IAPs As Novel Targets for Cancer Therapy

描述（由申请人提供）：细胞凋亡信号通路的缺陷和细胞凋亡抑制因子的上调赋予人癌细胞对治疗剂的抗性。克服耐药性的一个有希望的策略是直接靶向凋亡机制。越来越多的证据表明，凋亡通道蛋白（IAP）在人类肿瘤细胞中上调。我们发现，IAP抵消蛋白基因，如显性负性的survivinT 34 A突变体，XIAP相关因子1（XAF 1）和活性Smac的表达，诱导肿瘤细胞系中的细胞凋亡优先，但对正常细胞系没有明显的影响。为了确定肿瘤和正常细胞系中差异凋亡反应的分子基础，我们检测了人类癌症和正常细胞系中凋亡和抗凋亡因子的水平。我们发现，许多人癌细胞系具有组成性激活的半胱天冬酶活性，但没有发生凋亡。我们还在肿瘤细胞系中检测到高水平的IAP，如生存素和XIAP。基于这些观察结果，我们假设，在人肿瘤细胞中，而不是在正常细胞中，存在组成性激活的半胱天冬酶和上调的IAP，赋予选择性诱导肿瘤细胞中的凋亡细胞死亡的表达的生存素T34 A，XAF 1和/或活性Smac基因。IAP功能下调与化疗剂的组合进一步增强了肿瘤细胞系以及人肿瘤异种移植模型中的抗肿瘤作用。为了验证这一假设，我们将确定是否组成型激活的半胱天冬酶是必需的选择性诱导肿瘤细胞系中的细胞凋亡，通过在存在半胱天冬酶抑制剂或与自催化Rev-半胱天冬酶3基因的共表达的情况下表达抗凋亡蛋白基因。我们将进一步确定当细胞被工程化以表达高水平的活化的半胱天冬酶3和XIAP时，是否可以通过表达IAP抵消蛋白基因在正常细胞系中诱导凋亡。由于人肿瘤细胞表达高水平的存活素和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。在所提出的研究中，我们将确定IAP抵消蛋白基因的最有效组合，以在人肿瘤细胞中实现最大的凋亡诱导。最后，我们将确定IAP抵消蛋白基因的表达与化疗药物多西他赛或HSV-TK/GCV自杀基因治疗的组合是否增加了人乳腺癌和胰腺癌细胞系以及人肿瘤异种移植模型中的抗肿瘤作用。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.