title: The impact of KRAS mutations on VEGF-A production and tumour vascular network.
abstract: BACKGROUND
  The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile.
  
  
  METHODS
  Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results.
  
  
  RESULTS
  Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter.
  
  
  CONCLUSIONS
  Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis.
authors.affiliation: Translational Research Laboratory, Institut Català d'Oncologia-IDIBELL, Gran Via 199-203, 08908 L'Hospitalet del Llobregat, Barcelona, Spain.
authors.forename: Agnès
authors.initials: A
authors.lastname: Figueras
authors.forename: Maria Antonia
authors.initials: MA
authors.lastname: Arbos
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Quiles
authors.forename: Francesc
authors.initials: F
authors.lastname: Viñals
authors.forename: Josep Ramón
authors.initials: JR
authors.lastname: Germà
authors.forename: Gabriel
authors.initials: G
authors.lastname: Capellà
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003062:Codon; D004195:Disease Models, Animal; D015870:Gene Expression; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000098:blood supply* / Q000235:genetics* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; D018631:ras Proteins