title: Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies.
abstract: BACKGROUND
  Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.
  
  
  METHODS
  Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.
  
  
  RESULTS
  In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.
  
  
  CONCLUSIONS
  On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.
authors.affiliation: Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Fattore
authors.forename: Emanuele
authors.initials: E
authors.lastname: Marra
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Pisanu
authors.forename: Alessia
authors.initials: A
authors.lastname: Noto
authors.forename: Claudia
authors.initials: C
authors.lastname: de Vitis
authors.forename: Francesca
authors.initials: F
authors.lastname: Belleudi
authors.forename: Luigi
authors.initials: L
authors.lastname: Aurisicchio
authors.forename: Rita
authors.initials: R
authors.lastname: Mancini
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Torrisi
authors.forename: Paolo Antonio
authors.initials: PA
authors.lastname: Ascierto
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ciliberto
mesh_terms: D000906:Antibodies / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism* / Q000628:therapy*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020893:Receptor, ErbB-3 / Q000378:metabolism*; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D020893:Receptor, ErbB-3; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1
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