Transcriptional changes induced by KRAS mutants. Homo sapiens

The small GTPase KRAS transmits signals from cell surface receptors to various downstream signaling pathways, thereby regulating a multitude of cellular processes. Activating KRAS mutations occur in approximately 30% of human cancers and contribute to malignant transformation through constitutive activation of downstream signaling cascades. These mutations cluster in several hotspots, with codons 12 and 13 being most commonly. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. To address this hypothesis, we used MCF10A human mammary epithelial cells to establish isogenic cell lines that express eight different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological levels, and investigated the biochemical and functional consequences of the different KRAS variants in vitro. In addition, selected effects were evaluated in comparison to cells that overexpress mutant KRAS. The overall effects of mutants expressed at normal levels 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 or migratory abilities. KRAS-G12D, -G12V, -G13D, and -K117N were able to mediate EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by the K117N and Q61H variants. Analysis of known RAS effectors showed that none of the mutations led to increased phosphorylation of ERK, PDK1, or AKT. Both codon 13 mutations were associated with increased EGFR expression, which was not seen with other variants. Finally, comparative gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes, such as enhanced cytokine and p53 signaling, upon G13D expression. 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. Overall design: Total RNA was isolated using the RNeasy Mini Kit (Qiagen), and 500 ng of quality-checked total RNA was labeled and hybridized to HumanHT-12 v4 Expression BeadChips using whole genome BeadChip® Sentrix assays (Illumina). Measurements were performed in biological triplicates and data normalization and filtering was performed as previously reported using BRB Array Tools software package (available at http://linus.nci.nih.gov/BRB-ArrayTools.html).