Identification and functional analysis of a novel cancer related gene from lung cancer and malignant mesothelioma

我们在恶性间皮瘤细胞系NCI-H28中发现了染色体3p21.3纯合子缺失，并发现了β-连环蛋白和一个新基因MTSK1(暂定名)的缺失。为了确定β-连环蛋白本身是否对NCI-H28细胞株具有肿瘤抑制活性，我们将该基因转化到细胞中。用荧光素酶活性测定证实β-连环蛋白的转录活性后，进行克隆形成实验，发现NCI-H28细胞生长受到明显抑制。通过TUNEL实验，我们证实了这种抑制作用是由于诱导了细胞凋亡。由于NCI-H28缺乏β-连环蛋白，我们分析了γ-连环蛋白在β-连环蛋白缺失条件下的功能，发现γ-连环蛋白也具有Tcf/Lef依赖的转录活性。同时，虽然我们在9例恶性间皮瘤和63例肺癌中通过基因组PCR-SSCP分析搜索了MTSK1基因预测的开放阅读框中的突变，但我们没有发现任何体细胞突变。由于我们发现MTSK1有三种不同的剪接形式，我们构建了每个表达载体，将它们转化到NCI-H28中，并证实了它们的产物的表达。此外，我们还搜索了154例手术切除的原发性肺癌患者的RAS基因突变情况。11例发现有KRAS突变，显微切割技术发现其中9例肿瘤保留了野生型KRAS等位基因，提示突变的KRAS表达对KRAS的激活具有重要意义。

We identified a chromosome 3p21.3 homozygous deletion in a malignant mesothelioma cell line, NCI-H28,and found deletions of β-catenin and a novel gene, MTSK1 (tentative name). To determine whether β-catenin itself has a tumor suppressive activity against the NCI-H28 cell line, we transformed the gene into the cells. After confirmation of transcriptional activity of β-catenin with luciferase assay, we performed colony formation assay and found significant growth inhibition of the NCI-H28 cells. Using TUNEL assay, we confirmed that this inhibition was due to induction of apoptosis. Since NCI-H28 lacks β-catenin, we analyzed the function of γ-catenin under a null condition of β-catenin and found that γ-catenin also has TCF/LEF dependent transcriptional activity. Meanwhile, although we searched the predicted open reading frame of the MTSK1 gene for mutations with genomic PCR-SSCP analysis in 9 malignant mesotheliomas and 63 lung cancers, we did not find any somatic mutations. Since we discovered that MTSK1 has three alternative splicing forms, we constructed each expression vector, transformed them into NCI-H28,and confirmed expression of their products. Furthermore, we searched 154 primary lung cancers which were resected by surgery for mutations of RAS. 11 cases were found to have a KRAS mutation and, using microdissection technique, 9 tumors of them were shown to retain a wild-type KRAS allele, suggesting that expression of mutated KRAS is important for the activation of KRAS.