It has been shown that oncogenic HRAS is required for both induction and maintenance of EMT, mainly through its downstream effector ERK. A representative model for studying EMT has been devel oped in our lab following stable transfection HRASG12V in colon adenocarcinoma Caco 2 cells. The transformation process rendered mesenchymal like characteristics somehow to the cells as determined by their mor phology and global gene expression profile analysis. Many regulators and effectors have been described for the Rho family GTPases that may be implicated in their functions, including Focal Adhesion Kinase, a protein known to contribute to EMT, and fascin that is mainly involved with actin cytoskeletal organization as well as cell migration, downstream of Rho GTPases.

Fascin is an actin bundling protein normally upre gulated in several epithelial Inhibitors,Modulators,Libraries neoplasms and may have prognostic value as an early biomarker for more aggres sive colorectal adenocarcinomas, since it contributes to cancer cell migration in vitro and metastasis in vivo. Since KRASG12V and BRAFV600E mutations rarely coexist in human tumours, we aim to study their inde pendent and comparative contribution in migration and invasion of colorectal cancer Inhibitors,Modulators,Libraries cells through Rho GTPases signalling. Towards this end Caco 2 cells, that represent an intermediate adenoma of human colorectal cancer, were stably transfected to ectopically express KRASG12V and BRAFV600E. The doubling time and the cell cycle distribution by means of flow cytometry for each cell line have been examined.

Results obtained indicated Caco BR cells to have acquired a higher proliferation rate as compared to the parental cell line, Caco 2. For determining the transfor Inhibitors,Modulators,Libraries mation potential. a number of cell properties were ana lyzed following stable transfection. BRAFV600E induced cell properties, included altered morphology, colony for mation ability in Inhibitors,Modulators,Libraries soft agar, tumorigenicity in SCID mice. Here, we present evidence that BRAFV600E enhances migration and invasion properties in colon carcinoma cells through RhoA activation, while KRASG12V induces these properties less efficiently as compared to BRAFV600E, albeit through Cdc42 activation and filopodia formation. In parallel, HRASG12V induces high migration and invasion ability through Rac1. These results indicate that although KRAS and BRAF are members of the same pathway, different Rho dependent mechanisms are utilised by each oncogene to transform colon cancer cells.

These findings could be exploited towards targeted therapies to Rho pathway components depending on the genetic background of the cancer patient. Materials and methods Inhibitors,Modulators,Libraries Cell culture Caco 2, HT29 and Vandetanib cancer DLD 1 human colon adenoma carci noma cell lines were obtained from American Type Cul ture Collection and DKO 4 cells were kindly provided by Drs T. Sasazuki and S. Shirasawa.