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

基本信息

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
项目类别：
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

项目摘要

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）：远处转移性降解的甲状腺癌是一种致命的疾病，没有有效的治疗。这是从失去浓缩碘化物的能力，使其对放射性碘无反应，这是唯一已知的全身疗法。碘化物的摄取需要表达钠 - 碘对孢子（HNIS）。 HNIS启动子区域的高甲基化被认为是甲状腺癌细胞中HNIS表达丧失的可能机制。用脱甲基化和组蛋白脱乙酰基酶抑制剂处理这些细胞后，HNIS表达和功能将恢复。假定这些试剂可以通过放松染色质压实来转录机械进入基因。 5-氮杂丁胺（AZAC； DNA甲基转移酶抑制剂）和丁酸钠（NAB；推定的组蛋白脱乙酰基酶抑制剂），同时又一次地恢复了HNIS的表达和对甲状腺癌细胞系的功能。为了显示出宽松的HNIS染色质结构，当时AZAC和NAB恢复HNIS mRNA表达和HNIS功能时，我们评估了基因组HNIS区域的DNase I消化率。令人惊讶的是，这些治疗方法并未产生增强的DNase I敏感性，表明对染色质压实没有影响。用含有HNIS基因启动子的荧光素酶报告基因构建体转染的NAB处理的去分化的甲状腺癌细胞刺激了荧光素酶表达和天然HNIS表达。用蛋白质合成抑制剂（PSI；环己酰亚胺，苯胺，雌激素）治疗这些细胞，在剂量依赖性，时间疗程依赖性，细胞类型特异性和启动子特异性时尚中刺激荧光素酶表达。以及恢复的内源性HNIS基因mRNA表达。这表明存在跨作用的转录抑制剂NIS抑制器，导致HNIS表达丧失。我们绘制了负责此效应的HNIS启动子序列区域，即NIS抑制器结合位点（NRB）。 NRB用甲状腺癌细胞核提取物用作电泳迁移率转移测定法中的放射性标记探针，显示出序列特异性蛋白结合。用液相色谱与串联质谱法分析这些蛋白质，并揭示了NIS压缩器的重要成分的身份。我们假设甲状腺癌中碘化物转运活性的丧失是由于HNIS基因表达的丧失，这是NIS抑制与HNIS启动子的结合所致。这种新型机制可能是与各种药理剂报道的HNIS基因重新激活的推测的表观遗传机理的基础或补充。指示减少或拮抗NIS抑制剂成分的治疗应恢复负责碘化物转运的HNIS基因表达，从而使I-131 i-131治疗去分化的甲状腺癌的治疗。我们的目的是确认我们对NIS抑制蛋白的识别，并鉴定其他蛋白质复合成分，辨别其生理和病理生理学作用，并制定针对它的策略。这应该导致治疗以恢复去分化的甲状腺癌的NIS活性，从而恢复使用放射性碘来治疗甲状腺癌转移。公共卫生相关性声明：出于未知原因，甲状腺癌是美国男性和女性中癌症最快的癌症，这是20至30岁的白人妇女中最常见的癌症。尽管有75％的甲状腺癌患者对放射性碘治疗做出了充分的反应，但有10％的患者失去了对这种治疗的反应，并最终死于弥散性肿瘤。开发恢复对放射性碘的临床反应的新方法可以挽救生命。