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.

表面受体FAS介导的最佳定义凋亡途径之一是与FAS配体（FASL）结扎后诱导诱导死亡的信号传导复合物（DISC）的形成。该圆盘由FAS受体，衔接分子FADD和FLICE/CASPASE 8。caspase 8在椎间盘上激活，并触发下游caspases的蛋白水解激活，即直接（I型I细胞）（类型I细胞），或通过从线粒体和激活酶9（类型II II（类型）释放的细胞色素C而间接地（类型为I））。在II型细胞中，蛋白质的Bcl-2家族是凋亡的重要抑制剂，抑制了细胞色素C从线粒体中释放。各种肿瘤细胞类型表达FAS和FASL，但并非所有肿瘤细胞类型都具有功能性FAS/FASL途径。由于细胞毒性药物会诱导细胞凋亡，无论其细胞内靶点如何，并且耐FAS肿瘤在体外表现出对药物诱导的凋亡的交叉抗性，因此我们的假设是，FAS/FASL信号有缺陷的肿瘤对治疗和表现出更具攻击性的行为。我们已经研究了Ewings肉瘤家族（ESF）和神经母细胞瘤（NB）中FAS/FASL途径的信号传导成分，以了解导致凋亡受损的分子事件，并因此治疗失败。预计这种方法会导致在ESF肿瘤和NB中发现新型治疗剂。我们发现，许多ESF肿瘤在体外具有FAS敏感性，而NB副本高的NB细胞系具有FAS耐药性。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，并通过使用反义寡核苷酸下调Bcl-2，我们可以逆转它们对FAS介导的细胞凋亡的抗性。我们正在研究SCID小鼠中NB模型中反义BCL-2治疗和化学疗法的联合治疗功效。由于高N- MYC拷贝数与体内的侵略性NB生物学有关，并且侵略性NB对现有的治疗方式难治，因此与化学治疗剂和反义BCL2寡核苷酸的组合治疗可能在体内受益于体内。我们的数据还表明，在体外具有缺陷的FAS信号通路的NB细胞显示出蛋白质的表达，最近已证明该蛋白可以抑制椎间盘上的果/caspase 8的激活，并称为抗果冻抑制蛋白（FLIP）。我们已经显示翻转不仅存在​​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响应性ESFT细胞系中，我们发现我们不仅可以通过与Fas激活抗体结扎，而且可以通过诱导肿瘤细胞中Fas和Fasl的增强表达来诱导凋亡。这可以在用合成金属蛋白酶抑制剂（MMPI）处理后翻译后的后翻译来完成，该抑制全长FASL分子抑制了其可溶性形式的裂解，或通过化学疗法型剂量的合成FAS和FASL分子的合成来转录。我们通过FASL启动子激活实验证实了化学治疗剂在ESF肿瘤细胞中的FASL诱导。此外，我们发现化学治疗剂可以通过激活NF-KB转录因子诱导FASL表达。我们进一步证实了FASL分子对化学治疗剂诱导凋亡的需求，通过产生具有反义FASL序列的稳定转染剂，从而阻断FASL合成并抑制ESF肿瘤细胞中药物诱导的凋亡。这些发现支持化学治疗剂使用FAS/FASL途径在ESF肿瘤中诱导凋亡。我们还表明，MMPI治疗在体外与化学治疗剂具有协同作用，从而支持合成MMPI在治疗ESF肿瘤中的作用。 - 凋亡，Ewing的肉瘤家族（ESFT），神经母细胞瘤，FAS/FASL，MMPIS，金属蛋白酶抑制剂，人体组织，液体，细胞等。