Study on the intracellular Network of TBX3

TBX3细胞内网络的研究

• 批准号: 7686585
• 负责人: TAOSHENG HUANG
• 金额: $ 5.07万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686585
• 关键词: American; Basic Research Breast Cancer; Binding; Binding Sites; Biological; Biological Assay; Biological Markers; Boxing; Breast Cancer Cell; CDKN2A gene; Cancer cell line; Cells; Chromatin; Chromatin Structure; Clinical; Development; Embryo; Fibroblasts; Gene Expression; Gene Family; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Glutathione S-Transferase; Goals; Growth and Development function; HDAC1 gene; Histone Deacetylase; Human; Immunohistochemistry; In Situ Hybridization; In Vitro; Individual; Knock-out; Knockout Mice; Lead; Ligation; Luciferases; Malignant Neoplasms; Mammary gland; Mediating; Molecular; Molecular Biology; Mus; Mutation; Normal tissue morphology; Oligonucleotides; Outcome; Pathogenesis; Pathway interactions; Patients; Physiological; Play; Precipitation; Promoter Regions; Protein Overexpression; Proteins; Recruitment Activity; Research; Research Personnel; Role; Sensitivity and Specificity; Series; Serum; Syndrome; TP53 gene; Techniques; Testing; Tissues; Transcription Repressor/Corepressor; Transfection; Tumor Suppressor Genes; Tumor Suppressor Proteins; Woman; base; breast cancer diagnosis; chromatin immunoprecipitation; clinical application; cofactor; experience; human HDAC1 protein; human disease; in vitro Assay; in vivo; inhibitor/antagonist; malignant breast neoplasm; novel; novel therapeutics; programs; promoter; research study; therapeutic target; tool; transcription factor; tumor growth

DESCRIPTION (provided by applicant): Although breast cancer is one of the most common malignancies in American women, the genetic mechanisms responsible for its pathogenesis and clinical course remain largely unclear. TBX3 is a T-box transcription factor that may play a role in breast cancer and has not been previously investigated. Mutations of TBX3 in humans and mice show the clinical features of hypoplastic or absence of the mammary gland. Our earlier studies demonstrate that TBX3 is overexpressed in breast cancer cell lines, and can immortalize and transform mouse embryo fibroblast cells. TBX3 can also inhibit the expression of the p14ARF gene, a tumor suppressor and an inhibitor of MDM2-mediated degradation of p53. Therefore, overexpression of TBX3 decreases the stability of p53. Most recently, TBX3 was found to be elevated in serum of the patients with breast cancer. Collectively, these findings suggest that TBX3 plays an important role in breast cancer. In this proposal, we show that TBX3 interacts with histone deacetylase (HDAC)1, 2 and 5. Our central hypothesis is that TBX3 overexpression is associated with breast cancer and that TBX3 recruits HDACs to the p14ARF promoter and inhibits p14ARF tumor suppressor expression causing breast cancer. Aim 1, we will examine whether TBX3 is overexpressed in primary breast cancer tissues. Expression levels of TBX3 will be correlated with clinical outcomes and other established biomarkers. We anticipate that TBX3 could be a novel biomarker for breast cancer. TBX3 is mechanistically implicated in tumor growth, presumably by inhibiting the expression of p14ARF tumor suppressor gene. Although TBX3 has been characterized as a transcriptional repressor, the mechanism by which TBX3 represses gene expression is still unknown. Our preliminary results show that TBX3 interacts with HDACs and that the interaction is physiologically important. For Aim 2, we hypothesize that TBX3 recruits HDACs to p14ARF promoter and represses p14ARF tumor suppressor gene expression. We will test the interaction between TBX3 and HDACs by a series of in vivo and in vitro assays, including immunohistochemical analysis, Glutathione-S-transferase (GST) pulldown assay and chromatin immunoprecipitation (CHIP). Physiological significance of the TBX3-HDAC interaction will be further investigated in breast cancer cells and with the p14ARF promoter. In Aim 3, we propose to identify the TBX3 direct targets via chromatin immunoprecipitation-guided ligation selection (CHIP-GLAS). The CHIP-GLAS assay combines chromatin precipitation and microarray, which could be used to identify 20,000 promoter DNA/transcription factor interactions in one experiment with high sensitivity and specificity. Using this approach, we found that TBX3 binds to more than 600 promoters. We will verify those target genes involved in breast cancer and mammary gland development using various in vivo and in vitro assays. An important feature of the proposed research is translational for the development of a novel biomarker for breast cancer and basic biological information to clinical application will be optimized. Elucidation of the TBX3- HDAC interaction will deepen our understanding of the functions of TBX3 and may also lead to the identification of a novel therapeutic target for breast cancer.

