Regulation of the Fas Receptor and its Ligand in Pediatric Tumors

Fas 受体及其配体在小儿肿瘤中的调控

• 批准号: 6433413
• 负责人: MARIA TSOKOS
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433413
• 关键词: CD95 molecule; antineoplastics; antireceptor antibody; apoptosis; embryo neoplasm; human tissue; interferon gamma; neoplasm /cancer pharmacology; neuroblastoma; pediatric neoplasm /cancer; tissue /cell culture

One of the best defined apoptotic pathways is mediated by the surface receptor Fas, which upon ligation with the Fas ligand (FasL) induces the formation of a death-inducing signaling complex (DISC). The DISC consists of the Fas receptor, the adaptor molecule FADD and FLICE/caspase 8. Caspase 8 is activated at the DISC and triggers the proteolytic activation of downstream caspases, either directly (type I cells), or indirectly through release of cytochrome c from the mitochondria and activation of caspase 9 (type II cells). In type II cells, the bcl-2 family of proteins are important inhibitors of apoptosis inhibiting the release of cytochrome c from the mitochondria. Various tumor cell types express Fas and FasL, yet not all of them have a functional Fas/FasL pathway. Because cytotoxic drugs induce apoptosis, irrespective of their intracellular target, and Fas- resistant tumors show cross resistance to drug-induced apoptosis in vitro, we made the hypothesis that tumors with a defective Fas/FasL signaling pathway are refractory to treatment and exhibit a more aggressive behavior. We have investigated the signaling components of the Fas/FasL pathway in tumors of the Ewings sarcoma family (ESF) and in neuroblastoma (NB), in order to understand the molecular events that lead to their impaired apoptosis and hence treatment failures. This approach is expected to lead to the discovery of novel therapeutic agents in ESF tumors and NB.We have found that many ESF tumors are Fas- sensitive in vitro, whereas NB cell lines with high N-myc copy number are Fas-resistant. We also demonstrated that in NB cells, the Fas signaling pathway requires participation of mitochondria (type II cells) and by downregulating bcl-2 with antisense oligonucleotides we can reverse their resistance to Fas-mediated apoptosis. We are investigating the therapeutic efficacy of combined antisense bcl-2 treatment and chemotherapy in a NB model in SCID mice. Because high N- myc copy numbers have been associated with aggressive NB biology in vivo, and aggressive NB is refractory to existing therapeutic modalities, a combination treatment with chemotherapeutic agents and antisense bcl2 oligonucleotides may prove to be beneficial in vivo. Our data have also shown that NB cells with a defective Fas signaling pathway in vitro show expression of a protein that has been recently shown to inhibit the activation of FLICE/caspase 8 at the DISC and known as FLICE-inhibitory protein (FLIP). We have shown FLIP to be present not only in NB cell lines but also in tumor tissues from patients with aggressive NB. In the Fas-responsive ESFT cell lines, we found that we can induce apoptosis not only by ligation with a Fas activating antibody, but also by inducing enhanced expression of Fas and FasL in tumor cells. This can be accomplished either post- translationally after treatment with synthetic metalloproteinase inhibitors (MMPIs) which inhibit the cleavage of the full length FasL molecule to its soluble form, or transcriptionally by upregulation of the synthesis of the the Fas and FasL molecules by chemotherapeutic agents. We confirmed the induction of FasL by chemotherapeutic agents in ESF tumor cells by FasL promoter activation experiments. Furthermore, we found that the chemotherapeutic agents can induce FasL expression through activation of the NF-kB transcription factor. We further confirmed the requirement of the FasL molecule for the induction of apoptosis by chemotherapeutic agents by generating stable transfectants with antisense FasL sequences which blocked FasL synthesis and inhibited the drug-induced apoptosis in ESF tumor cells. These findings support that chemotherapeutic agents use the Fas/FasL pathway to induce apoptosis in ESF tumors. We also showed that MMPI- treatment had a synergistic effect with chemotherapeutic agents in vitro, thus supporting a role for synthetic MMPIs in the treatment of ESF tumors. - apoptosis, Ewing's sarcoma family of tumors (ESFT), neuroblastoma, Fas/FasL, MMPIs, metalloproteinase inhibitors, - Human Tissues, Fluids, Cells, etc.

