Transcriptional Repression of Sodium-Iodide Symporter in Thyroid Carcinoma

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命疾病。这是由于丧失了浓缩碘的能力，使其对放射性碘无反应，而放射性碘是唯一已知的全身疗法。碘的摄取需要碘化同调蛋白（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。用去甲基化和组蛋白去乙酰酶抑制剂处理这些细胞后，hNIS的表达和功能恢复。据推测，这些药物允许转录机制通过放松染色质压实来进入基因。5-氮杂胞苷（AzaC； DNA甲基转移酶抑制剂）和丁酸钠（NaB；假定的组蛋白去乙酰化酶抑制剂）单独或共同恢复甲状腺癌细胞hNIS的表达和功能。为了证明其表观遗传效应表明的hNIS染色质结构松弛，我们评估了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，这表明对染色质压实没有影响。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结构，刺激荧光素酶的表达和天然hNIS的表达。用蛋白合成抑制剂（PSIs，环己亚胺，大霉素，艾美汀）处理这些细胞，以剂量依赖性，时间依赖性，细胞类型特异性和启动子特异性的方式刺激荧光素酶的表达；并恢复内源性hNIS基因mRNA的表达。这表明存在一种反式作用的转录抑制剂，nis抑制因子，负责hNIS表达的丧失。我们绘制了hNIS启动子序列区域负责这种作用，nis抑制因子结合位点（NRBS）。NRBS作为放射性标记探针用于甲状腺癌细胞核提取物的电泳迁移迁移测定，显示序列特异性蛋白结合。用液相色谱-串联质谱法对这些蛋白进行了分析，发现了nis抑制因子的一个重要成分。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，这是由于nis抑制因子与hNIS启动子结合造成的。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。针对减少或拮抗NIS抑制成分的治疗应恢复负责碘转运的hNIS基因表达，从而使I-131治疗去分化甲状腺癌。我们的目标是确认我们鉴定的nis抑制蛋白和鉴定其他蛋白质复合物组分，了解其生理和病理生理作用，并制定针对它的策略。这将导致在去分化甲状腺癌中恢复NIS活性的治疗，恢复使用放射性碘治疗甲状腺癌转移。

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.