title: Genetic targeting of B-RafV600E affects survival and proliferation and identifies selective agents against BRAF-mutant colorectal cancer cells.
abstract: BACKGROUND
  Colorectal cancers carrying the B-Raf V600E-mutation are associated with a poor prognosis. The purpose of this study was to identify B-RafV600E-mediated traits of cancer cells in a genetic in vitro model and to assess the selective sensitization of B-RafV600E-mutant cancer cells towards therapeutic agents.
  
  
  METHODS
  Somatic cell gene targeting was used to generate subclones of the colorectal cancer cell line RKO containing either wild-type or V600E-mutant B-Raf kinase. Cell-biologic analyses were performed in order to link cancer cell traits to the BRAF-mutant genotype. Subsequently, the corresponding tumor cell clones were characterized pharmacogenetically to identify therapeutic agents exhibiting selective sensitivity in B-RafV600E-mutant cells.
  
  
  RESULTS
  Genetic targeting of mutant BRAF resulted in restoration of sensitivity to serum starvation-induced apoptosis and efficiently inhibited cell proliferation in the absence of growth factors. Among tested agents, the B-Raf inhibitor dabrafenib was found to induce a strong V600E-dependent shift in cell viability. In contrast, no differential sensitizing effect was observed for conventional chemotherapeutic agents (mitomycin C, oxaliplatin, paclitaxel, etoposide, 5-fluorouracil), nor for the targeted agents cetuximab, sorafenib, vemurafenib, RAF265, or for inhibition of PI3 kinase. Treatment with dabrafenib efficiently inhibited phosphorylation of the B-Raf downstream targets Mek 1/2 and Erk 1/2.
  
  
  CONCLUSIONS
  Mutant BRAF alleles mediate self-sufficiency of growth signals and serum starvation-induced resistance to apoptosis. Targeting of the BRAF mutation leads to a loss of these hallmarks of cancer. Dabrafenib selectively inhibits cell viability in B-RafV600E mutant cancer cells.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hirschi
authors.forename: Eike
authors.initials: E
authors.lastname: Gallmeier
authors.forename: Andreas
authors.initials: A
authors.lastname: Ziesch
authors.forename: Maximilian
authors.initials: M
authors.lastname: Marschall
authors.affiliation: Department of Internal Medicine II, University of Munich, Marchioninistr, 15, D-81377 Munich, Germany. frank.kolligs@med.uni-muenchen.de.
authors.forename: Frank T
authors.initials: FT
authors.lastname: Kolligs
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010091:Oximes / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D000970:Antineoplastic Agents; D016895:Culture Media, Serum-Free; D007093:Imidazoles; D010091:Oximes; D034741:RNA, Small Interfering; C482128:MAP2K2 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C561627:dabrafenib