最明确的凋亡途径之一是由表面受体Fas介导的，其在与Fas配体（FasL）连接后诱导形成死亡诱导信号复合物（DISC）。DISC由Fas受体、衔接分子FADD和FLICE/半胱天冬酶8组成。胱天蛋白酶8在DISC处被激活，并直接（I型细胞）或通过从线粒体释放细胞色素c和激活胱天蛋白酶9（II型细胞）间接触发下游胱天蛋白酶的蛋白水解激活。在II型细胞中，bcl-2蛋白家族是抑制细胞色素c从线粒体释放的凋亡的重要抑制剂。多种肿瘤细胞表达Fas和FasL，但并非所有肿瘤细胞都具有功能性Fas/FasL通路。因为细胞毒性药物诱导细胞凋亡，而不管它们的细胞内靶点如何，并且Fas抗性肿瘤在体外显示出对药物诱导的细胞凋亡的交叉抗性，所以我们假设具有缺陷的Fas/FasL信号传导途径的肿瘤对治疗是难治的并且表现出更具侵袭性的行为。我们研究了尤因氏肉瘤家族（ESF）和神经母细胞瘤（NB）中Fas/FasL通路的信号传导成分，以了解导致其细胞凋亡受损并因此导致治疗失败的分子事件。这种方法有望导致ESF肿瘤和NB中新的治疗剂的发现。我们已经发现，许多ESF肿瘤是Fas敏感的体外，而具有高N-myc拷贝数的NB细胞系是Fas抗性的。我们还证明，在NB细胞中，Fas信号通路需要线粒体（II型细胞）的参与，并且通过用反义寡核苷酸下调bcl-2，我们可以逆转它们对Fas介导的凋亡的抗性。我们正在研究联合反义bcl-2治疗和化疗在SCID小鼠NB模型中的治疗效果。因为高N-myc拷贝数与体内侵袭性NB生物学相关，并且侵袭性NB对现有治疗方式是难治的，所以用化疗剂和反义bcl 2寡核苷酸的联合治疗可以证明在体内是有益的.我们的数据还表明，体外Fas信号通路缺陷的NB细胞显示出最近显示出抑制DISC处FLICE/半胱天冬酶8活化的蛋白质的表达，该蛋白质被称为FLICE抑制蛋白（FLIP）。我们已经证明FLIP不仅存在于NB细胞系中，而且存在于侵袭性NB患者的肿瘤组织中。在Fas应答的ESFT细胞系中，我们发现我们不仅可以通过与Fas活化抗体连接来诱导凋亡，而且可以通过诱导肿瘤细胞中Fas和FasL的表达增强来诱导凋亡。这可以在用抑制全长FasL分子裂解成其可溶形式的合成金属蛋白酶抑制剂（MMPIs）处理后进行，或者通过化疗剂上调Fas和FasL分子的合成进行转录。我们通过FasL启动子激活实验证实了化疗药物在ESF肿瘤细胞中诱导FasL。此外，我们发现化疗药物可以通过激活NF-κ B转录因子诱导FasL表达。我们通过用反义FasL序列产生稳定的转染子进一步证实了FasL分子对化疗药物诱导凋亡的需要，所述反义FasL序列阻断FasL合成并抑制ESF肿瘤细胞中药物诱导的凋亡。这些发现支持化疗药物使用Fas/FasL途径诱导ESF肿瘤细胞凋亡。我们还表明，MMPI治疗与化疗药物在体外具有协同效应，从而支持合成MMPI在治疗ESF肿瘤中的作用。- 细胞凋亡、尤文氏肉瘤家族肿瘤（ESFT）、神经母细胞瘤、Fas/FasL、MMPI、金属蛋白酶抑制剂、人类组织、液体、细胞等。