title: Coexpression of NRASQ61R and BRAFV600E in human melanoma cells activates senescence and increases susceptibility to cell-mediated cytotoxicity.
abstract: Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in G(0)-G(1) phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(61ON)) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/Cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" cells.
authors.affiliation: Unit of Immunobiology of Human Tumors, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Petti
authors.forename: Alessandra
authors.initials: A
authors.lastname: Molla
authors.forename: Claudia
authors.initials: C
authors.lastname: Vegetti
authors.forename: Soldano
authors.initials: S
authors.lastname: Ferrone
authors.forename: Andrea
authors.initials: A
authors.lastname: Anichini
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Sensi
mesh_terms: D000483:Alleles; D017951:Antigen Presentation; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000502:physiology; D003602:Cytotoxicity, Immunologic; D004318:Doxycycline / Q000494:pharmacology; D004789:Enzyme Activation; D015972:Gene Expression Regulation, Neoplastic; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000096:biosynthesis* / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis* / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D015395:Histocompatibility Antigens Class I; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras); D004318:Doxycycline