Transcriptional Repression of Sodium-Iodide Symporter in Thyroid Carcinoma

甲状腺癌中碘化钠同向转运体的转录抑制

• 批准号: 7367324
• 负责人: Kenneth Bruce Ain
• 金额: $ 12.6万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367324
• 关键词: ADP Ribose Transferases; Accounting; Affinity Chromatography; Age-Years; Anisomycin; Aortic Aneurysm; Azacitidine; Binding; Binding Proteins; Binding Sites; Biological Assay; Bos taurus; Butyrates; C-terminal; CREB1 gene; Calcium; Calmodulin; Cancer Patient; Cancer cell line; Catalytic Domain; Cattle; Cell Line; Cells; Chromatin; Chromatin Structure; Chromosomes; Clinical; Clinical Trials; Clinical Trials, Other; Co-Immunoprecipitations; Complementary DNA; Complex; Condition; Consensus; Coupled; CpG Islands; Cross-Linking Reagents; Cycloheximide; DNA Binding; DNA Binding Domain; DNA Methylation; DNA Methyltransferase Inhibitor; DNA Repair; DNA ligase III; Data; Deoxyribonuclease I; Depsipeptides; Development; Digestion; Disease; Distal; Distant; Distantly Metastatic; Dominant-Negative Mutation; Dose; Drug usage; Effectiveness; Egtazic Acid; Electrophoretic Mobility Shift Assay; Elements; Emetine; Enhancers; Epigenetic Process; Fractionation; Functional disorder; Gel; Gel Chromatography; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Globulins; Goals; Grant; Histone Deacetylase; Histone Deacetylase Inhibitor; Human; Hypermethylation; I131 isotope; Immunoglobulin G; Immunohistochemistry; In Vitro; Incubated; Iodides; Iodine; Isotopically-Coded Affinity Tagging; Label; Life; Ligands; Light; Liquid Chromatography; Luciferases; Lung; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of thyroid; Maps; Measures; Membrane; Messenger RNA; Methodology; Methods; Molecular Conformation; Molecular Weight; Mus; Neoplasm Metastasis; Nuclear; Nuclear Extract; Nude Mice; Numbers; Oligonucleotides; Operative Surgical Procedures; Orphan Drugs; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Physiological; Physiology; Plasmids; Poly(ADP-ribose) Polymerases; Polymerase Chain Reaction; Precipitation; Probability; Production; Promoter Regions; Protein Binding; Protein Structure Initiative; Protein Synthesis Inhibitors; Proteins; Public Health; Publishing; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Proteins; Radiation; Radioactive; Radioactive Iodine; Radiolabeled; Range; Rate; Rattus; Reagent; Recombinants; Regulatory Element; Relative (related person); Reporter; Reporting; Repression; Repressor Proteins; Research; Run-On Assays; Running; Score; Site; Small Interfering RNA; Sodium Butyrate; Streptavidin; Systemic Therapy; TEV protease; TNFSF11 gene; Techniques; Testing; Thinking; Thyroglobulin; Thyroid Gland; Thyroid carcinoma; Thyrotropin; Time; Tissues; Transcription Initiation Site; Transcription factor genes; Transfection; Translations; Treatment Failure; Tretinoin; Trichostatin A; Trypsin; Type I DNA Topoisomerases; United States; United States Food and Drug Administration; Upstream Enhancer; Validation; Valproic Acid; Vorinostat; Weight; Western Blotting; Woman; activating transcription factor; base; butyrate; cancer cell; cell type; chelation; expectation; functional restoration; gene repression; human SLC5A5 protein; human tissue; in vivo; inhibitor/antagonist; mRNA Expression; men; mutant; novel; pre-clinical; promoter; radiotracer; receptor; repaired; research study; response; restoration; sodium-iodide symporter; tandem mass spectrometry; therapeutic target; thyroid transcription factor 1; transcription factor; tumor; tumor xenograft; uptake

