Transcriptomic output of endogenous KRasG12D and modestly overexpressed MYC in MEFs
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ABSTRACT: We sought to compare the transcriptomic output of Kras activation with that of modestly overexpressed cMYC, to better understand the mechanism of oncogenic cooperation. We isolated fibrobalsts from mouse embryos carrying CRE-inducible alleles for KRasG12D (LSL-KRasG12D) and MYC (Rosa26-LSL-MYC), alongside double positive and wild-type control MEFs. All MEFs were infected at early passage with Adeno-CMV-CRE, to induced expression of conditional alleles, where present, and RNA was isolated 24hrs post infection.
Project description:Microarray expression data generated to compare the biological impact of KrasG12D allelic duplication in p53null mouse embryonic fibroblasts (MEFs). The RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through activating mutations in KRAS. Mouse models demonstrated that activation of a single endogenous mutant Kras allele is sufficient to promote lung tumour formation, but acquisition of other genetic alterations is required for malignant progression. Using a well-established lung cancer mouse model we recently demonstrated that advanced KrasG12D-driven spontaneous tumours frequently exhibit enhanced MAPK signalling and KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1), implying that mutant Kras copy gains are positively selected during lung cancer progression. To compare the oncogenic impact of a single mutant allele versus additional mutant Kras copy gain, we carried out a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells and show that these genotypes are phenotypically distinct. Title: Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities Authors: Emma M Kerr, Edoardo Gaude, Frances K Turrell, Christian Frezza and Carla P Martins For MEF generation, KrasLSL-G12D/+ ;p53Fx/Fx mixed background (C57Bl/6/129/Sv) animals were interbred and embryos collected at day E12.5 to overcome KrasLSL-G12D/G12D embryonic lethality and Cre-mediated recombination performed immediately after MEF generation. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-Glutamine for one passage and then infected with adenovirus-Cre (5âÃâ107âplaque-forming units/1 x 106 cells). Recombination of LoxP sites was confirmed by PCR analysis. Three independent embryos per genotype were analysed using GPL6887 Illumina MouseWG-6 v2.0 expression beadchip.
Project description:A transgenic mouse model of Kras-driven lung adenocarcinoma, with reversible activation of Myc, was used to explore the effect of Myc activity on lipid metabolism in lung tumours. Gene expression analysis links lipid metabolism, transport and storage to Myc activity. Lung cells from adeno-cre infected R26LSL-CMER and LSL-KrasG12D; R26LSL-CMER mice were isolated 14 days post-treatment with (LSL-KrasG12D; R26LSL-CMER and R26LSL-CMER) or without (R26LSL-CMER only) tamoxifen. GFP positive cells were FACs sorted. To increase RNA yield, cells from two mice of the same genotype and treatment were combined. There were two biological replicates for each condition. Gene expression was compared between lung cells with inactive MycER and lung cells expressing active MycER plus or minus KRasG12D.
Project description:We constructed LSL-KrasG12D/+ and Spc-Cre;LSL-KrasG12D/+ mice to investigate the role of microRNA in the pathogenesis of lung cancer
Project description:Transcriptional profiling of mouse Pancreatic cancer cells comparing Pdx1-Cre LSL-KrasG12D TGIF1L/L P53L/L cells with Pdx1-Cre LSL-KrasG12D P53L/L cells, and to determine the effects of TGIF1 deletion on PDAC gene expression.
Project description:Purpose: Emerging evidence in the field of RAS biology is beginning to illustrate that RAS mutations are functionally distinct. To elucidate functional differences between NRAS mutants we performed RNA-sequencing on mouse embryonic fibroblasts (MEFs) expressing different NRAS codon 61 variants from the endogenous gene locus Methods: MEFs were isolated from Tyr::CRE-ERT2 LSL-Nras mouse models containing different Nras variants in the endogeous gene locus. NRAS expression was induced in MEFs using an Adenoviral Cre delivery system. RNA was isolated from MEFs six days post-NRAS induction and submitted for RNA-sequencing Results: Differential expression and gene set enrichment analysis (GSEA) between NRAS variants revealed enrichment of transcripts associated with cellular proliferation between all mutant Nras alleles versus wild-type Nras. Further, MEFs expressing NRAS mutants capable of driving melanoma formation were enriched for transcripts associated with RAS-MYC signaling axis. Conclusion: While all NRAS mutants are capable of enhancing cellular proliferation versus wild-type NRAS, the melanomagenic potential of these mutants correlates with the ability to enhance activation of the RAS-MYC signaling axis
Project description:To investigate the role of SHP2 (Ptpn11) in pancreatic carcinogenesis, murine pancreatic whole tissue RNA samples of 9 week old mice with the genotypes Ptf1a-Cre;LSL-KrasG12D (ID-labels Kxxx) and Ptf1a-Cre;LSL-KrasG12D;Ptpn11fl/fl (ID-labels Mxxxx) were analyzed by microarray.
Project description:Pooled KRC (LSL-KrasG12D; Rb1L/L; Pdx1-Cre: oncogenic Kras and deleted Rb1 in the pancreas) cells derived from 2 month old mice were compared to pooled KC (LSL-KrasG12D; Pdx1-Cre: oncogenic Kras in the pancreas) cells derived from 8 month old mice.
Project description:RNA-sequencing of cancer cell lines derived from genetically engineered mouse models KPC (Ptf1a-Cre; LSL-KrasG12D; p53f/+) vs KPCAf/f (Ptf1a-Cre; LSL-KrasG12D; p53f/+; Arid1af/f) followed by GSEA revealed that gene signatures for EMT and stem cells were enriched in KPCAf/f, consistent with phenotypic observations of increased migration, invasion and anchorage-independent growth.
Project description:Transcriptional profiling of mouse Prostate cancer cells comparing Pbsn-Cre LSL-KrasG12D P53L/L cells with Pbsn-Cre LSL-BrafV600E P53L/L cells, and to determine the effects of Kras or Braf mutantion on murine PCa gene expression.