title: Effect of SMURF2 targeting on susceptibility to MEK inhibitors in melanoma.
abstract: BACKGROUND
  The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.
  
  
  METHODS
  We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.
  
  
  RESULTS
  Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).
  
  
  CONCLUSIONS
  Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.
authors.affiliation: Molecular Cancer Studies, Wellcome Trust Centre for Cell Matrix Research, University of Manchester, Oxford Rd, Manchester, UK.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Smith
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ferguson
authors.forename: Imanol
authors.initials: I
authors.lastname: Arozarena
authors.forename: Robert
authors.initials: R
authors.lastname: Hayward
authors.forename: Richard
authors.initials: R
authors.lastname: Marais
authors.forename: Anna
authors.initials: A
authors.lastname: Chapman
authors.forename: Adam
authors.initials: A
authors.lastname: Hurlstone
authors.forename: Claudia
authors.initials: C
authors.lastname: Wellbrock
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000201:enzymology; D051379:Mice; D008819:Mice, Nude; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D058990:Molecular Targeted Therapy / Q000379:methods*; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases / Q000187:drug effects / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays; D015027:Zebrafish
chemical_list: C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; C517975:AZD 6244; D000970:Antineoplastic Agents; D001549:Benzamides; D001562:Benzimidazoles; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; C471641:SMURF2 protein, human; D044767:Ubiquitin-Protein Ligases; D020928:Mitogen-Activated Protein Kinases