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

Abstract

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-3β (GSK3β), a multifunctional kinase whose role in tumor cell survival is not defined. Experimental Design: We used molecular, genetic, and pharmacologic antagonists of GSK3β 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 GSK3β resulted in sustained elevation of p53, with up-regulation of p21Waf1/Cip1 and loss of survivin levels. Molecular targeting of GSK3β by overexpression of a GSK3β dominant-negative mutant, or acutesilencing of GSK3β 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 p14ARF, and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3β 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 GSK3β in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorectal cancer patients retaining wild-type p53. © 2005 American Association for Cancer Research.