Dissecting the contribution of Mad2 to oncogenic transformation in cells lacking p107 and p130

Resumen

The first evidence of Mad2 as a target of p107 and p130 was provided by Dynlacht and coworkers (Ren et al., 2002), showing that the loss of these proteins led to dramatic derepression in E2F4 targets such as Mad2. Later, Hernando et al. found that an aberrant expression of Mad2 protein in pRb-deficient cells was associated with the acquisition of chromosomal instability (Hernando et al., 2004). Previously, our laboratory has found that p27-/-, p130-/-, and p107 +/- (5/6KO) mice showed significantly reduced viability and a broader tumor spectrum, compared to p27-/- or p27-/-; p130-/- (DKO) animals. Moreover, in contrast to tumors present in p107-/-; p27-/-, DKO or p27-/- mice that were adenomas well encapsulated, without signs of malignancy, the 5/6KO cohort displayed adenocarcinoma-like tumors with invasion of the surrounding tissues. Interestingly, these tumors showed Mad2 expression significantly upregulated. Considering these results, the aim of my dissertation has been to determine whether Mad2 upregulation contributes to the transformation of cells deficient for p107 and p130 proteins. For that purpose, we selected a non-transformed cell line (NMuMG) where the expression of p107 and p130 was silenced by lentiviral infection (DKD), leading to Mad2 upregulation. The main consequence of Mad2 overexpression was the induction of chromosomal instability. We found that the DKD cells displayed a higher frequency of events associated with CIN and this increment was due to Mad2 upregulation. Moreover, the presence of CIN was linked to the acquisition of migratory and invasive properties. In our study, the DKD cells grew forming invasospheres in 3D culture, a morphology described for metastatic breast cancer cells, and had a more migratory behavior. We were able to rescue both phenotypes by normalizing Mad2 levels, confirming its implication in the acquisition of aggressive characteristics. Altogether, these results indicate that the increment of Mad2 expression in cells lacking p107 and p130 contributes to the malignant transformation of the cells.