Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

In addition to the tumor suppressor p53 protein, also termed p53 alpha, the TP53 gene produces p53 beta and p53 gamma through alternative splicing of exons 9 beta and 9 gamma located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53 beta and p53 gamma at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9 beta/9 gamma. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and alpha variant, supporting our experimental data. Using siRNA specifically targeting exons 9 beta/9 gamma, we demonstrate that cell growth can be driven by modulating p53 beta and p53 gamma expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53 beta and p53 gamma promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53 beta enhanced p53 alpha transcriptional activity on the p21 and Bax promoters, while p53 gamma increased p53 alpha transcriptional activity on the Bax promoter only. Moreover, p53 beta and p53 gamma co-immunoprecipitate with p53 alpha only in the presence of p53-responsive promoter. Interestingly, although p53 beta and p53 gamma promote apoptosis in MCF7 cells, p53 beta and p53 gamma maintain cell growth in response to TG003 in a p53 alpha-dependent manner. The dual activities of p53 beta and p53 gamma isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53 beta and p53 gamma regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.