Ciribilli Yari, Singh Prashant, Inga Alberto, Borlak Jürgen
Centre for Integrative Biology (CIBIO), University of Trento, 38123 Povo (TN), Italy.
Centre for Pharmacology and Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Oncotarget. 2016 Oct 4;7(40):65514-65539. doi: 10.18632/oncotarget.11804.
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.
c-Myc在肺癌代谢中的作用尚不确定。因此，我们研究了c-Myc在肺乳头状腺癌（PLAC）中的活性。基因组学揭示了90个显著上调的基因（>3倍），这些基因编码细胞生长、DNA代谢、RNA加工和核糖体生物合成相关蛋白，并且生物信息学分析确定了超过95%的上调基因上存在c-Myc结合位点（TFBS）。在33个新的TFBS位点进行的电泳迁移率变动分析（EMSA）证实了DNA结合活性，从公共数据库中检索到的染色质免疫沉淀测序（ChIP-seq）数据也证实这些位点与c-Myc结合。针对RNA末端磷酸环化酶样蛋白1（RCL1）、核糖体蛋白SA（RPSA）、核磷蛋白/核仁素3（NPM3）和己糖激酶1（HK1）开发的双荧光素酶基因报告分析证实了c-Myc的功能相关性，并且对过表达异位c-Myc的HEK293T细胞进行的ChIP分析表明，在RCL1、NPM3、HK1和RPSA的预测TFBS位点上，c-Myc的占据率增加。注意，c-Myc在染色质上的募集与阳性对照CCND2和CDK4相当。计算分析确定了主要调节因子（MR），即不均一核核糖核蛋白A1、核仁素嘌呤/嘧啶内切酶1、磷酸丙糖异构酶1、叶酸转运蛋白（SLC19A1）和核磷蛋白，它们影响高达90%的PLAC调节基因的活性。它们的表达分别被诱导了3倍、3倍、6倍、3倍、11倍和7倍。STRING分析证实了受调节基因之间的蛋白质-蛋白质相互作用，对脂肪酸合酶、丝氨酸羟甲基转移酶1、精氨酸1和己糖激酶2进行的蛋白质免疫印迹分析显示出肿瘤特异性诱导。已发表的敲低研究证实，这些蛋白质通过破坏肿瘤性脂肪生成、促进尿嘧啶积累以及抑制精氨酸代谢和葡萄糖衍生的核糖核苷酸生物合成来诱导细胞凋亡。最后，转化研究表明，MR和47个PLAC上调基因的高表达与肺腺癌患者的不良生存相关（风险比3.2，p<0.001），因此为PLAC的分子靶向治疗提供了理论依据。
