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: 8090451
• 负责人: Kenneth Bruce Ain
• 金额: $ 22.65万
• 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 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090451
• 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; 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; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; 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; liquid chromatography mass spectrometry; mRNA Expression; men; mutant; novel; pre-clinical; preclinical study; 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（申请人提供）： 远处转移性去分化甲状腺癌是一种致命疾病，如果没有有效的治疗方法。这是由于失去了集中碘化物的能力，使其对放射性碘（唯一已知的全身疗法）没有反应。碘化物的吸收需要碘化钠同向转运体 (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中 hNIS 表达缺失的可能机制。用去甲基化剂和组蛋白脱乙酰酶抑制剂处理这些细胞后，hNIS 表达和功能得以恢复。据推测，这些药物允许转录机制通过放松染色质压缩来接近基因。 5-氮杂胞苷（AzaC；DNA 甲基转移酶抑制剂）和丁酸钠（NaB；推定的组蛋白脱乙酰酶抑制剂）无论单独还是一起，都能恢复甲状腺癌细胞系的 hNIS 表达和功能。为了显示推测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 DNAse I 消化率。令人惊讶的是，这些处理并没有产生增强的 DNAse I 敏感性，表明对染色质压缩没有影响。用含有 hNIS 基因启动子的荧光素酶报告构建体转染 NaB 处理的去分化甲状腺癌细胞，刺激荧光素酶表达和天然 hNIS 表达。用蛋白质合成抑制剂（PSIs；放线菌酮、茴香霉素、依米丁）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。这表明存在反式作用转录抑制剂 NIS 阻遏物，它是 hNIS 表达缺失的原因。我们绘制了负责这种效应的 hNIS 启动子序列区域，即 NIS 阻遏物结合位点 (NRBS)。 NRBS 在甲状腺癌细胞核提取物的电泳迁移率变动测定中用作放射性标记探针，显示出序列特异性的蛋白质结合。使用液相色谱法和串联质谱法对这些蛋白质进行分析，揭示了 NIS 阻遏物的重要成分的身份。我们假设甲状腺癌中碘化物转运活性的丧失是由于 NIS 阻遏蛋白与 hNIS 启动子结合而导致 hNIS 基因表达丧失。这种新机制可能是各种药物报道的 hNIS 基因再激活的假定表观遗传机制的基础或补充。旨在减少或拮抗 NIS 阻遏物成分的治疗应恢复负责碘转运的 hNIS 基因表达，从而使 I-131 能够治疗去分化甲状腺癌。我们的目标是确认我们对 NIS 阻遏蛋白的鉴定，并鉴定其他蛋白质复合物成分，辨别其生理和病理生理效应，并制定针对它的策略。这应该会导致恢复去分化甲状腺癌中 NIS 活性的治疗，恢复使用放射性碘来治疗甲状腺癌转移。 公共健康相关性声明：由于不明原因，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充分反应，但 10% 的患者对此治疗失去反应并最终死于播散性肿瘤。开发恢复放射性碘临床反应的新方法可以挽救生命。