Abstract
Abstract Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). The APC protein is an integral part of the destruction complex that controls cytoplasmic β-catenin levels by promoting ubiquitin-mediated degradation of β-catenin. Loss-of-function mutations in APC are commonly found in CRC, leading to constitutively high levels of β-catenin which then associates with TCF4, inducing expression of target genes that are important in cell growth. The KRAS/BRAF/MEK/ERK signaling pathway is also a major transforming growth regulatory signaling pathway. Indeed, gain-of-function mutations of KRAS and BRAF are detected in up to 60% of CRCs. Although both the Wnt/β-catenin and the ERK pathways are major pathways for tumorigenesis of intestinal epithelial cells (IECs), no significant interactions between these pathways have been identified. Methods. Retrovirus encoding the HA-tagged wild-type MEK1 (wtMEK) or activated MEK1 (MEK1S218D/S222D, caMEK) or oncogenic form of KRASG12V were used to infect and transform normal IECs in culture. The transcriptional activity of β-catenin/TCF4 complex was analyzed by using the luciferase reporter systems TOPFLASH and c-myc promoter (mutated or not for TCF4 binding elements). Expression levels of Wnt/β-catenin targets axin2, lef-1 and c-myc were evaluated by qPCR and protein localisation by immunofluorescence. A dominant-negative form of TCF4 (TCF4-DN) was used to verify the contribution of β-catenin/TCF4 complex in transformation induced by oncogenic activation of KRAS or MEK1. β-catenin transcriptional activity was analyzed in human CRC cells exhibiting APC mutation, treated or not with the MEK inhibitor U0126. Results. 1- Phase-contrast microscopy revealed that KRASG12V and caMEK-expressing IECs underwent a morphological change from an epithelial morphology to an elongated morphology. Cells clearly loose cell-to-cell contacts when compared to control cells. E-cadherin and β-catenin proteins disappeared from cell-cell junctions. 2- β-catenin/TCF4 transcriptional activity as well as mRNA levels of c-myc, axin2 and lef1 were markedly increased in IECs transformed by oncogenic MEK1 or KRAS. 3- Expression of the dominant-negative form of TCF4 severely attenuated IEC transformation induced by oncogenic MEK1 or KRAS. 4- Treatment of human CRC cell lines with U0126 significantly reduced axin2, lef-1 and c-myc mRNA levels, indicating that expression of these β-catenin target genes is likely dependent on MEK/ERK activity. Accordingly, β-catenin/TCF4 transcriptional activity was inhibited following MEK inhibition by U0126 or ERK dephosphorylation by DUSP4 expression. Conclusion. Our data indicate that the oncogenic activation of the KRAS/ERK signalling pathway in IECs up-regulates β-catenin/TCF4 transcriptional activity which contributes to the tumorigenic potential of this signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4169. doi:1538-7445.AM2012-4169
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