English abstract
Colorectal cancer (CRC) is a disease caused by a serial of genetic mutations and epigenetic aberrations including the signal transduction pathways. The MAPK pathway regulated by the Ras proteins upon activation by growth factors is the most commonly mutated. This causes a cascade of downstream protein phosphorylation and activation causing gene expression. In CRC, the MAPK pathway carries gain-of-function mutations in both KRAS and BRAF genes, generally activated by the upstream Sos1/2 proteins, which have not been well explored in CRCs especially in the K-Ras and B-Raf mutations carriers. The aim of this study was to observe the effect of Sosi and So$2 proteins on the regulation of the MAPK pathway when KRAS and BRAF mutations are present by monitoring the expression of the downstream proteins, MEK and ERK and their related phosphorylation forms. We knocked down Sosl and Sos2 proteins in HT-29, Caco-2 and MDA-MB-231 cells using Sosl siRNA. Specific exons of KRAS, BRAF, SOSI and SOS2 genes were amplified and subjected to sequencing to confirm the previously reported mutations. Western bolt was performed using anti-Sosl, anti-Sos2, anti-MEK, anti-pMĘK, anti-ERK and anti-PERK antibodies. All mutations (KRAS (G13D), BRAF (G464V) in MDA-MB-231 cells; BRAF (V600E) in HT-29 cells) reported in the literature about cell lines used in this study were confirmed by sequencing in this study. Knockdown of Sosl protein was observed when HT-29, Caco-2 and MDA-MB-231 cells were treated with siRNA directed against Sosl. In Caco-2 cells, Sos1 expressions as well as p-MEK and p-ERK were reduced after 18 hours of transfection. Whereas, a gradual knockdown of Sos1 protein was observed in MDA-MD 231 cells at 18 and 24 hours after transfection without affecting the expression of the downstream effectors. In HT-29 cells, siRNA Sosi transfection did not cause any change in the expression of the Sosi protein, indicating the inefficient shutdown of the protein. In conclusion this study demonstrated that Sos1/2 proteins are not necessary for the regulation of MAPK pathway when gain-of function mutations of KRAS and BRAF are present in a tumour cell and the regulation of the pathway could be coupled with proteins of other pathways to support tumourogenesis.
