Activation of p53-dependent apoptosis by acute ablation of glycogen synthase kinase-3β in colorectal cancer cells

Purpose: The restoration of checkpoint mechanisms may provide a rational anticancer approach, but the molecular circuitries of how this can be achieved therapeutically are poorly understood. A pivotal signaling network in colorectal cancer cells involves glycogen synthase kinase-3 (GSK3 beta), a multifunctional kinase whose role in tumor cell survival is not defined. Experimental Design: We used molecular, genetic, and pharmacologic antagonists of GSK3 beta in P53+/+ or P53(-/-) colorectal cancer cells. We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, and cell death by multiparametric flow cytometry. A xenograft colorectal cancer model was used to study antitumor activity in vivo. Results: Treatment of P53(+/+) colorectal cancer cells with pharmacologic inhibitors of GSK3 beta resulted in sustained elevation of p53, with up-regulation of p21(Waf1/Cip1) and loss of survivin levels. Molecular targeting of GSK3 beta by overexpression of a GSK3 beta dominant-negative mutant, or acutesilencing of GSK3 beta by RNA interference, reproduced the induction of transcriptionally active p53 in colorectal cancer cells. This pathway was recapitulated by deregulated Wnt/T-cell factor signaling, with elevation of the tumor suppressor p14(ARF), and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3 beta blockade resulted in p53-dependent apoptosis, which was contributed by acute loss of survivin and inhibition of colorectal cancer growth in mice. Conclusions: Acute ablation of GSK3 beta in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorecial cancer patients retaining wild-type p53.