Purpose: Genetic instability is considered as one of the main pathways in human carcinogenesis. Since aberrant HGF/c-Met signaling causes human cancers, we investigated whether activation of aberrant c-Met signaling induced genetic instability and what the underlying mechanisms were.
Methods: Aneuploidy was assessed by Giemsa staining. The number of centrosome was determined by staining with ?-tubulin antibody. To analyze causes of centrosome amplification, we performed immunostaing for Cep-170 protein as a mature centrosome marker. Abnormality of chromosome segregation was measured by counting lagging and/or bridge chromosomes. Activation of Akt, Erk, JNK and p38 was analyzed by western blot analysis using corresponding phospho-specific antibodies. To investigate signaling pathways involved in centrosome amplification, we used signaling inhibitors, siRNA and dominant-negative mutants. To activate mitotic checkpoint, we used microtubule stabilizing drug, Taxol. Aurora-A and Polo-like kinase-1 (Plk-1) kinase activities were determined by immune complex kinase assay.
Results: Both stable and transient expression of M1268T, a constitutively active form of Met, increased the cell population with aneuploidy. In addition, supernumerary centrosomes and multinucleated cells were increased, indicating that aberrant HGF/c-Met signaling indeed caused genomic instability. Among the possible down-stream signaling molecules, phospho-Erk and phospho-Akt levels were found to be elevated in M1268T expressing cells compared to MOCK-transfected cells. LY294002, a PI3K inhibitor, but not U0126, an MEK inhibitor, could abolish both centrosome hyperamplification and multinucleated cell formation. Moreover, Akt gene knockdown by Akt siRNA as well as expression of dominant-negative mutant forms of Akt or PTEN significantly inhibited both phenotypes. Interestingly, ectopic expression of wild-type Akt promoted supernumerary centrosomes indicating that activation of PI3K-Akt axis is both necessary and sufficient. Furthermore, we observed that the increase in aneuploidy by M1268T was found in p53-/- HCT116 cells, but not in p53+/+ HCT116 cells, which strongly suggests that M1268T induces chromosomal instability depending on p53 status. We further checked the CIN (chromosomal instability)-inducing effect of M1268T in mouse embryonic fibroblasts from p53-/- background. As expected, M1268T expression also resulted in the increase of aneuploidy and polyploidy as well as multi- and micronuclei formation, thus expanding our observation to non-transformed cells.
Conclusion: Taken together, the data demonstrated that the aberrant Met signaling induces centrosome hyperamplification and the resultant increase of chromosomal instability via PI3K pathway depending on p53 status.
