Abstract
Studies focused on the killing of activated B-RAF melanoma cells by the histone deacetylase (HDAC) inhibitor AR42. Compared to other tumor cell lines, PDX melanoma isolates were significantly more sensitive to AR42-induced killing. AR42 and the multi-kinase inhibitor pazopanib interacted to activate: an eIF2α-Beclin1 pathway causing autophagosome formation; an eIF2α-DR4/DR5/CD95 pathway; and an eIF2α-dependent reduction in the expression of c-FLIP-s, MCL-1 and BCL-XL. AR42 did not alter basal chaperone activity but increased the ability of pazopanib to inhibit HSP90, HSP70 and GRP78. AR42 and pazopanib caused HSP90/HSP70 dissociation from RAF-1 and B-RAF that resulted in reduced 'RAF' expression. The drug combination activated a DNA-damage-ATM-AMPK pathway that was associated with: NFκB activation; reduced mTOR S2448 and ULK-1 S757 phosphorylation; and increased ULK-1 S317 and ATG13 S318 phosphorylation. Knock down of PERK, eIF2α, Beclin1, ATG5 or AMPKα, or expression of IκB S32A S36A, ca-mTOR or TRX, reduced cell killing. AR42, via lysosomal degradation, reduced the protein expression of HDACs 2/5/6/10/11. In vivo, a 3-day exposure of dabrafenib/trametinib resistant melanoma cells to the AR42 pazopanib combination reduced tumor growth and enhanced survival from ~25 to ~40 days. Tumor cells that had adapted through therapy exhibited elevated HGF expression and the c-MET inhibitor crizotinib enhanced AR42 pazopanib lethality in this evolved drug-resistant population.
Keywords:
ER stress; autophagy; chaperone; death receptor.
MeSH terms
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Angiogenesis Inhibitors / administration & dosage
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Angiogenesis Inhibitors / pharmacology
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Animals
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Antineoplastic Combined Chemotherapy Protocols / pharmacology
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Autophagosomes / drug effects
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Autophagosomes / metabolism
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Blotting, Western
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Cell Line, Tumor
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Cell Survival / drug effects
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Drug Resistance, Neoplasm / drug effects
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Drug Synergism
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Endoplasmic Reticulum Chaperone BiP
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Eukaryotic Initiation Factor-2 / genetics
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Eukaryotic Initiation Factor-2 / metabolism
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Histone Deacetylase 6
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Histone Deacetylase Inhibitors / administration & dosage
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Histone Deacetylase Inhibitors / pharmacology
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Histone Deacetylases / genetics
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Histone Deacetylases / metabolism
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Humans
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Imidazoles / pharmacology*
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Indazoles
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Kaplan-Meier Estimate
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Male
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Melanoma / drug therapy*
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Melanoma / genetics
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Melanoma / metabolism
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Mice, Nude
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Microscopy, Fluorescence
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Oximes / pharmacology*
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Phenylbutyrates / administration & dosage
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Phenylbutyrates / pharmacology*
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Pyridones / pharmacology*
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Pyrimidines / administration & dosage
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Pyrimidines / pharmacology*
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Pyrimidinones / pharmacology*
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RNA Interference
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Sulfonamides / administration & dosage
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Sulfonamides / pharmacology*
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Xenograft Model Antitumor Assays
Substances
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Angiogenesis Inhibitors
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Endoplasmic Reticulum Chaperone BiP
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Eukaryotic Initiation Factor-2
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HSPA5 protein, human
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Histone Deacetylase Inhibitors
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Hspa5 protein, mouse
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Imidazoles
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Indazoles
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OSU-HDAC42 compound
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Oximes
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Phenylbutyrates
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Pyridones
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Pyrimidines
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Pyrimidinones
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Sulfonamides
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trametinib
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pazopanib
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HDAC6 protein, human
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Histone Deacetylase 6
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Histone Deacetylases
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dabrafenib