HOME
Return to Table of Contents
CONSTITUTIVELY ACTIVE MEK1 CAUSES TRANSFORMATION AND XENOGRAFT GROWTH OF IMMORTALIZED INTESTINAL EPITHELIAL CELLS, AND IS ACCOMPANIED BY AN EPITHELIAL TO MESENCHYMAL TRANSITION
E Lemieux, V Durand, N Rivard
CIHR Team on Digestive Epithelium, University of Sherbrooke
Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Strong evidences exist for the critical involvement of Ras/Raf/MEK/ERK cascade in the regulation of intestinal epithelial cell proliferation. K-rasV12G is the most frequently mutated oncogene in colorectal cancer and targeted expression of K-rasV12G in the intestinal epithelium causes activation of the MEK/ERK pathway and tumorigenesis in mice. However, the precise role of MEK/ERK signaling pathway in EMT has not been elucidated.
METHODS: Retrovirus encoding the HA-tagged MEK1wild type (wtMEK) or constitutively active mutant of MEK1 (MEK1-S218D/S222D, caMEK) were used to infect normal intestinal epithelial cells (IEC-6). Modifications of gene expression were determined with Affymetrix rat microarrays. Expression of specific genes was confirmed by western blot and RT-PCR. Matrix metalloprotease (MMP) activities were determined by zymography and protein localisation by immunofluorescence. Cell morphology was verified by electron microscopy.
RESULT: 1- Expression of caMEK in IEC-6 cells led to their transformation as judged by their fibroblastic morphology, increased sensitivity to growth factors for DNA synthesis and autonomous cell cycling. 2- Microarray data showed that caMEK1 expression induces genes involved in cell adhesion, migration and resistance to anoikis/apoptose. 3- A significant induction of EMT markers including vimentin (5-fold), EGR-1 (3-fold), Snail1 (3-fold), Slug (4-fold), SIP1 (2-fold), serpinE2 (12-fold), integrinalpha6 (4-fold), MMP2 (4-fold) and MMP9 (3-fold) was observed in caMEK-expressing cells; reduction of E-cadherin (5-fold), ZO-1 (6-fold) and occludin (5-fold) was also noted. 4- Zymography studies confirmed the elevated MMP2 and MMP9 activities in media of caMEK-expressing cells. 5- Expression levels of Bcl-2, Bcl-XL and phosphorylated Bad were enhanced correlating with anoikis resistance of caMEK-expressing cells. 6- ChIP assays confirmed the increased binding of EGR-1 to Snail1 promoter in caMEK-expressing cells. 7- Injection of caMEK-expressing cells in nude mice led to rapid tumor formation; interestingly, the tumors were positive for PECAM-31 staining indicating their vascularization.
CONCLUSION: Constitutive activation of MEK/ERK pathway induces an epithelial to mesenchymal transition which confers a growth advantage, survival and capacity of invasion to intestinal epithelial cells.
CIHR MT144-05 to NR