title: Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.
abstract: BACKGROUND
  Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses.
  
  
  METHODS
  Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.
  
  
  RESULTS
  We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses.
  
  
  CONCLUSIONS
  These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.
authors.forename: Lin
authors.initials: L
authors.lastname: Jiang
authors.forename: Cécile
authors.initials: C
authors.lastname: Campagne
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Sundström
authors.forename: Pedro
authors.initials: P
authors.lastname: Sousa
authors.forename: Saima
authors.initials: S
authors.lastname: Imran
authors.forename: Monika
authors.initials: M
authors.lastname: Seltenhammer
authors.forename: Gerli
authors.initials: G
authors.lastname: Pielberg
authors.forename: Mats J
authors.initials: MJ
authors.lastname: Olsson
authors.forename: Giorgia
authors.initials: G
authors.lastname: Egidy
authors.forename: Leif
authors.initials: L
authors.lastname: Andersson
authors.affiliation: Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden. Anna.Golovko@imbim.uu.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Golovko
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D014644:Genetic Variation; D005838:Genotype; D006734:Horse Diseases / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D006801:Humans; D020935:MAP Kinase Signaling System; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000662:veterinary*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics
chemical_list: D015513:Oncogene Proteins; D048493:Proto-Oncogene Proteins B-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016283:Proto-Oncogene Proteins p21(ras)