title: Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human MCF10A isogenic cell lines.
abstract: KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.
authors.affiliation: Department of Internal Medicine III, Ulm University, Ulm, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Stolze
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Reinhart
authors.affiliation: Department of Internal Medicine III, Ulm University, Ulm, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Bulllinger
authors.affiliation: 1] Department of Translational Oncology, National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany [2] Heidelberg University Hospital, Heidelberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Fröhling
authors.affiliation: 1] Department of Internal Medicine III, Ulm University, Ulm, Germany [2] Department of Translational Oncology, National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Scholl
mesh_terms: D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D015398:Signal Transduction; D015854:Up-Regulation; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C000631429:PDK1 protein, human; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins