Activation of p53-dependent apoptosis by acute ablation of glycogen synthase kinase-3beta in colorectal cancer cells.
OBJECTIVE
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-3beta (GSK3beta), a multifunctional kinase whose role in tumor cell survival is not defined.


METHODS
We used molecular, genetic, and pharmacologic antagonists of GSK3beta 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 GSK3beta resulted in sustained elevation of p53, with up-regulation of p21(Waf1/Cip1) and loss of survivin levels. Molecular targeting of GSK3beta by overexpression of a GSK3beta dominant-negative mutant, or acute-silencing of GSK3beta 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, GSK3beta 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 GSK3beta 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.
Ghosh
Jagadish C
JC
Department of Cancer Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Altieri
Dario C
DC
D017209:Apoptosis; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D045325:HCT116 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D051034:Inhibitor of Apoptosis Proteins; D008301:Maleimides / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D011687:Purines / Q000494:pharmacology; D034622:RNA Interference; D000077022:Survivin; D051820:TCF Transcription Factors; D013830:Thiadiazoles / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin
C494356:4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione; C471843:6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine; C107489:BIRC5 protein, human; C493956:CDKN1A protein, human; C495270:CTNNB1 protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003598:Cytoskeletal Proteins; D007211:Indoles; D051034:Inhibitor of Apoptosis Proteins; D008301:Maleimides; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011687:Purines; C417521:SB 216763; D000077022:Survivin; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; C544895:Tcf7l2 protein, mouse; D013830:Thiadiazoles; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D051176:beta Catenin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D016203:CDC2 Protein Kinase; D038362:Glycogen Synthase Kinase 3