title: Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition.
abstract: OBJECTIVE
  Metastatic melanoma is characterized by extremely poor survival rates and hence novel therapies are urgently required. The ability of many anticancer drugs to activate autophagy, a lysosomal-mediated catabolic process which usually promotes cell survival, suggests targeting the autophagy pathway may be a novel means to augment therapy.
  
  
  METHODS
  Autophagy and apoptosis were assessed in vitro in human melanoma cell lines in response to clinically achievable concentrations of the endoplasmic reticulum (ER) stress-inducing drugs fenretinide or bortezomib, and in vivo using a s.c. xenograft model.
  
  
  RESULTS
  Autophagy was activated in response to fenretinide or bortezomib in B-RAF wild-type cells, shown by increased conversion of LC3 to the autophagic vesicle-associated form (LC3-II) and redistribution to autophagosomes and autolysosomes, increased acidic vesicular organelle formation and autophagic vacuolization. In contrast, autophagy was significantly reduced in B-RAF-mutated melanoma cells, an effect attributed partly to oncogenic B-RAF. Rapamycin treatment was unable to stimulate LC3-II accumulation or redistribution in the presence of mutated B-RAF, indicative of de-regulated mTORC1-dependent autophagy. Knockdown of Beclin-1 or ATG7 sensitized B-RAF wild-type cells to fenretinide- or bortezomib-induced cell death, demonstrating a pro-survival function of autophagy. In addition, autophagy was partially reactivated in B-RAF-mutated cells treated with the BH3 mimetic ABT737 in combination with fenretinide or bortezomib, suggesting autophagy resistance is partly mediated by abrogated Beclin-1 function.
  
  
  CONCLUSIONS
  Our findings suggest inhibition of autophagy in combination with ER stress-inducing agents may represent a means by which to harness autophagy for the therapeutic benefit of B-RAF wild-type melanoma.
authors.affiliation: Dermatological Sciences, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle Upon Tyne, UK.
authors.forename: Jane L
authors.initials: JL
authors.lastname: Armstrong
authors.forename: Marco
authors.initials: M
authors.lastname: Corazzari
authors.forename: Shaun
authors.initials: S
authors.lastname: Martin
authors.forename: Vittoria
authors.initials: V
authors.lastname: Pagliarini
authors.forename: Laura
authors.initials: L
authors.lastname: Falasca
authors.forename: David S
authors.initials: DS
authors.lastname: Hill
authors.forename: Nicola
authors.initials: N
authors.lastname: Ellis
authors.forename: Salim
authors.initials: S
authors.lastname: Al Sabah
authors.forename: Christopher P F
authors.initials: CP
authors.lastname: Redfern
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: Fimia
authors.forename: Mauro
authors.initials: M
authors.lastname: Piacentini
authors.forename: Penny E
authors.initials: PE
authors.lastname: Lovat
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D001713:Biphenyl Compounds / Q000008:administration & dosage / Q000494:pharmacology; D015153:Blotting, Western; D001897:Boronic Acids / Q000008:administration & dosage / Q000494:pharmacology; D000069286:Bortezomib; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D017313:Fenretinide / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009596:Nitrophenols / Q000008:administration & dosage / Q000494:pharmacology; D010879:Piperazines / Q000008:administration & dosage / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000008:administration & dosage / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D013449:Sulfonamides / Q000008:administration & dosage / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: C501332:ABT-737; D001713:Biphenyl Compounds; D001897:Boronic Acids; D008164:Luminescent Proteins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D009596:Nitrophenols; D010879:Piperazines; D011719:Pyrazines; D013449:Sulfonamides; D017313:Fenretinide; D000069286:Bortezomib; D048493:Proto-Oncogene Proteins B-raf