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C-Myc targeted regulators of cell metabolism in a transgenic mouse model of papillary lung adenocarcinoma.

ABSTRACT: c-Myc's role in pulmonary cancer metabolism is uncertain. We therefore investigated c-Myc activity in papillary lung adenocarcinomas (PLAC). Genomics revealed 90 significantly regulated genes (> 3-fold) coding for cell growth, DNA metabolism, RNA processing and ribosomal biogenesis and bioinformatics defined c-Myc binding sites (TFBS) at > 95% of up-regulated genes. EMSA assays at 33 novel TFBS evidenced DNA binding activity and ChIP-seq data retrieved from public repositories confirmed these to be c-Myc bound. Dual-luciferase gene reporter assays developed for RNA-Terminal-Phosphate-Cyclase-Like-1(RCL1), Ribosomal-Protein-SA(RPSA), Nucleophosmin/Nucleoplasmin-3(NPM3) and Hexokinase-1(HK1) confirmed c-Myc functional relevance and ChIP assays with HEK293T cells over-expressing ectopic c-Myc demonstrated enriched c-Myc occupancy at predicted TFBS for RCL1, NPM3, HK1 and RPSA. Note, c-Myc recruitment on chromatin was comparable to the positive controls CCND2 and CDK4. Computational analyses defined master regulators (MR), i.e. heterogeneous nuclear ribonucleoprotein A1, nucleolin, the apurinic/apyrimidinic endonuclease 1, triosephosphate-isomerase 1, folate transporter (SLC19A1) and nucleophosmin to influence activity of up to 90% of PLAC-regulated genes. Their expression was induced by 3-, 3-, 6-, 3-, 11- and 7-fold, respectively. STRING analysis confirmed protein-protein-interactions of regulated genes and Western immunoblotting of fatty acid synthase, serine hydroxyl-methyltransferase 1, arginine 1 and hexokinase 2 showed tumor specific induction. Published knock down studies confirmed these proteins to induce apoptosis by disrupting neoplastic lipogenesis, by endorsing uracil accumulation and by suppressing arginine metabolism and glucose-derived ribonucleotide biosynthesis. Finally, translational research demonstrated high expression of MR and of 47 PLAC up-regulated genes to be associated with poor survival in lung adenocarcinoma patients (HR 3.2 p < 0.001) thus, providing a rationale for molecular targeted therapies in PLACs.

SUBMITTER: Ciribilli Y

PROVIDER: S-EPMC5323172 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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c-Myc targeted regulators of cell metabolism in a transgenic mouse model of papillary lung adenocarcinoma.

Ciribilli Yari Y Singh Prashant P Inga Alberto A Borlak Jürgen J

Oncotarget 20161001 40

c-Myc's role in pulmonary cancer metabolism is uncertain. We therefore investigated c-Myc activity in papillary lung adenocarcinomas (PLAC). Genomics revealed 90 significantly regulated genes (> 3-fold) coding for cell growth, DNA metabolism, RNA processing and ribosomal biogenesis and bioinformatics defined c-Myc binding sites (TFBS) at > 95% of up-regulated genes. EMSA assays at 33 novel TFBS evidenced DNA binding activity and ChIP-seq data retrieved from public repositories confirmed these to ...[more]

