Down regulation of Wip1 phosphatase in MCF-7 breast cancer cells enhances doxorubicin-induced DNA damage and apoptosis

B48

MCF-7 breast cancer cells harbor an amplified PPM1D/Wip1 gene and over express Wip1 phosphatase. We show first that modulating Wip1 expression has both positive and negative affects on the growth of MCF-7 cells. Stable isogenic MCF-7 sublines designated as MCF-clones were established in which Wip1 protein expression was significantly down regulated by introduction of a plasmid-based PPM1D antisense RNA. Decreased Wip1 expression sensitized the MCF-clones to doxorubicin-mediated DNA damage-induced apoptosis. The enhanced apoptotic response was correlated with an increase in total p53 protein and N-terminal phosphoforms of p53-Ser15 and p53-Ser46. In addition increased expression of the pro-apoptotic Bax gene occurred at both the mRNA and protein level. Bax-siRNA knock down abrogated the enhanced apoptotic response mediated by increased Bax protein expression. Assays using the Waf1 and Bax promoters to drive a luciferase reporter gene revealed that luciferase activity driven by the Bax promoter was enhanced in MCF-clones while luciferase activity driven by the Waf1 promoter was decreased relative to parental MCF-7 cells. The study reveals a novel molecular mechanism involving Wip1 phosphatase knock-down, increased N-terminal p53 phosphorylation and an enhanced apoptotic response mediated by transcriptional activation of the Bax gene. Our results suggest that Wip1 phosphatase should be considered as a potential therapeutic target in breast tumors and other tumor types that over express it and provides a rationale for combining Wip1 knock-down strategies with anticancer drugs that induce DNA damage.