this was not associated with morbidity. 30% of subcutaneous tumors regressed spontaneously, and this was dependent on CD8+ CTLs. These mice showed lasting anti-tumor-immunity against parental C26 tumor cell challenges. When C26-KRKD cells were injected in an athymic background, subcutaneous tumors were formed in 100% of the mice and none of these tumors regressed. C26-KRKD cells showed enhanced expression of MHC class I molecules at the cell surface. Conclusion. Sequence specific suppression of mutant KRAS by RNA interference in a highly aggressive and metastatic CRC cell line leads to reversion of malignant characteristics in vitro and to complete abrogation of the metastatic potential in vivo. KRAS knockdown facilitates the development of an effective CTL-mediated anti-tumor immune response.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]