Sa1921 Molecular Detection of Colorectal Neoplasia: Do Markers That Target Acquired DNA Alterations in Sporadic Cases Also Discriminate Lynch Syndrome Cases?

IRS-4 was constructed. RKO cells were transfected i) with the recombinant plasmid pcDNA3IRS-4 expressing the protein, or ii) with the empty plasmid pcDNA3 as a control. The impact of IRS-4 overexpression was analyzed on cell cycle and IGF-1 signaling. Sixteen colon tumor samples and paired tumor-adjacent normal tissue were collected during surgery and processed after obtaining informed consent. We evaluated the relationship between IRS-4 level and the proteins expression involved in G1/S checkpoint in the tumor and paired normal of all the reported cases by western blot. RESULTS: As we expected, RKO cells overexpressing IRS-4 showed higher levels of this protein than the control cells. The high levels of IRS-4 caused an increase of pRb, Rb, E2F, Cyclin E and Cyclin D1 expression in RKO cells. IRS-4, pRb, Rb and E2F were highly expressed in the cancerous tissue, in comparison with the normal epithelium. We evaluated the correlations between IRS-4 expression and the level of proteins involved in the control of cell cycle. The levels of IRS4 correlated positively with pRb (rpearson=0.745, P value <0.0001), Rb (rpearson=0.810, P value <0.0001) and E2F (rpearson=0.856, P value <0.0001). Regarding to the IGF-1 pathway, the levels of IRS-4 showed a positive correlation with pIGF-1R (rpearson=0.657, P value <0.0001), pAKT (rpearson=0.537, P value <0.01), AKT (rpearson=0.520, P value <0.01) and GSK-3 (rpearson=0.911, P value <0.0001) expression. Our general finding showed that IRS4 stimulates the expression of the proteins that unlock the G1/S restriction point in the cell cycle in RKO cells. Interestingly, the proteins involved in the cell cycle increased especially in the cancerous tissue of the patients overexpressing IRS-4. CONCLUSION: Our results suggest that IRS-4 is an important protein involved in the cell cycle regulation in colon cancer cells and it could be a key protein in the CRC development.