描述（由申请人提供）：尽管乳腺癌是美国女性最常见的恶性肿瘤之一，但其发病机制和临床过程的遗传机制仍不清楚。 TBX3 是一种 T-box 转录因子，可能在乳腺癌中发挥作用，但之前尚未被研究过。人和小鼠中TBX3的突变表现出乳腺发育不全或缺失的临床特征。我们早期的研究表明，TBX3 在乳腺癌细胞系中过度表达，并且可以使小鼠胚胎成纤维细胞永生化和转化。 TBX3 还可以抑制 p14ARF 基因的表达，p14ARF 基因是一种肿瘤抑制因子，也是 MDM2 介导的 p53 降解的抑制剂。因此，TBX3 的过度表达会降低 p53 的稳定性。最近，发现乳腺癌患者血清中的 TBX3 升高。总的来说，这些发现表明 TBX3 在乳腺癌中发挥着重要作用。在本提案中，我们证明 TBX3 与组蛋白脱乙酰酶 (HDAC)1、2 和 5 相互作用。我们的中心假设是 TBX3 过度表达与乳腺癌相关，并且 TBX3 将 HDAC 招募到 p14ARF 启动子并抑制 p14ARF 肿瘤抑制基因的表达，从而导致乳腺癌。目标1，我们将检查TBX3在原发性乳腺癌组织中是否过度表达。 TBX3 的表达水平将与临床结果和其他已建立的生物标志物相关。我们预计 TBX3 可能成为乳腺癌的新型生物标志物。 TBX3 在机制上与肿瘤生长有关，可能是通过抑制 p14ARF 肿瘤抑制基因的表达来实现的。尽管 TBX3 已被定性为转录抑制因子，但 TBX3 抑制基因表达的机制仍不清楚。我们的初步结果表明 TBX3 与 HDAC 相互作用，并且这种相互作用具有重要的生理意义。对于目标 2，我们假设 TBX3 将 HDAC 招募到 p14ARF 启动子并抑制 p14ARF 肿瘤抑制基因的表达。我们将通过一系列体内和体外测定来测试 TBX3 和 HDAC 之间的相互作用，包括免疫组织化学分析、谷胱甘肽-S-转移酶 (GST) Pulldown 测定和染色质免疫沉淀 (CHIP)。将在乳腺癌细胞中和 p14ARF 启动子中进一步研究 TBX3-HDAC 相互作用的生理意义。在目标 3 中，我们建议通过染色质免疫沉淀引导的连接选择 (CHIP-GLAS) 来鉴定 TBX3 直接靶标。 CHIP-GLAS 检测结合了染色质沉淀和微阵列，可在一项实验中鉴定 20,000 个启动子 DNA/转录因子相互作用，具有高灵敏度和特异性。使用这种方法，我们发现 TBX3 与 600 多个启动子结合。我们将使用各种体内和体外测定来验证那些涉及乳腺癌和乳腺发育的靶基因。该研究的一个重要特点是可转化为乳腺癌新型生物标志物的开发，并且将优化临床应用的基本生物学信息。阐明 TBX3-HDAC 相互作用将加深我们对 TBX3 功能的理解，也可能导致乳腺癌新治疗靶点的确定。