SATB2 and Nickel Carcingenesis

描述（由申请人提供）：SATB 2是2003年首次发现的同源框转录因子，其结合富含AT的序列，并可能结合染色质修饰酶如组蛋白乙酰转移酶和脱乙酰酶以调节基因表达。最近的一些论文已经描述了SATB 2在多种癌症中的过表达，并强调了这种过表达基因在驱动肿瘤发生中的重要性。我们研究了致癌金属如镍（Ni）、六价铬（Cr+6）、砷（As）和钒酸盐（V）对正常人支气管上皮细胞（BEAS 2B）的恶性转化。虽然这些金属中的每一种在转化BEAS 2B细胞时都有其独特的基因表达特征，但这种特征在金属之间有很大的不同。然而，SATB 2在每个转化的克隆中被这些金属中的任何一种增加。SATB 2在亲本BEAS 2B细胞中不表达。进一步的研究表明，慢性Ni离子处理可诱导BEAS 2B细胞中SATB 2 mRNA和蛋白的表达。我们假设SATB 2是正常哺乳动物发育所需的转录因子，但其在慢性镍暴露期间的不适当表达是细胞转化的驱动因素。我们想研究其在BEAS 2B和16 HB细胞中过表达的机制和后果，暴露于Ni并被Ni转化。我们将在正常BEAS-2B和16 HBE细胞中过表达SATB 2，并研究其对细胞转化特性的影响，并使用基因芯片研究这些细胞中其他基因的表达。还将在镍暴露的存在下研究SATB 2过表达和导致的细胞转化。我们将通过在镍转化的BEAS 2B和16 HBE细胞中用siRNA瞬时敲低和用小发夹RNA稳定敲低来降低SATB 2的水平，并研究SATB 2损失对其转化性质的后果，并在镍转化的细胞中使用基因芯片研究这种敲低对其他基因表达的影响。我们将研究SATB 2过表达的机制，重点是其启动子，增强子，上游调控因子和靶向SATB 2的miRNA，慢性镍处理BEAS 2B和16 HBE细胞和镍转化细胞。此外，SATB 2在BEAS 2B和16 HBE细胞和Ni转化细胞中过表达，将进行免疫沉淀，并通过凝胶电泳和质谱法鉴定相互作用的蛋白伴侣。为了解决镍是否能够在体内诱导SATB 2表达，我们将通过吸入或摄入将A/J小鼠暴露于不同剂量的镍，并分析几种靶组织中的SATB 2表达。为了探索SATB 2在镍诱导的肿瘤发生中的作用，我们将分析由亚硫化镍暴露诱导的大鼠肺肿瘤中的SATB 2表达水平（从国家毒理学计划档案获得）。

DESCRIPTION (provided by applicant): SATB2 is a homobox transcription factor first discovered in 2003 that binds to AT rich sequences and likely binds chromatin modifying enzymes such as histone acetyltranferases and deacetylases for the regulation of gene expression. A number of recent papers have described SATB2 overexpression in a variety of cancers and have emphasized the importance of this overexpressed gene in driving tumorigenesis. We have studied the malignant transformation of normal human bronchial epithelial cells (BEAS2B) by carcinogenic metals such as nickel (Ni), hexavalent chromium (Cr+6), arsenic (As) and vanadate (V). While each of these metals has its own unique signature of gene expression when they transform BEAS2B cells, the signature is vastly different from metal to metal. However, SATB2 is increased in every transformed clone by any one of these metals. SATB2 is not expressed in parental BEAS2B cells. Further studies have shown that SATB2 mRNA and protein are induced in BEAS2B cells by chronic Ni ion treatment. We hypothesize that SATB2 is a transcription factor needed for normal mammalian development but its inappropriate expression during chronic Ni exposure is a driver of cell transformation. We want to investigate the mechanisms and consequences of its overexpression in BEAS2B and 16HB cells exposed to and transformed by Ni. We will overexpress SATB2 in normal BEAS-2B and 16HBE cells and investigate the effect this has on the cell's transformed properties and study the expression of other genes in these cells using gene chips. SATB2 overexpression and resulting cell transformation will also be studied in the presence of nickel exposure. We will lower the levels of SATB2 by transient knockdown with siRNA and stable knockdown with small hairpin RNA in nickel transformed BEAS2B and 16HBE cells and study the consequences of SATB2 loss on their transformed properties and investigate the effect this knockdown has on the expression of other genes using gene chips in the nickel transformed cells. We will study the mechanism of SATB2 overexpression focusing on its promoter, enhancer, upstream regulators and miRNA that target SATB2 following chronic nickel treatment of BEAS2B and 16HBE cells and in nickel transformed cells. In addition, SATB2 overexpressed in BEAS2B and 16HBE cells and in Ni-transformed cells will be immunoprecipitated, and interacting protein partners will be identified by gel electrophoresis and mass spectrometry. To address whether nickel is able to induce SATB2 expression in vivo, we will expose A/J mice to various doses of nickel by inhalation or ingestion, and analyze SATB2 expression in several target tissues. To explore the role of SATB2 in nickel-induced tumorigenesis, we will analyze the levels of SATB2 expression in rat lung tumors induced by nickel subsulfide exposure (obtained from National Toxicology Program archive).