La down-regolazione del microRNA 1 e l'over-espressione di MET caratterizzano i carcinomi del colon-retto invasivi.

Colorectal carcinoma is frequently characterized by over-expression of the MET oncogene that encodes the tyrosine kinase receptor for the hepatocyte growth factor (HGF). MET over-expression leads to aberrant activation of the receptor. It elicits the activation of the ¿invasive growth program¿, in which proliferative responses are integrated by biological activities such as extracellular matrix degradation, cell polarization, migration and survival. Previous studies performed by our and other groups in MET over-expressing cancer cell lines, revealed that some microRNAs, namely miR-1, miR- 34b, miR-34c and miR-199a*, down-regulate MET expression at post- transcriptional level, thus reducing the invasive proprieties due to aberrant MET activation. MET over-expression in primary colorectal tumors is not due to gene amplification. Since many microRNAs are frequently down-regulated in tumors, we evaluated the expression of MET targeting microRNAs in 78 primary colorectal adenocarcinomas, in order to investigate if they could control MET expression. Quantitative Real-time PCR analyses showed that MET was significantly over-expressed in 84,6% of tumors, in absence of gene amplification, and revealed that miR-1 was also significantly down- regulated in 88,5% of cases. We tested if miR-1 could negatively regulate MET in colon cancer cell lines. Upon miR-1 transfection, HT29 and DLD1 cells showed a 75% and 50% reduction of MET mRNA expression level, respectively. MiR-1 over-expression led to a strong reduction of the MET protein and impaired the scattering, the viability and the ¿wound healing¿ ability of HT29 cells, in response to HGF. In addition, miR-1 transfected cells showed a 70% reduction of the directional migration in response to HGF. These results led us to consider miR-1 as an oncosuppressor gene for its ability to impair the ¿invasive growth program¿ in colon cancer cells, by directly acting on MET activated pathways. To verify if miR-1 reduction could be a possible mechanism of MET increase in colorectal cancers, we evaluated the ¿concordance¿ between miR-1 down-regulation and MET over-expression in primary colorectal tumors. We found a concordance miR-1/MET in 76,9% of tumors, at different stages of progression. In relation to the node invasion, the concordance miR-1/MET was present in 69,6% of patients without node invasion (N0) and in 87,1% of patients with node infiltrations (N1+N2) (p= 0,05). Analyzing the invasiveness of the tumors, the concordance miR-1/MET was present in 68,4% of the not invasive tumors (Dukes' A,B) and in the 85% of the invasive and metastatic ones (Dukes' C,D) (p= 0,07). Finally we investigated the existence of a correlation between miR-1 down-regulation and MET over- expression in primary tumors in relation to the presence or absence of metastases; we also evaluated the correlation between the MET over-expression and the up-regulation of MACC-1 (a known transcriptional activator of MET). Only in metastatic colorectal tumors MET showed a significant anti-correlation with miR-1 expression (Pearson= -0,85) and a positive correlation, even if less strong, with the increase of MACC-1 (Pearson= +0,66). In conclusion, this work demonstrates that miR-1 and MET are two important molecules that are functionally altered in colorectal tumors with invasive-metastatic proprieties. These evidences render MET and miR- 1 particularly attractive molecules as candidates for therapeutic treatment of colorectal cancer.