SATB2 and Nickel Carcingenesis

描述（由申请人提供）：SATB2是一种同源盒转录因子，于2003年首次发现，可与富含AT的序列结合，可能与组蛋白乙酰转移酶、去乙酰化酶等染色质修饰酶结合，调控基因表达。最近的一些论文描述了SATB2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（BEAS2B）的恶性转化。虽然这些金属中的每一种在转化BEAS2B细胞时都有自己独特的基因表达特征，但金属之间的特征却大不相同。然而，这些金属中的任何一种都会使SATB2在每个转化的克隆中增加。SATB2在亲本BEAS2B细胞中不表达。进一步研究表明，慢性Ni离子处理可诱导BEAS2B细胞中SATB2 mRNA和蛋白的表达。我们假设SATB2是正常哺乳动物发育所需的转录因子，但其在慢性Ni暴露中的不适当表达是细胞转化的驱动因素。我们想要研究它在暴露于Ni和Ni转化的BEAS2B和16HB细胞中过表达的机制和后果。我们将在正常的BEAS-2B和16HBE细胞中过表达SATB2，研究其对细胞转化特性的影响，并利用基因芯片研究这些细胞中其他基因的表达。SATB2过表达和由此产生的细胞转化也将在镍暴露的情况下进行研究。我们将在镍转化的BEAS2B和16HBE细胞中通过siRNA瞬时敲低和小发夹RNA稳定敲低SATB2水平，研究SATB2缺失对其转化特性的影响，并在镍转化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。我们将重点研究慢性镍处理BEAS2B和16HBE细胞以及镍转化细胞中SATB2过表达的机制，重点研究其启动子、增强子、上游调节因子和靶向SATB2的miRNA。此外，在BEAS2B和16HBE细胞以及ni转化细胞中过表达的SATB2将被免疫沉淀，并通过凝胶电泳和质谱鉴定相互作用的蛋白伴侣。为了研究镍是否能够诱导SATB2在体内的表达，我们将通过吸入或摄入不同剂量的镍暴露于A/J小鼠，并分析SATB2在几个靶组织中的表达。为了探讨SATB2在镍诱导肿瘤发生中的作用，我们将分析暴露于亚硫化镍诱导的大鼠肺肿瘤中SATB2的表达水平（来自国家毒理学计划档案）。

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).