Transcriptional Repression of Sodium-Iodide Symporter in Thyroid Carcinoma

甲状腺癌中碘化钠同向转运蛋白的转录抑制

• 批准号: 7638616
• 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/7638616
• 关键词: ADP Ribose Transferases; Accounting; Affinity Chromatography; Age-Years; Anisomycin; Aortic Aneurysm; Azacitidine; Binding; Binding Proteins; Binding Sites; Biological Assay; 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; Co-Immunoprecipitations; Complementary DNA; Complex; 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; 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; Oligonucleotides; Operative Surgical Procedures; Orphan Drugs; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Physiological; Physiology; Plasmids; Poly(ADP-ribose) Polymerases; Precipitation; Probability; Production; Promoter Regions; Protein Binding; Protein Structure Initiative; Protein Synthesis Inhibitors; Proteins; Publishing; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Proteins; Radiation; Radioactive; Radioactive Iodine; Radiolabeled; Rattus; Reagent; Recombinants; Regulatory Element; Relative (related person); Reporter; Reporting; Repression; Repressor Proteins; Research; Run-On Assays; Running; Site; Small Interfering RNA; Sodium Butyrate; Streptavidin; Systemic Therapy; TEV protease; TNFSF11 gene; Techniques; Testing; 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; Upstream Enhancer; Validation; Valproic Acid; Vorinostat; Weight; Western Blotting; Woman; activating transcription factor; base; cancer cell; cell type; chelation; effective therapy; expectation; functional restoration; gene repression; human tissue; in vivo; inhibitor/antagonist; mRNA Expression; men; mutant; novel; pre-clinical; promoter; protein complex; public health relevance; 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（由申请人提供）：远处转移性去分化甲状腺癌是一种无法有效治疗的致死性疾病。这是由于缺乏浓缩碘的能力，使其对放射性碘（唯一已知的全身治疗）无反应。碘摄取需要钠-碘同向转运体（hNIS）的表达。hNIS基因启动子区甲基化可能是甲状腺癌细胞hNIS基因表达缺失的机制之一。用去甲基化和组蛋白去乙酰化酶抑制剂处理这些细胞后，hNIS表达和功能恢复。据推测，这些代理人允许转录机器通过放松染色质压缩的基因。5-氮杂胞苷（AzaC; DNA甲基转移酶抑制剂）和丁酸钠（NaB;推定的组蛋白去乙酰化酶抑制剂）两者单独和一起恢复甲状腺癌细胞系的hNIS表达和功能。为了显示由其假定的表观遗传效应所暗示的松弛的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DNA酶I消化率。令人惊讶的是，这些处理没有产生增强的DNA酶I敏感性，表明对染色质致密化没有影响。NaB处理的去分化甲状腺癌细胞，转染含有hNIS基因启动子的荧光素酶报告构建体，刺激荧光素酶表达和天然hNIS表达。用蛋白质合成抑制剂（PSI;放线菌酮、茴香霉素、恩替卡韦）处理这些细胞，以剂量依赖性、时间过程依赖性、细胞类型特异性和启动子特异性的方式刺激荧光素酶表达;以及恢复内源性hNIS基因mRNA表达。这表明存在一种反式作用的转录抑制剂，NIS-阻遏物，负责hNIS表达的损失。我们绘制了hNIS启动子序列区域负责这种效果，NIS-阻遏物结合位点（NRBS）。在甲状腺癌细胞核提取物的电泳迁移率变动分析中，NRBS用作放射性标记探针，显示序列特异性蛋白结合。这些蛋白质进行了分析与液相色谱串联质谱法，并揭示了一个重要组成部分的NIS-阻遏物的身份。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，从而导致NIS阻遏物与hNIS启动子的结合。这种新的机制可能是基础或除了推测的hNIS基因再激活的表观遗传机制与各种药物的报告。针对减少或拮抗NIS-阻遏物组分的治疗应恢复负责碘转运的hNIS基因表达，从而使去分化甲状腺癌的I-131治疗成为可能。我们的目标是确认我们的NIS-阻遏蛋白的鉴定，并确定其他蛋白质复合物的成分，辨别其生理和病理生理作用，并制定针对它的策略，这将导致治疗，以恢复NIS活性在去分化甲状腺癌，恢复使用放射性碘治疗甲状腺癌转移。 公共卫生相关性声明：甲状腺癌，由于未知的原因，是美国男性和女性中增长最快的癌症，并且是20至30岁之间的白色女性中最常见的癌症。虽然75%的甲状腺癌患者对放射性碘治疗有充分的反应，但10%的患者对这种治疗失去反应，最终死于扩散性肿瘤。开发新的方法来恢复对放射性碘的临床反应可以挽救生命。