Study on the intracellular Network of TBX3

虽然乳腺癌是美国女性最常见的恶性肿瘤之一，但遗传因素 其发病机制和临床过程仍不清楚。TBX 3是一个T-box 转录因子可能在乳腺癌中发挥作用，但之前尚未进行过研究。突变 人和小鼠中的TBX 3显示出乳腺发育不全或缺失的临床特征。我们 早期的研究表明，TBX 3在乳腺癌细胞系中过表达，并且可以永生化， 转化小鼠胚胎成纤维细胞。TBX 3还可以抑制p14 ARF基因的表达， MDM 2介导的p53降解的抑制剂和抑制剂。因此，TBX 3的过表达 降低p53的稳定性。最近，发现TBX 3在乳腺癌患者的血清中升高， 癌总的来说，这些发现表明TBX 3在乳腺癌中起着重要作用。在这一提议中， 我们显示TBX 3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TBX 3 过表达与乳腺癌相关，TBX 3将HDAC募集到p14 ARF启动子， 抑制p14 ARF肿瘤抑制因子表达，导致乳腺癌。 目的1，我们将检查TBX 3是否在原发性乳腺癌组织中过表达。表达水平 TBX 3的表达将与临床结果和其他已建立的生物标志物相关。我们预计TBX 3可以 成为乳腺癌的新生物标志物。TBX 3在机制上与肿瘤生长有关，推测是通过 抑制抑癌基因p14 ARF的表达。虽然TBX 3被描述为 尽管TBX 3是转录抑制因子，但TBX 3抑制基因表达的机制仍不清楚。我们 初步结果表明TBX 3与HDAC相互作用，并且这种相互作用在生理上是重要的。为 目的2：我们假设TBX 3募集HDACs到p14 ARF启动子，并抑制p14 ARF肿瘤抑制因子， 基因表达。我们将通过一系列体内和体外测定来测试TBX 3和HDAC之间的相互作用， 包括免疫组化分析、谷胱甘肽-S-转移酶（GST）下拉分析和染色质 免疫沉淀（CHIP）。TBX 3-HDAC相互作用的生理意义将进一步研究 在乳腺癌细胞和p14 ARF启动子。在目标3中，我们建议通过以下方式识别TBX 3直接目标： 染色质免疫沉淀引导的连接选择（CHIP-GLAS）。CHIP-GLAS检测结合了 染色质沉淀和微阵列，可用于鉴定20，000个启动子DNA/转录因子 在一个实验中具有高灵敏度和特异性的相互作用。使用这种方法，我们发现TBX 3结合 超过600名赞助商。我们将验证那些与乳腺癌和乳腺癌相关的靶基因 使用各种体内和体外测定进行开发。 拟议研究的一个重要特征是转化为一种新的生物标志物的发展， 乳腺癌和基本生物信息的临床应用将得到优化。TBX 3的解析- HDAC的相互作用将加深我们对TBX 3功能的理解，也可能导致识别 乳腺癌的新治疗靶点。

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 represser, 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.