Ferguson Deborah A, Muenster Matthew R, Zang Qun, Spencer Jeffrey A, Schageman Jeoffrey J, Lian Yun, Garner Harold R, Gaynor Richard B, Huff J Warren, Pertsemlidis Alexander, Ashfaq Raheela, Schorge John, Becerra Carlos, Williams Noelle S, Graff Jonathan M
Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas 75390-9039, USA.
Cancer Res. 2005 Sep 15;65(18):8209-17. doi: 10.1158/0008-5472.CAN-04-3726.
Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST). For CAST, we constructed a plasmid in which the signal sequence of beta-lactamase was deleted such that it does not confer ampicillin resistance. Eukaryotic cDNA libraries cloned into pCAST produced tens of thousands of ampicillin-resistant clones, 80% of which contained cDNA fragments encoding secreted and membrane spanning proteins. We identified 2,708 unique sequences from cDNA libraries made from surgical breast cancer specimens. We analyzed the expression of 1,287 of the 2,708 genes and found that 166 were overexpressed in breast cancers relative to normal breast tissues. Eighty-five percent of these genes had not been previously identified as markers of breast cancer. Twenty-three of the 166 genes (14%) were relatively tissue restricted, suggesting use as cancer-specific targets. We also identified several new markers of ovarian cancer. Our results indicate that CAST is a robust, rapid, and low cost method to identify cell surface and secreted proteins and is applicable to a variety of relevant biological questions.
分泌蛋白和细胞表面蛋白在癌症中发挥着重要作用，是潜在的药物靶点和肿瘤标志物。在此，我们描述了一项对乳腺癌中编码分泌蛋白和细胞表面蛋白的基因进行的大规模分析。为了鉴定这些基因，我们开发了一种名为大肠杆菌氨苄青霉素分泌捕获（CAST）的新型信号序列捕获方法。对于CAST，我们构建了一个质粒，其中β-内酰胺酶的信号序列被删除，因此它不赋予氨苄青霉素抗性。克隆到pCAST中的真核cDNA文库产生了数万个氨苄青霉素抗性克隆，其中80%包含编码分泌蛋白和跨膜蛋白的cDNA片段。我们从手术切除的乳腺癌标本制备的cDNA文库中鉴定出2708个独特序列。我们分析了2708个基因中的1287个基因的表达，发现相对于正常乳腺组织，有166个基因在乳腺癌中过表达。这些基因中85%以前未被鉴定为乳腺癌标志物。166个基因中的23个（14%）相对组织受限，提示可作为癌症特异性靶点。我们还鉴定出了几种新的卵巢癌标志物。我们的结果表明，CAST是一种强大、快速且低成本的方法，可用于鉴定细胞表面和分泌蛋白，适用于各种相关生物学问题。
