Identification of Mutant K-Ras-dependent Phenotypes Using a Panel of Isogenic Cell Lines*

Background: Many studies analyzing K-Ras function rely on overexpression. Results: Knock-out and knock-in of endogenous mutant K-ras have modest effects on downstream signaling but strong effects on gene expression and transformation. Conclusion: Genomic manipulation allows physiological determination of WT and mutant K-Ras consequences. Significance: Gene expression patterns can be used to monitor inhibition of mutant K-Ras. To assess the consequences of endogenous mutant K-Ras, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. These include the cancer cell lines DLD1, HCT116, and Hec1A, in which either the WT or mutant K-ras allele has been disrupted, and SW48 colorectal cancer cells and human mammary epithelial cells in which a single copy of mutant K-ras was introduced at its endogenous genomic locus. We find that single copy mutant K-Ras causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and N-Ras occurs in some, but not all, of these cell lines. Mutant K-Ras also had relatively minor effects on cell proliferation and cell migration but more dramatic effects on cell transformation as assessed by growth in soft agar. Surprisingly, knock-out of the wild type K-ras allele consistently increased growth in soft agar, suggesting tumor-suppressive properties of this gene under these conditions. Finally, we examined the effects of single copy mutant K-Ras on global gene expression. Although transcriptional programs triggered by mutant K-Ras were generally quite distinct in the different cell lines, there was a small number of genes that were consistently overexpressed, and these could be used to monitor K-Ras inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant K-Ras signaling and phenotypes but that many depend on cell context and environmental cues.

[1]  V. Corces,et al.  Loss of the normal N-ras allele in a mouse thymic lymphoma induced by a chemical carcinogen. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Shirasawa,et al.  Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. , 1993, Science.

[3]  M. Wigler,et al.  Multiple ras functions can contribute to mammalian cell transformation , 1995, Cell.

[4]  D. Shalloway,et al.  Cell cycle-dependent activation of Ras , 1996, Current Biology.

[5]  S. Lowe,et al.  Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a , 1997, Cell.

[6]  V. Ferrans,et al.  Ras Proteins Induce Senescence by Altering the Intracellular Levels of Reactive Oxygen Species* , 1999, The Journal of Biological Chemistry.

[7]  Ming You,et al.  Wildtype Kras2 can inhibit lung carcinogenesis in mice , 2001, Nature Genetics.

[8]  Elizabeth M. Smigielski,et al.  dbSNP: the NCBI database of genetic variation , 2001, Nucleic Acids Res..

[9]  T. Jacks,et al.  Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. , 2001, Genes & development.

[10]  M. Malumbres,et al.  The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene. , 2002, Cancer research.

[11]  Manuel Serrano,et al.  Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. , 2003, Cancer cell.

[12]  E. Petricoin,et al.  Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. , 2003, Cancer cell.

[13]  R. DePinho,et al.  Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. , 2004, Cancer cell.

[14]  R. Del Maestro,et al.  Spheroid Preparation from Hanging Drops: Characterization of a Model of Brain Tumor Invasion , 2004, Journal of Neuro-Oncology.

[15]  R. Perez-soler,et al.  Complex effects of Ras proto-oncogenes in tumorigenesis. , 2003, Carcinogenesis.

[16]  T. Jacks,et al.  Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Waldman,et al.  B-Raf Is Dispensable for K-Ras-Mediated Oncogenesis in Human Cancer Cells 1 , 2004, Cancer Research.

[18]  A. Rosenthal,et al.  Transcriptional basis of KRAS oncogene-mediated cellular transformation in ovarian epithelial cells , 2004, Oncogene.

[19]  M Paesmans,et al.  The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis , 2004, British Journal of Cancer.

[20]  J. Mesirov,et al.  An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis , 2005, Nature Genetics.

[21]  Holger Sondermann,et al.  Regulation of Ras Signaling Dynamics by Sos-Mediated Positive Feedback , 2006, Current Biology.

[22]  Jeffrey T. Chang,et al.  Oncogenic pathway signatures in human cancers as a guide to targeted therapies , 2006, Nature.

[23]  A. Bardelli,et al.  Knock-in of oncogenic Kras does not transform mouse somatic cells but triggers a transcriptional response that classifies human cancers. , 2007, Cancer research.

[24]  B. Park,et al.  Knock-in of mutant K-ras in nontumorigenic human epithelial cells as a new model for studying K-ras mediated transformation. , 2007, Cancer research.

[25]  Suzanne Schubbert,et al.  Hyperactive Ras in developmental disorders and cancer , 2007, Nature Reviews Cancer.

[26]  Robert A. Weinberg,et al.  Ras oncogenes: split personalities , 2008, Nature Reviews Molecular Cell Biology.

[27]  D. Zecchin,et al.  Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses , 2008, Proceedings of the National Academy of Sciences.

[28]  F. Siannis,et al.  Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. , 2008, The Lancet. Oncology.

[29]  Patricia Greninger,et al.  A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival. , 2009, Cancer cell.

[30]  J. Reis-Filho,et al.  Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma , 2009, Pigment cell & melanoma research.

[31]  C. Sander,et al.  V600EBRAF is associated with disabled feedback inhibition of RAF–MEK signaling and elevated transcriptional output of the pathway , 2009, Proceedings of the National Academy of Sciences.

[32]  K. Kinzler,et al.  Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells , 2009, Science.

[33]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[34]  P. V. van Diest,et al.  Oncogenic KRAS desensitizes colorectal tumor cells to epidermal growth factor receptor inhibition and activation. , 2010, Neoplasia.

[35]  Jin Li,et al.  KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies. , 2010, Lung cancer.

[36]  A. Bardelli,et al.  Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. , 2010, JAMA.

[37]  Youngho Seo,et al.  Selective activation of p53-mediated tumour suppression in high-grade tumours , 2010, Nature.

[38]  L. Yin,et al.  RNAi‐mediated CD73 suppression induces apoptosis and cell‐cycle arrest in human breast cancer cells , 2010, Cancer science.

[39]  M. DePristo,et al.  A framework for variation discovery and genotyping using next-generation DNA sequencing data , 2011, Nature Genetics.

[40]  T. Jacks,et al.  Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus. , 2011, Blood.

[41]  Mingming Jia,et al.  COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer , 2010, Nucleic Acids Res..

[42]  Scott E. Kern,et al.  Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis , 2011, Nature.

[43]  J. Minna,et al.  Knockdown of Oncogenic KRAS in Non–Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy , 2011, Molecular Cancer Therapeutics.

[44]  T. Eberlein.  Association of KRAS p.G13D Mutation With Outcome in Patients With Chemotherapy-Refractory Metastatic Colorectal Cancer Treated With Cetuximab , 2012 .

[45]  Min Yu,et al.  TAK1 Inhibition Promotes Apoptosis in KRAS-Dependent Colon Cancers , 2012, Cell.