Tumor Selective Apoptosis by TRAIL

描述（由申请人提供）：TRAIL是癌症治疗的候选物，其选择性地诱导转化/肿瘤细胞而非正常细胞的凋亡。然而，TRAIL诱导的细胞凋亡的肿瘤选择性的机制知之甚少。FLICE抑制蛋白（c-FLIP）是程序性细胞死亡的调节因子。最近，我们已经证明，全长c-FLIPL相互作用的TRAIL受体（DR 5）在配体的情况下，以防止细胞凋亡。在通过TRAIL诱导细胞凋亡后，在TRAIL DISC（死亡诱导信号复合物）处的主要c-FLIP变体是c-FLIPp 43（半胱天冬酶切割的产物）和c-FLIP。我们的研究进一步表明，c-FLIPL通过蛋白水解裂解的消除是TRAIL诱导的细胞凋亡的早期步骤，并且c-FLIPL、c-FLIPs和c-FLIPp 43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-FLIPp 43促进c-Akt存活途径的激活。本申请的总体目标是阐明c-FLIP变体调节TRAIL诱导的细胞凋亡的分子机制，以及c-FLIP变体在Bcr-Abl诱导的慢性髓性白血病的体外和体内模型中对TRAIL诱导的细胞凋亡的肿瘤选择性中的不同作用。 在目的1中，我们将确定c-FLIP变体的特定功能和c-FLIPL的半胱天冬酶切割在TRAIL诱导的细胞凋亡中的作用。这将通过使用稳定表达每种c-FLIP变体的c-FLIP（-/-）胚胎成纤维细胞和通过特异性靶向c-FLIP变体的siRNA在体外实现。此外，我们将确定c-FLIP变体通过表征它们调节的不同信号复合物来调节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存活通路在介导c-FLIP的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-FLIP变体在介导TRAIL选择性肿瘤死亡中的特定作用，以及它们作为TRAIL治疗的预测标志物的用途。这些研究将在体外使用Bcr-Abl转化细胞和Bcr-Abl诱导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.