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.