IAPs As Novel Targets for Cancer Therapy

描述(申请人提供)：凋亡信号通路的缺陷和凋亡抑制因子的上调使人类癌细胞对治疗药物产生耐药性。克服这种耐药性的一个有希望的策略是直接靶向细胞凋亡机制。越来越多的证据表明，凋亡抑制蛋白(IAPs)在人类肿瘤细胞中表达上调。我们发现，显性阴性Survivin T34A突变体、XIAP相关因子1(XAF1)和活性Smac等IAP拮抗蛋白基因的表达在肿瘤细胞系中优先诱导细胞凋亡，而对正常细胞系无明显影响。为了确定肿瘤和正常细胞系中不同的凋亡反应的分子基础，我们检测了人类肿瘤和正常细胞系中凋亡和抗凋亡因子的水平。我们发现，许多人类癌细胞株都具有结构性激活的caspase活性，但并未发生凋亡。我们还在肿瘤细胞系中检测到高水平的IAP，如Survivin和XIAP。基于这些观察，我们推测，在人类肿瘤细胞中存在结构性激活的caspase和上调的IAP，而在正常细胞中不存在，通过表达SurvivinT34A、XAF1和/或活性Smac基因来选择性地诱导肿瘤细胞的凋亡。IAP功能的下调与化疗药物的结合进一步增强了肿瘤细胞系和人类肿瘤异种移植模型的抗肿瘤效果。为了验证这一假设，我们将确定是否需要结构性激活的caspase来选择性地诱导肿瘤细胞系的凋亡，方法是在caspase抑制剂存在的情况下表达LAP抵消蛋白基因，还是在自催化的REV-caspase3基因的共同表达的情况下。我们将进一步确定，当细胞同时高水平表达激活的caspase3和XIAP时，是否可以通过表达IAP对抗蛋白基因来诱导正常细胞系的凋亡。由于人类肿瘤细胞表达高水平的Survivin和XIAP，因此有必要抑制这两种蛋白的功能，以释放IAP对凋亡途径的阻断。在这项拟议的研究中，我们将确定最有效的LAP抑制蛋白基因组合，以实现最大限度地诱导人类肿瘤细胞的凋亡。最后，我们将确定IAP抑制蛋白基因的表达与化疗药物多西他赛或HSV-TK/GCV自杀基因治疗相结合是否增加了人乳腺和胰腺癌细胞系以及人类肿瘤异种移植模型的抗肿瘤效果。这项研究的结果应该使我们能够确定IAP是否是开发癌症特异性治疗方法的新的分子靶点。

DESCRIPTION (provided by applicant): Defects in apoptosis signaling pathway and upregulation of apoptosis inhibitory factors confer resistance of human cancer cells to therapeutic agents. A promising strategy to overcome the resistance is to target apoptosis machinery directly. Increasing evidence indicates that inhibitor-of-apoptosis proteins (IAPs) are upregulated in human tumor cells. We found that expression of IAP counteracting protein genes, such as dominant negative survivinT34A mutant, XIAP associated factor 1 (XAF1) and active Smac, induce apoptotic cell death preferentially in tumor cell lines but have no apparent effect on normal cell lines. To determine the molecular bases of the differential apoptotic response in tumor and normal cell lines, we examined the levels of apoptotic and anti-apoptotic factors in human cancer and normal cell lines. We found that many human cancer cell lines have constitutively activated caspase activities and yet are not undergoing apoptosis. We also detected high levels of IAPs, such as survivin and XIAP, in the tumor cell lines. Based on these observations, we hypothesize that the presence of constitutively activated caspases and upregulated IAPs in human tumor cells, but not in normal cells, confers selectivity in induction of apoptotic cell death in tumor cells by expression of survivinT34A, XAF1 and/or active Smac genes. A combination of downregulation of IAP function with chemotherapy agents further enhances anti-tumor effects in tumor cell lines as well as in human tumor xenograft models. To test the hypothesis, we will determine whether constitutively activated caspases are required for selective induction of apoptosis in tumor cell lines by expression of LAP counteracting protein genes in the presence of caspase inhibitors or with co-expression of an autocatalytic Rev-caspase 3 gene. We will further determine whether apoptosis can be induced in normal cell lines by expressing IAP counteracting protein genes when the cells are engineered to express high levels of both activated caspase 3 and XIAP. Since human tumor cells express high levels of survivin and XIAP, it is necessary to inhibit the function of both proteins to release the blockage of IAPs on the apoptotic pathway. In the proposed study, we will determine the most effective combination of lAP counteracting protein genes to achieve maximum apoptosis induction in human tumor cells. Finally, we will determine whether a combination of expression of IAP counteracting protein genes with chemotherapy drug docetaxel or HSV-TK/GCV suicidal gene therapy increases the anti-tumor effect in human breast and pancreatic cancer cell lines as well as in human tumor xenograft models. Results from this research should allow us to determine if IAPs are novel molecular targets for the development of cancer-specific therapeutic approaches.