title: Inhibition of melanoma growth by small molecules that promote the mitochondrial localization of ATF2.
abstract: OBJECTIVE
  Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCε in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality.
  
  
  METHODS
  We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was conducted using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2.
  
  
  RESULTS
  We identified two compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, and anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the 12-O-tetradecanoylphorbol-l3-acetate (TPA)-induced membrane translocation of protein kinase C (PKC) isoforms, whereas both compounds decreased ATF2 phosphorylation by PKCε and ATF2 transcriptional activity. Overexpression of either constitutively active PKCε or phosphomimic mutant ATF2(T52E) attenuated the cellular effects of the compounds.
  
  
  CONCLUSIONS
  The imaging-based high-throughput screen provides a proof-of-concept for the identification of small molecules that block the oncogenic addiction to PKCε signaling by promoting ATF2 nuclear export, resulting in mitochondrial membrane leakage and melanoma cell death.
authors.affiliation: Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.
authors.forename: Tal
authors.initials: T
authors.lastname: Varsano
authors.forename: Eric
authors.initials: E
authors.lastname: Lau
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Feng
authors.forename: Marine
authors.initials: M
authors.lastname: Garrido
authors.forename: Loribelle
authors.initials: L
authors.lastname: Milan
authors.forename: Susanne
authors.initials: S
authors.lastname: Heynen-Genel
authors.forename: Christian A
authors.initials: CA
authors.lastname: Hassig
authors.forename: Ze'ev A
authors.initials: ZA
authors.lastname: Ronai
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001549:Benzamides / Q000737:chemistry / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D009281:Naphthalenes / Q000737:chemistry / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010627:Phenethylamines / Q000737:chemistry / Q000494:pharmacology; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
chemical_list: C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D000970:Antineoplastic Agents; D001549:Benzamides; D009281:Naphthalenes; D010627:Phenethylamines; C584940:SBI-0087702; C584939:SBI-0089410; D054852:Small Molecule Libraries; D013449:Sulfonamides; D049452:Green Fluorescent Proteins; D051744:Protein Kinase C-epsilon