Based in the need to develop more precise therapies, bladder cancer has been classified related to its gene expression pattern, in three main groups, similar to those in breast cancer: luminal like, basal like and p53 like bladder tumors (Choi, 2014; Dambauer, 2014). The biological and clinical significance of these signatures remains unclear. p63 characterize basal like bladder tumors, however, the p63- related molecular pathways in this pathology are not well known yet. Our hypothesis is that p63 acts as a protumoral factor, by activation of invasive signaling pathways. Knocking down of p63 in human bladder cancer cell lines MGHU3 and UMUC14 was developed using a TET-ON system where p63 depletion is induced externally under doxycycline (DOX). Cells were treated duringr 96 hours with DOX and p63 expression was analyzed by western blot. We observed an almost complete abrogation of p63 expression under DOX in MGHU3 and a 60% of depletion in UMUC14 cells. Gelatin degradation assay was performed in both cell lines. While DOX had no effect in the degradation ability of Non-Targeting cells, shp63_i cells under DOX degraded 60 percent less compared to without DOX. (p≤0,01; ANOVA
Tukey between with and without DOX). Migratory ability was tested using the wound healing assay in UMUC14 cells. p63 depleted cells presented a significantly lower potential of 70% to close the wound 24 h after initiation of the wound healing assay (p < 0.0001; t test Mann Whitney, DOXY vs untreated shp63_i cells). Spheroids of both cell lines were grown during 30 days and diameter was compared between with and without DOX (ANOVA with Tukey comparisons ***p<0.001 day 4; and between DOX 30 days and DOX 4 days *p<0.001 at day 16). Immunofluorescence of spheroids were performed and the results showed less intensity and number of positive cells not only for p63 but also for E-cadherin expression. Based on the results previously exposed we can conclude that p63 is an essential protein through which the cell mediates its progression mechanism affecting fundamental processes such as migration, matrix degradation and 3D growth capacity.