Abstract 4418: Oncogenic activation of RAS and RAF induce distinct ERK activation in the colon

Abstract

Abstract Background and aims: Activation of the RAS/RAF/MEK/ERK pathway occurs frequently in colorectal cancer (CRC), typically a result of somatic mutations to either KRAS or BRAF. These mutations are mutually exclusive, however, indicating that either alteration is sufficient to deregulate the mitogen-activated protein kinase (MAPK) pathway. The phenotypic properties of CRCs harboring either KRAS or BRAF mutation, are dissimilar, particularly with respect to their histological progression. In precancerous lesions, KRAS mutations are common to serrated and non-serrated hyperplastic aberrant crypt foci (ACF), whereas BRAF mutations are rarely found in non-serrated hyperplastic ACF. These earlier observations suggest that, despite sharing a common pathway, somatic activation of KRAS or BRAF may differentially affect downstream signaling events. In the following study, we examine the affects of KRAS and BRAF mutations on downstream signaling in genetically engineered mouse models. Methods: Transgenic mice were generated that express mutant forms of KRAS or BRAF within the colonic epithelium by crossing LSL-KRASG12D and LSL-BRAFV600E mice to Carbonic anhydrase 1 (Car1)-Cre mice. Colons were harvested from both KRAS and BRAF mutant mice at 8 weeks of age for histological and molecular evaluation; Car1-Cre mice served as a control for subsequent histological and molecular analyses. Results: Both KRAS and BRAF mutant mice developed widespread hyperplasia throughout the colonic epithelium by 8 weeks of age. Hyperplastic morphology is typified by an extreme lengthening of colonic crypts, and by the development of large, prominent goblet cells when compared with control colon. No such changes are found in the small intestine. Notably, crypt length is shorter in BRAF compared to KRAS mutant mice. Western blot analysis of colonic mucosa revealed that both KRAS and BRAF mutant mice have high levels of phosphorylated MEK and ERK expression compared to controls. However, the levels of phosphorylated ERK are dramatically lower in BRAF compared to KRAS mutant colons. Furthermore, CRAF phosphorylation was increased only in the KRAS colon. To evaluate the activation states of ERK in greater detail, the relative amounts of non-phosphorylated, singly phosphorylated and dually phosphorylated ERK-2 isoforms were measured in freshly scraped colonic mucosa using a nanofluidic proteomic immunoassay. Interestingly, up-regulation of singly phosphorylated ERK-2 was observed only in KRAS mutant mice, but not in BRAF mice, suggesting that a negative feedback loop is present in the BRAF colon, but not in KRAS mice. Conclusion: These observations suggest that KRAS and BRAF mutations have distinct effects on ERK phosphorylation during early stages of colon carcinogenesis, perhaps in part because CRAF is not fully activated in BRAF mutant mice. Further analyses of underlying mechanisms that may contribute to differential ERK phosphorylation will be investigated. Citation Format: Shingo Miyamoto, David A. Drew, Daniel W. Rosenberg. Oncogenic activation of RAS and RAF induce distinct ERK activation in the colon. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4418. doi:10.1158/1538-7445.AM2014-4418