Regulation of the Fas Receptor and its Ligand in Pediatric Tumors

其中一个最明确的凋亡途径是由表面受体Fas介导的，它与Fas配体（FasL）连接后诱导死亡诱导信号复合物（DISC）的形成。DISC由Fas受体、接头分子FADD和FLICE/caspase 8组成。Caspase 8在DISC处被激活，直接（I型细胞）或间接通过线粒体释放细胞色素c和激活Caspase 9 （II型细胞）触发下游Caspase的蛋白水解激活。在II型细胞中，bcl-2家族蛋白是凋亡的重要抑制剂，可抑制线粒体释放细胞色素c。多种肿瘤细胞类型均表达Fas和FasL，但并非所有肿瘤细胞都具有功能性Fas/FasL通路。由于细胞毒性药物诱导细胞凋亡，而不考虑其细胞内靶点，并且Fas耐药肿瘤在体外对药物诱导的细胞凋亡表现出交叉耐药，因此我们假设具有Fas/FasL信号通路缺陷的肿瘤难以治疗，并且表现出更具攻击性的行为。我们研究了尤文氏肉瘤家族（ESF）和神经母细胞瘤（NB）肿瘤中Fas/FasL通路的信号传导成分，以了解导致其凋亡受损和治疗失败的分子事件。这种方法有望导致ESF肿瘤和NB的新治疗药物的发现。我们发现许多ESF肿瘤在体外对Fas敏感，而具有高N-myc拷贝数的NB细胞系则具有Fas抗性。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，通过反义寡核苷酸下调bcl-2，我们可以逆转它们对Fas介导的凋亡的抗性。我们正在研究反义bcl-2联合化疗对SCID小鼠NB模型的治疗效果。由于高N- myc拷贝数与体内侵袭性NB生物学有关，并且侵袭性NB对现有的治疗方式具有难治性，因此化疗药物和反义bcl2寡核苷酸的联合治疗可能在体内有益。我们的数据还显示，体外Fas信号通路缺陷的NB细胞表达了一种蛋白质，该蛋白质最近被证明可以抑制DISC上FLICE/caspase 8的激活，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连接Fas激活抗体来诱导凋亡，还可以通过诱导肿瘤细胞中Fas和FasL的表达增强来诱导凋亡。这可以在翻译后使用合成金属蛋白酶抑制剂（MMPIs）来抑制全长FasL分子的裂解到其可溶性形式，或者通过化疗药物上调Fas和FasL分子的合成来实现。我们通过FasL启动子激活实验证实了化疗药物对ESF肿瘤细胞FasL的诱导作用。此外，我们发现化疗药物可以通过激活NF-kB转录因子诱导FasL表达。我们进一步证实了FasL分子在化疗药物诱导细胞凋亡中的作用，通过制备具有反义FasL序列的稳定转染物，阻断了FasL的合成，抑制了ESF肿瘤细胞药物诱导的细胞凋亡。这些发现支持化疗药物利用Fas/FasL通路诱导ESF肿瘤细胞凋亡。我们还发现MMPI-治疗在体外与化疗药物具有协同作用，从而支持合成MMPI在ESF肿瘤治疗中的作用。-细胞凋亡，尤文氏肉瘤家族肿瘤（ESFT），神经母细胞瘤，Fas/FasL, MMPIs，金属蛋白酶抑制剂，-人体组织，液体，细胞等。

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.