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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。开发新的治疗选择是前列腺癌治疗的紧迫问题。我们最近证明GSK-3 β抑制消除前列腺癌细胞中的TRAIL抗性，表明GSK-3 β是前列腺癌中TRAIL介导的凋亡的负调节因子。此外，我们发现GSK-3 β抑制通过转录机制导致蛋白酪氨酸磷酸酶SHP-2表达的显著增加。为了确定增加的SHP-2表达是否是前列腺源性上皮细胞中TRAIL致敏的原因，我们建立了几种SHP-2过表达的前列腺癌细胞系，然后评估它们对TRAIL介导的细胞死亡的反应性。用基于MTT的测定评估细胞存活。正如预期的那样，亲本非恶性RWPE-1细胞与其他两种恶性细胞系CWR 22 Rv 1和LAPC-4相比显示出显著更高的对TRAIL的抗性。然而，在所有三个SHP-2过表达稳定系中发现了对TRAIL诱导的细胞死亡的类似增强作用。接下来，我们确定过表达SHP-2是否增强前列腺癌LAPC-4细胞中TRAIL介导的死亡诱导信号复合物（DISC）的形成。使用生物素标记的重组TRAIL（Bio-TRAIL）进行DISC沉淀。我们发现过表达SHP-2显著增强了TRAIL刺激的FADD和caspase-8向TRAIL受体DR 4的募集。Caspase-8裂解（由于活化）也增加。这些结果表明，SHP-2在增强TRAIL介导的DISC组装和随后的凋亡细胞死亡中起着积极的作用。明年，我们将确定敲低SHP-2对前列腺癌细胞中TRAIL介导的凋亡的影响。

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.