Tumor Selective Apoptosis by TRAIL

描述(申请人提供)：TRAIL是癌症治疗的候选药物，它选择性地诱导转化/肿瘤细胞的凋亡，但不能诱导正常细胞的凋亡。然而，TRAIL诱导细胞凋亡的肿瘤选择性机制尚不清楚。Flice抑制蛋白(c-FLIP)是细胞程序性死亡的调节因子。最近，我们已经证明了全长c-flipl在没有配体的情况下与其中一个TRAIL受体(DR5)相互作用以防止细胞凋亡。在TRAIL诱导细胞凋亡时，TRAIL盘(死亡诱导信号复合体)上主要的c-flip变异体是caspase裂解产物c-flipp43和c-flips。我们的研究进一步表明，c-FLIPL的蛋白水解性切割是TRAIL诱导细胞凋亡的早期步骤，c-FLIPL、c-Flips和c-FLIPp43在介导TRAIL功能中具有不同的作用。为了支持这一假设，我们观察到c-Flipl而不是c-Flipp43促进了c-Akt生存通路的激活。本应用的总体目标是阐明c-flip变异体调控TRAIL诱导的细胞凋亡的分子机制，以及在体外和体内bcr-Abl诱导的慢性髓系白血病模型中c-flip变异体在TRAIL诱导的细胞凋亡的肿瘤选择性中的不同作用。 在目标1中，我们将确定c-flip变异体的特定功能以及c-flipl的caspase裂解在TRAIL诱导的细胞凋亡中的作用。这将在体外通过使用稳定表达每个c-flip变体的c-flip(-/-)胚胎成纤维细胞和专门针对c-flip变体的siRNA来实现。此外，我们将通过表征c-flip变体调控的不同信号复合体来确定c-flip变体调控TRAIL诱导的细胞凋亡的分子机制。在目标2中，我们将描述c-Akt生存通路在介导c-FLIP抗凋亡活性中的新作用。我们还将研究c-flip诱导Akt激活的分子机制。在目标3中，我们将确定c-翻转变异体在TRAIL介导选择性肿瘤死亡中的特定作用，并将其用作TRAIL治疗的预测标记物。这些研究将在体外使用BCR-Abl转化的细胞进行，并在BCR-Abl诱导的白血病的体内模型中进行。

DESCRIPTION (provided by applicant): TRAIL is a candidate for cancer therapy that selectively induces apoptosis in transformed/tumor cells but not in normal cells. However, the mechanism for the tumor selectivity of TRAIL-induced apoptosis is poorly understood. FLICE Inhibitory Protein (c-FLIP) is a regulator of programmed cell death. Recently, we have demonstrated that full-length c-FLIPL interacts with one of the TRAIL receptors (DR5) in the absence of ligand to prevent apoptosis. Upon induction of apoptosis by TRAIL, the major c-FLIP variants at the TRAIL DISC (Death Inducing Signaling Complex) are c-FLIPp43 (product of caspase cleavage) and c-FLIPs. Our studies further indicate that elimination of c-FLIPL by proteolytic cleavage is an early step in TRAIL-induced apoptosis and that c-FLIPL, c-FLIPs and c-FLIPp43 have distinct roles in mediating TRAIL function. In support of this hypothesis, we have observed that c-FLIPL, but not c-FLIPp43, promotes activation of the c-Akt survival pathway. The overall goal of this application is to elucidate the molecular mechanisms by which the c-FLIP variants regulate TRAIL-induced apoptosis and the distinct roles of the c-FLIP variants in the tumor selectivity of TRAIL-induced apoptosis in vitro and in an in vivo model for Bcr-Abl-induced chronic myeloid leukemia. In Aim 1, we will determine the specific functions of the c-FLIP variants and the role of caspase cleavage of c-FLIPL in TRAIL-induced apoptosis. This will be achieved in vitro by using c-FLIP (-/-) embryonic fibroblasts that stably express each of the c-FLIP variants, and by siRNAs that specifically target the c-FLIP variants. Furthermore, we will determine the molecular mechanism by which the c-FLIP variants regulate TRAIL-induced apoptosis by characterizing the distinct signaling complexes that they regulate. In Aim 2, we will delineate the novel role of the c-Akt survival pathway in mediating the anti-apoptotic activity of c-FLIP. We will also investigate the molecular mechanism by which c-FLIP induces Akt activation. In Aim 3, we will determine the specific roles of the c-FLIP variants in mediating selective tumor death by TRAIL and their utilization as a predictive marker for TRAIL-therapy. These studies will be performed in vitro using Bcr-Abl transformed cells and in an in vivo model for Bcr-Abl-induced leukemia.