PMID: 27602772

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Project description:To study a complex network of gene expression changes underlying c-Myc-triggered carcinogenesis in lung we performed oligonucleotide array analysis of solid lung adenocarcinomas which developed in transgenic mice due to constitutive expression of c- Myc in alveolar type II epithelial cells and compared them with the non-transgenic lung as a control. The microarray analysis yielded 162 up regulated and 301 down regulated genes. The genes were further analyzed with regard to their biological functions, to known responsiveness to c-Myc and to the presence of potential c-Myc -binding sites in their promoters. We identified inappropriate expression of a variety of genes involved in the several pathways critical for the development of lung tumor: cell cycle, cell growth, cell death, adhesion, cytoskeleton organization, invasive and angiogenic capacity. Some genes identified in this study were known to be similarly changed in human malignancies and specifically in lung tumors which makes the SPC/c-Myc-transgenic mouse model useful for investigating human lung cancer. Many other genes differently expressed in lung tumors are novel targets for mechanism based therapies and serve as useful tumor markers. New c- Myc-responsive genes and putative c-Myc -targets identified in this study contributed to the collective examination of distinct c-Myc transcriptomes that are partly cell type- and species- specific The molecular rules defining c-Myc’s activity in lung cancer remain elusive. We therefore investigated the c-Myc gene networks in a transgenic mouse model of lung cancer. Genome wide gene expression profiling applied to histological well-defined tumors revealed 162 up and 301 repressed genes and identified novel c-Myc targeted cell cycle and apoptosis genes. To better understand transcriptional responses the co-occupancy of different transcription factors (TF) were analyzed. This defined composite modules with 64, 68, 96 and 92, 88 and 96% of tumor associated down-regulated genes having TF-binding sites for either c-Myc, Klf7, Gata3 or c-Myc, Sox18 and P53, respectively. The composite modules fitted 48% and 80% of regulated genes. Likewise, 75, 96, 89 and 44, 59 and 54% of up-regulated genes in tumor or non-tumor transgenic tissue were enriched for Myc, Elf5,Cebp and c-Myc, Hbp1 and Hif1 TF-binding sites, respectively to suggest diverse and distinct TF-clusters at different stages of disease. Importantly, the genes coding for TFs were regulated as well and electrophoretic mobility shift assay (EMSA) confirmed c-Myc DNA-binding activity at targeted promoters of regulated genes. Furthermore, computational biology defined Gata3, mortalin and moesin to function in master regulatory gene networks and experimental validation of the constructed networks was achieved by EMSA, RT-PCR, Western blotting and gene reporter assay. Lastly, evidence for c-Myc DNA binding activity at targeted gene promoters was also derived from published Chip-sequence data of 7 human cell lines and the clinical significance of findings can be demonstrated by their regulation in human lung cancer. In conclusion, novel c-Myc targeted cell cycle and apoptosis genes were identified to function in gene regulatory networks. The results of the present study improve an understanding of c-Myc transforming capacity and provide a molecular rationale for the development of molecularly targeted therapies.

Project description:BackgroundLung adenocarcinoma is a common malignant tumor, and its early diagnosis and treatment are key to improving patient survival rates. However, due to the non-specific early symptoms, many patients are already at an advanced stage when diagnosed. Non-targeted metabolomics analysis, as a method for comprehensive analysis of metabolites in the body, has been shown to have potential in the early diagnosis of cancer. This study aims to identify early-stage lung adenocarcinoma-specific biomarkers using non-targeted metabolomics analysis in an established mouse model. The intervention mechanism of indoleamine 2,3-dioxygenase (IDO) inhibitor in early-stage lung adenocarcinoma is explored to provide evidence for clinical disease treatment.MethodsTwenty specific-pathogen-free-grade female Kunming mice were divided into control group, experimental group, Epacadostatlow group, and Epacadostathigh group. After modeling, immune therapy intervention (epacadostat) was administered to the mice, and plasma and urine samples were collected from all mice on day 7 and day 28. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was performed to identify potential biomarkers for diagnosing early-stage lung adenocarcinoma. Cluster analysis and correlation analysis were used to explore the differential expression patterns of metabolites in different samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify enriched pathways of differentially expressed metabolites.ResultsA total of 348 metabolites were identified after merging the positive and negative ion modes. Among them, organic acids and derivatives (16.954%) and lipids and lipid-like molecules (15.517%) were the two major classes of metabolites in the early-stage lung adenocarcinoma mice. Anthranilic acid (vitamin L1), 1-methylhistidine, 12(R)-HETE, and hippuric acid were the major differentially expressed metabolites on both day 7 and day 28, and they showed correlations with each other. Metabolic pathway analysis revealed multiple dysregulated pathways in lung adenocarcinoma mice.ConclusionsUPLC-QTOF-MS analysis is a feasible method for identifying biomarkers of lung adenocarcinoma. Epacadostat, a novel and promising IDO inhibitor, may exert its therapeutic effect by modulating 1-methylhistidine and anthranilic acid (vitamin L1).

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C-Myc targeted regulators of cell metabolism in a transgenic mouse model of papillary lung adenocarcinoma.

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c-Myc targeted regulators of cell metabolism in a transgenic mouse model of papillary lung adenocarcinoma.

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