DESCRIPTION (provided by applicant): Distantly metastatic dedifferentiated thyroid cancer is a fatal disease without effective treatment. This is from loss of ability to concentrate iodide, rendering it unresponsive to radioactive iodine, the only known systemic therapy. Iodide uptake requires expression of sodium-iodide symporter (hNIS). Hypermethylation of hNIS promoter region was thought to be a likely mechanism for loss of hNIS expression in thyroid cancer cells. hNIS expression and function is restored after treating these cells with demethylating and histone deacetylase-inhibiting agents. It was presumed that these agents permit transcriptional machinery access to the gene by relaxing chromatin compaction. 5-azacytidine (AzaC; DNA methyltransferase inhibitor) and sodium butyrate (NaB; putative histone deacetylase inhibitor) both, singly and together, restore hNIS expression and function to thyroid cancer cell lines. To show relaxed hNIS chromatin structures suggested by their presumed epigenetic effects, we assessed DNAse I digestion rate of genomic hNIS regions when AzaC and NaB restored hNIS mRNA expression and hNIS function. Surprisingly, these treatments did not produce enhanced DNAse I sensitivity suggesting the absence of effect on chromatin compaction. NaB-treated dedifferentiated thyroid cancer cells, transfected with a luciferase reporter construct containing hNIS gene promoter, stimulated both luciferase expression and native hNIS expression. Treating these cells with protein synthesis inhibitors (PSIs; cycloheximide, anisomycin, emetine), stimulated luciferase expression in a dose-dependent, time course-dependent, cell type-specific, and promoter-specific fashion; as well as restored endogenous hNIS gene mRNA expression. This suggested presence of a trans-acting transcriptional inhibitor, NIS-repressor, responsible for loss of hNIS expression. We mapped the hNIS promoter sequence region responsible for this effect, the NIS-repressor binding site (NRBS). NRBS, used as a radiolabeled probe in electrophoretic mobility shift assays with thyroid cancer cell nuclear extract, shows sequence-specific protein binding. These proteins were analyzed with liquid chromatography coupled to tandem mass spectrometry and revealed identity of a significant component of NIS-repressor. We hypothesize that loss of iodide transport activity in thyroid cancer results from loss of hNIS gene expression, consequent to NIS-repressor binding to the hNIS promoter. This novel mechanism may underlie or be in addition to presumed epigenetic mechanisms of hNIS gene reactivation reported with various pharmacologic agents. Treatments directed to reduce or antagonize NIS- repressor components should restore hNIS gene expression responsible for iodide transport enabling I-131 therapy of dedifferentiated thyroid cancers. Our aims are to confirm our identification of NIS-repressor protein and identify other protein complex components, discern its physiological and pathophysiological effects, and develop strategies targeting it. This should result in treatments to restore NIS activity in dedifferentiated thyroid cancers, restoring use of radioactive iodine to treat thyroid cancer metastases. Public Health Relevance Statement: Thyroid cancer, for unknown reasons, is the most rapidly increasing cancer in the United States in both men and women and it is the most common cancer in white women between 20 and 30 years of age. Although 75% of thyroid cancer patients respond adequately to radioactive iodine therapy, 10% of patients lose response to this treatment and eventually die from disseminated tumor. Development of new methods of restoring clinical response to radioactive iodine could save lives.

描述(申请人提供)：远处转移性去分化甲状腺癌是一种致命的疾病，没有有效的治疗方法。这是由于失去了浓缩碘的能力，使其对放射性碘失去了反应，放射性碘是唯一已知的系统疗法。碘摄取需要钠碘同向转运体(HNIS)的表达。HNIS启动子区域的高甲基化被认为是甲状腺癌细胞hNIS表达缺失的可能机制。在用去甲基化和组蛋白脱乙酰酶抑制剂处理这些细胞后，HNIS的表达和功能得到恢复。据推测，这些试剂允许转录机械通过放松染色质压缩来获得基因。5-氮胞苷(AzaC；DNA甲基转移酶抑制剂)和丁酸钠(NAB；可能的组蛋白去乙酰化酶抑制剂)单独或联合使用，可恢复甲状腺癌细胞hNIS的表达和功能。为了显示其可能的表观遗传效应所暗示的宽松的hNIS染色质结构，我们评估了当AzaC和NAB恢复hNIS mRNA表达和hNIS功能时基因组hNIS区域的DNase I消化速度。令人惊讶的是，这些处理并没有提高DNase I的敏感性，这表明对染色质压缩没有影响。用含hNIS基因启动子的荧光素酶报告载体转染NAB处理的去分化甲状腺癌细胞，可同时刺激荧光素酶和天然hNIS的表达。用蛋白质合成抑制剂(PSI；环己酰亚胺、茴香霉素、埃米汀)处理这些细胞，以剂量依赖、时间依赖、细胞类型特异和启动子特异的方式刺激荧光素酶的表达，并恢复内源性hNIS基因的mRNA表达。这表明存在一种反式作用的转录抑制因子，即NIS抑制因子，负责hNIS表达的丢失。我们定位了与这一效应有关的hNIS启动子序列区域，即NIS-抑制物结合位点(NRBS)。NRBS在甲状腺癌细胞核提取液的凝胶迁移率变化分析中用作放射性标记的探针，显示出序列特异性的蛋白质结合。用液相色谱-串联质谱仪对这些蛋白质进行了分析，发现它们是NIS抑制物的一个重要成分。我们假设，甲状腺癌中碘转运活性的丧失是由于hNIS基因表达的丧失，导致NIS抑制物与hNIS启动子结合。这一新的机制可能是各种药物报道的hNIS基因重新激活的假定表观遗传机制的基础或补充。针对减少或拮抗NIS抑制物成分的治疗应恢复负责碘转运的hNIS基因的表达，使I-131治疗去分化的甲状腺癌成为可能。我们的目标是确认我们鉴定的NIS-阻遏蛋白，并鉴定其他蛋白质复合体成分，识别其生理和病理生理学效应，并开发针对它的策略。这应该会导致在去分化的甲状腺癌中恢复NIS活性的治疗，恢复使用放射性碘治疗甲状腺癌转移。 公共卫生相关声明：由于未知原因，甲状腺癌是美国男性和女性中增长最快的癌症，也是20至30岁白人女性中最常见的癌症。尽管75%的甲状腺癌患者对放射性碘治疗有足够的反应，但10%的患者对这种治疗失去反应，最终死于播散性肿瘤。开发恢复临床对放射性碘反应的新方法可以拯救生命。