Study on the intracellular Network of TBX3

描述(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病程仍很不清楚。TBX3是一种T-box转录因子，可能在乳腺癌中发挥作用，以前还没有研究过。人和小鼠的TBX3基因突变表现为乳腺发育不良或缺失的临床特征。我们早期的研究表明，TBX3在乳腺癌细胞系中过表达，并能永生化和转化小鼠胚胎成纤维细胞。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降解的抑制因子。因此，Tbx3的过表达降低了P53的稳定性。最近发现乳腺癌患者血清中TBX3水平升高。总而言之，这些发现表明，TBX3在乳腺癌中起着重要作用。在这个方案中，我们证明了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的过度表达与乳腺癌有关，并且TBX3招募HDAC到p14ARF 促进和抑制导致乳腺癌的p14ARF抑癌基因的表达。目的1，我们将检测TBX3在乳腺癌组织中是否过表达。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可能成为乳腺癌的一种新的生物标志物。Tbx3可能通过抑制p14ARF抑癌基因的表达而参与肿瘤生长。尽管TBX3已被鉴定为转录抑制因子，但其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，并且这种相互作用在生理上是重要的。对于目的2，我们假设TBX3招募HDAC到p14ARF启动子并抑制p14ARF抑癌基因的表达。我们将通过一系列的体内和体外实验，包括免疫组织化学分析、谷胱甘肽-S转移酶下拉实验和染色质免疫沉淀(ChIP)来检测Tbx3与HDACs的相互作用。在乳腺癌细胞和p14ARF启动子作用下，将进一步研究TBX3-HDAC相互作用的生理学意义。在目标3中，我们建议通过染色质免疫沉淀引导的结扎选择(CHIP-GLAS)来识别TBX3的直接靶标。CHIP-GLAS法结合染色质沉淀和微阵列技术，可以在一次实验中鉴定20,000个启动子DNA/转录因子的相互作用，具有很高的敏感性和特异性。利用这种方法，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实验来验证那些与乳腺癌和乳腺发育有关的靶基因。这项研究的一个重要特点是为开发一种新的乳腺癌生物标志物提供翻译，并将基础生物学信息优化到临床应用。阐明TBX3-HDAC的相互作用将加深我们对TBX3功能的理解，也可能有助于确定乳腺癌的新治疗靶点。

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.