Identification of Anticancer Drug Targets

越来越多的证据表明，遗传不稳定是许多癌症，特别是实体瘤发病机制的基础。最近的研究表明，大多数散发性结直肠癌中发生的染色体不稳定(CIN)可由有丝分裂纺锤体检查点基因突变引起。微卫星不稳定性(MIN)发生在大多数遗传性非息肉病性结直肠癌(HNPCCs)中，由错配修复基因突变引起。导致CIN和MIN的突变增加了肿瘤的异质性，这被认为是推动肿瘤进展并使抗癌药物治疗复杂化的原因。然而，由于这些突变也将肿瘤细胞与正常细胞区分开来，它们可能为识别抗癌药物靶点提供关键途径。酿酒酵母基因bub1和msh2与已知在人类肿瘤中引起CIN和MIN的基因同源。在癌症发病机制中具有基本作用的基因的酵母同源基因的存在，应该允许使用合成致死分析来识别抗癌药物靶标。合成致死分析是酵母遗传学家用来识别基因的一种技术，当这些基因发生突变时，会在先前表征的基因突变的背景下导致细胞致死。已知具有Bub1或MSH2突变的酵母是可行的。使用合成致死分析，人们可以识别当突变时，导致Bub1或MSH2突变的合成致命性的基因。由导致合成致命性的人类同源基因编码的蛋白质代表着hBUB1或hMSH2突变癌症的潜在药物靶点。其具体目标是：1)通过合成致死分析确定缺乏Bub1或MSH2的酿酒酵母菌株生存所需的基因。将使用分别由Bub1-ADE3和MSH2 ADE3质粒拯救的适当构建的Bub1和MSH2菌株进行合成致死性分析。带有Bub1和MSH2的突变基因的合成致死性将得到证实，野生型版本将通过互补克隆。2)检测由hBUB1或hMSH2突变引起的CIN和MIN细胞中AIM 1中鉴定的人类同源基因突变的合成致死性。目标1中确定的基因的人类同源基因将被克隆，并将确定它们在适当细胞系中突变的后果。

There is increasing evidence that genetic instability underlies the pathogenesis of many cancers, particularly solid tumors. It was recently demonstrated that chromosomal instability (CIN), which occurs in most sporadic colorectal cancers, can be caused by mutations in mitotic-spindle-checkpoint genes. Microsatellite instability (MIN) occurs in most hereditary non-polyposis colorectal cancers (HNPCCs), and is caused by mutations in mismatch-repair genes. Mutations causing CIN and MIN increase tumor heterogeneity, which is thought to drive tumor progression and complicate anticancer drug therapies. However, because these mutations also distinguish tumor cells from normal cells, they may provide critical avenues for the identification of anticancer drug targets. The S. cerevisiae genes BUB1 and MSH2 are homologous to genes known to cause CIN and MIN in human tumors. The existence of yeast homologs of genes with fundamental roles in cancer pathogenesis should allow the identification of anticancer drug targets using synthetic lethal analysis. Synthetic lethal analysis is a technique used by yeast geneticists to identify genes that, when mutated, result in lethality to the cell in the context of mutations in previously characterized genes. Yeast with mutations in BUB1 or MSH2 are known to be viable. Using synthetic lethal analysis, one can identify genes that, when mutated, result in synthetic lethality with BUB1 or MSH2 mutations. The proteins encoded by the human homologs of genes that bring about synthetic lethality represent potential drug targets for cancers with mutations in hBUB1 or hMSH2. The Specific Aims are: 1) To identify genes by synthetic lethal analysis that are required for the viability of S. cerevisiae strains lacking BUB1 or MSH2. Synthetic lethal analysis will be performed using appropriately constructed bub1 and msh2 strains rescued by BUB1-ADE3 and MSH2 ADE3 plasmids, respectively. Synthetic lethality of mutated genes with bub1 and msh2 will be confirmed and the wild-type versions will be cloned by complementation. 2) To test the synthetic lethality of mutations in the human homologs of genes identified in Aim 1 in cells with CIN and MIN due to mutations in hBUB1 or hMSH2. The human homologs of genes identified in Aim 1 will be cloned, and the consequences of their mutation in appropriate cell lines will be determined.