Hucl Tomas, Brody Jonathan R, Gallmeier Eike, Iacobuzio-Donahue Christine A, Farrance Iain K, Kern Scott E
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD 21231, USA.
Cancer Res. 2007 Oct 1;67(19):9055-65. doi: 10.1158/0008-5472.CAN-07-0474.
Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies.
鉴定具有癌症特异性过表达的基因，为以无偏见的方式有效发现癌症特异性活性提供了可能性。我们应用这一模式来研究间皮素（MSLN）的过表达，这是胰腺癌几乎普遍存在的、在诊断和治疗上有用的特征。我们鉴定出一个18bp的上游增强子，称为CanScript，它能强烈激活原本较弱的组织非特异性启动子的转录，并在癌症特异性MSLN转录异常升高的细胞中选择性发挥作用。对CanScript引入突变显示出两个功能不同的位点：一个Sp1样位点和一个MCAT元件。凝胶阻滞和染色质免疫沉淀分析表明，MCAT元件在体外和体内均与转录增强因子（TEF）-1（TEAD1）结合。通过TEF-1敲低实验确定，MSLN蛋白过表达需要TEF-1的存在。癌症特异性似乎由TEF-1的一个假定的限制性辅因子提供，只有在MSLN过表达的细胞系中，外源性TEF-1才能与之竞争。CanScript串联体具有增强的活性。这些结果确定了一个TEF家族成员是MSLN过表达的主要调节因子，MSLN过表达是胰腺癌和其他癌症的一个基本特征，这可能是由于上游高度频繁的异常细胞活动所致。CanScript序列代表了一个用于癌症特异性靶向的模块化元件，可能适用于近三分之一的人类恶性肿瘤。
