ROLE OF SHP-2 IN GSK-3BETA INHIBITOR-INDUCED TRAIL SENSITIZATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 开发新的治疗方案是前列腺癌治疗的一个紧迫问题。我们最近证实GSK-3β的抑制消除了前列腺癌细胞对TRAIL的抵抗，表明GSK-3β是TRAIL介导的前列腺癌细胞凋亡的负调控因子。此外，我们发现GSK-3β的抑制通过转录机制导致蛋白酪氨酸磷酸酶SHP-2的表达显著增加。为了确定SHP-2表达增加是否与前列腺上皮细胞中TRAIL的增敏有关，我们建立了几个SHP-2过表达的前列腺癌细胞系，然后评估了它们对TRAIL介导的细胞死亡的反应性。采用四甲基偶氮唑盐比色法测定细胞存活率。正如预期的那样，亲代非恶性RWPE-1细胞对TRAIL的抵抗力显著高于另外两个恶性细胞系CWR22Rv1和LAPC-4。然而，在所有三个过表达SHP-2的稳定株系中，都发现了类似的促进TRAIL诱导的细胞死亡的作用。接下来，我们确定过表达SHP-2是否促进了前列腺癌LAPC-4细胞中TRAIL介导的死亡诱导信号复合体(DISC)的形成。用生物素标记的重组TRAIL(Bio-TRAIL)进行圆盘沉淀。我们发现，过表达SHP-2显著增强TRAIL刺激的FADD和caspase-8对TRAIL受体DR4的募集。Caspase-8裂解(由于激活)也增加。这些结果表明，SHP-2在促进TRAIL介导的椎间盘组装和随后的细胞凋亡中起着积极的作用。明年，我们将确定SHP-2基因敲除对TRAIL介导的前列腺癌细胞凋亡的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The development of novel therapeutic options is an urgent issue for prostate cancer treatment. We recently demonstrated that GSK-3beta suppression eliminates TRAIL resistance in prostate cancer cells, indicating GSK-3beta is a negative regulator of TRAIL-mediated apoptosis in prostate cancer. Moreover, we found that GSK-3beta suppression resulted in a dramatic increase of protein tyrosine phosphatase SHP-2 expression via a transcriptional mechanism. To determine if increased SHP-2 expression is responsible for TRAIL sensitization in prostate-derived epithelial cells, we established several SHP-2 over-expressing prostate cancer cell lines and then evaluated their responsiveness to TRAIL-mediated cell death. Cellular survival was assessed with a MTT-based assay. As expected, parental non-malignant RWPE-1 cells showed a significant higher resistance to TRAIL compared to other two malignant cell lines CWR22Rv1 and LAPC-4. However, a similar enhancing effect was found in all three SHP-2 over-expressing stable lines to TRAIL-induced cell death. Next, we determined if over-expressing SHP-2 enhances the formation of TRAIL-mediated death-inducing signaling complex (DISC) in prostate cancer LAPC-4 cells. DISC precipitation was performed using biotin-tagged recombinant TRAIL (Bio-TRAIL). We found that over-expressing SHP-2 significantly enhanced TRAIL-stimulated recruitment of FADD and caspase-8 to TRAIL receptor DR4. Caspase-8 cleavage (due to activation) was also increased. These results suggest that SHP-2 plays a positive role in enhancing TRAIL-mediated DISC assembly and subsequent apoptotic cell death. Next year, we will determine the effect of knocking down of SHP-2 on TRAIL-mediated apoptosis in prostate cancer cells.