| The p53 homolog p63 encodes multiple protein isoforms either with (TAp63) or without (DeltaNp63) the N-terminal transactivation domain. Gene targeting of p63 shows that it is essential for epithelial stem cell renewal and for the development of stratified epithelia. Emerging evidence suggests that DeltaNp63 functions as a tumor suppressor, although the underlying mechanism is not well-understood. We sought to explore the molecular basis by which p63 executes its tumor suppressor function, particularly the suppression of metastasis. Ectopic expression of DeltaNp63alpha inhibits human breast cancer Hs578T cell invasion and Matrigel outgrowth. We analyzed the gene expression profile of Hs578T cells ectopically expressing DeltaNp63alpha using Affymetrix arrays. Notably, wild-type DeltaNp63alpha, but not its mutant derivatives, up-regulates Cluster of Differentiation 82 (CD82), a documented metastasis suppressor. Inhibition of cell invasion by DeltaNp63alpha is largely restored by elimination of CD82. On the other hand, knockdown of p63 leads to reduced CD82 expression concomitant with increased cell invasion, which is partially reversed by ectopic expression of CD82. Interestingly, inhibition of glycogen synthase kinase-3 beta (G5K3beta) by a pharmacological inhibitor or shRNA reduces DeltaNp63alpha and CD82 expression independent of beta-catenin. GSK3beta ablation promotes cell invasion, which is blocked by overexpression of CD82. These data suggest that DeltaNp63alpha functions as a tumor suppressor through up-regulation of CD82, and that GSK3beta regulates cell invasion by modulating the DeltaNp63alpha-CD82 pathway.;In addition, we investigated how TAp63 is regulated. We demonstrate that Nuclear Factor-kappa B (NF-kappaB) induces TAp63 gene expression. Inhibition of NF-kappaB by inhibitor of kappa B alpha (IkappaBalpha) super-repressor or a chemical inhibitor leads to down-regulation of TAp63 mRNA expression. The responsible elements for NF-kappaB-mediated TAp63 induction are located within the region from -784 to -296 in the TAp63 promoter, which contains two NF-kappaB binding sites. RelA/p50 complexes bind to both sites as shown by electrophoretic mobility shift assays. Furthermore, we show that an Sp1 site between the two NF-kappaB sites is important in NF-kappaB-mediated upregulation of TAp63. Together, these data reveal that TAp63 is a transcriptional target of NF-kappaB, and may play an important role in cell proliferation, differentiation and survival upon NF-kappaB activation by various stimuli. |
