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Activation of p53-Dependent Apoptosis by Acute Ablation of Glycogen Synthase Kinase-3ß in Colorectal Cancer Cells

Authors' Affiliation: Department of Cancer Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts

Requests for reprints: Dario C. Altieri, Department of Cancer Biology, LRB428, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. Phone: 508-856-5775; Fax: 508-856-5792; E-mail: dario.altieri{at}umassmed.edu.

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-3ß (GSK3ß), a multifunctional kinase whose role in tumor cell survival is not defined.

Experimental Design: We used molecular, genetic, and pharmacologic antagonists of GSK3ß 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 GSK3ß resulted in sustained elevation of p53, with up-regulation of p21^Waf1/Cip1 and loss of survivin levels. Molecular targeting of GSK3ß by overexpression of a GSK3ß dominant-negative mutant, or acutesilencing of GSK3ß 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, GSK3ß 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 GSK3ß 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.

Key Words: Apoptosis • GSK3ß • p53 • survivin • colorectal cancer

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