title: Differential modulatory effects of GSK-3β and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma.
abstract: BACKGROUND
  GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319.
  
  
  RESULTS
  MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis.
  
  
  CONCLUSIONS
  Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.
authors.affiliation: Division of Hematology-Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.
authors.forename: Qingjun
authors.initials: Q
authors.lastname: Liu
authors.forename: James W
authors.initials: JW
authors.lastname: Mier
authors.forename: David J
authors.initials: DJ
authors.lastname: Panka
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001557:Benzenesulfonates / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004357:Drug Synergism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000378:metabolism; D009336:Necrosis; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D021381:Protein Transport / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology* / Q000627:therapeutic use; D034622:RNA Interference; D000077157:Sorafenib; D013141:Spiro Compounds / Q000494:pharmacology; D047368:Tumor Burden / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: C494829:AIFM1 protein, human; D000970:Antineoplastic Agents; D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D001557:Benzenesulfonates; D050759:Cyclin-Dependent Kinase Inhibitor p21; D007211:Indoles; C548961:MI 319; D010671:Phenylurea Compounds; D011725:Pyridines; D013141:Spiro Compounds; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D009536:Niacinamide; D000077157:Sorafenib; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3