Identification of Anticancer Drug Targets

越来越多的证据表明，遗传不稳定性是许多癌症，特别是实体瘤发病机制的基础。最近的研究表明，在大多数散发性结直肠癌中发生的染色体不稳定性（CIN）可能是由有丝分裂纺锤体检查点基因的突变引起的。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。引起CIN和MIN的突变增加了肿瘤的异质性，这被认为会推动肿瘤的进展并使抗癌药物治疗复杂化。然而，由于这些突变也能区分肿瘤细胞和正常细胞，它们可能为鉴定抗癌药物靶点提供重要途径。酿酒葡萄球菌基因BUB1和MSH2与已知引起人类肿瘤CIN和MIN的基因同源。酵母同源基因在癌症发病机制中具有重要作用，这将使我们能够利用合成致死分析来鉴定抗癌药物靶点。合成致死分析是酵母遗传学家使用的一种技术，用于识别在先前特征基因突变的背景下突变时导致细胞致死的基因。已知具有BUB1或MSH2突变的酵母菌是活的。利用合成致死性分析，人们可以识别出突变后会导致BUB1或MSH2突变的合成致死性的基因。由人类同源基因编码的蛋白质带来合成致死，代表了hBUB1或hMSH2突变癌症的潜在药物靶点。具体目的是：1)通过合成致死分析鉴定缺乏BUB1或MSH2的酿酒葡萄球菌菌株生存所需的基因。合成致死分析将分别使用由bub1 -ADE3和msh2 ADE3质粒拯救的适当构建的bub1和msh2菌株进行。bub1和msh2突变基因的合成致死率将得到证实，野生型基因将通过互补克隆出来。2)检测hBUB1或hMSH2基因突变对CIN和MIN细胞中Aim 1基因人类同源基因突变的合成致死性。在Aim 1中鉴定的人类同源基因将被克隆，并确定其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.