TRAIL Therapy for Rhabdomyosarcoma

该提案将阐明TRAIL信号通路的关键调节因子，该调节因子决定了4/7例小儿横纹肌肉瘤（RMS）对浓度< 1 ng/ml的TRAIL诱导的细胞毒性和细胞凋亡的灵敏度。所有培养的细胞系都表达高水平的受体DR 5，但不表达DR 4或诱饵受体DcR 1或DcR 2，并表达FADD和半胱氨酸天冬氨酸蛋白酶原-8，Rh 36除外。c-FLIP的表达在TRAIL敏感性和抗性系中是高的，从而与TRAIL敏感性不直接相关。前半胱氨酸蛋白酶-8和-10的表达在TRAIL敏感的RD、Rh 1和Rh 30中最高。TRAIL敏感的Rh 18在没有半胱天冬酶原-10和c-FLIP的情况下表达半胱天冬酶原-8，而半胱天冬酶原-10在TRAIL抗性的Rh 41中不表达。基于这些细胞特征，将测试以下假设：1）在TRAIL与DR 5连接后，在RMS细胞系中在DR 5、FADD、半胱氨酸天冬氨酸蛋白酶原-8和c-FLIP之间形成DISC，导致I型（直接）或II型（线粒体依赖性和半胱氨酸天冬氨酸蛋白酶原-10依赖性）细胞死亡，和2）Rh 41中的TRAIL抗性是由于半胱氨酸天冬氨酸蛋白酶-10的缺陷。第二个焦点是阐明可以使TRAIL抗性RMS对TRAIL敏感或可以引起与TRAIL的协同相互作用的试剂。TRAIL与放线菌素-D或阿霉素的组合已经在TRAIL敏感的RMS系中显示出>累加效应，并且与重组人干扰素-γ（IFN-γ）的组合已经在TRAIL抗性HT 29人结肠癌细胞中诱导了细胞毒性应答。因此，我们将测试以下假设：1）TRAIL抗性Rh 28和Rh 41可以通过放线菌酮、放线菌素-D或IFN-γ对TRAIL敏化，和2）可以鉴定在TRAIL敏感性RMS细胞系中引发与TRAIL的协同相互作用的特异性化疗剂。第三，我们将基于组织培养中获得的数据，检验TRAIL单独给药或与化疗药物联合给药时在人RMS异种移植物中诱导抗肿瘤反应的假设。该提案的长期目标是开发针对儿童RMS转移性疾病的高效治疗方法：1）了解参与细胞死亡和凋亡调节的特定信号通路，2）识别新的分子靶点，3）基于特定分子特征开发新的治疗策略。

The proposal will elucidate critical regulators of the TRAIL signaling pathway that determine the exquisite sensitivity of 4/7 pediatric rhabdomyosarcomas (RMS) to TRAIL-induced cytotoxicity and apoptosis at concentrations < 1 ng/ml. All cultured cell lines express high levels of receptor DR5, but not DR4 or the decoy receptors DcR1 or DcR2, and express FADD and procaspase-8, with the exception of Rh36. Expression of c-FLIP is high in TRAIL-sensitive and -resistant lines, thereby not correlating directly with TRAIL sensitivity. Expression of procaspases-8 and -10 is highest in TRAIL-sensitive RD, Rh1 and Rh30. TRAIL-sensitive Rh18 expresses procaspase-8 in the absence of procaspase-10 and c-FLIP, and procaspase-10 is not expressed in TRAIL-resistant Rh41. Based upon these cellular characteristics the following hypotheses will be tested: 1) Following ligation of TRAIL to DR5, a DISC is formed among DR5, FADD, procaspase-8 and c-FLIP in RMS cell lines resulting in type I (direct) or type II (mitochondria- and procaspase-10- dependent) cell death, and 2) TRAIL resistance in Rh41 is due to deficiency in caspase-10. The second focus is to elucidate agents that can sensitize TRAIL- resistant RMS to TRAIL, or that can elicit synergistic interactions with TRAIL. Combination of TRAIL with actinomycin-D or doxorubicin has demonstrated > additive effects in TRAIL-sensitive RMS lines, and with recombinant human interferon-gamma (IFN-gamma) has induced cytotoxic response in TRAIL-resistant HT29 human .colon carcinoma cells. We will therefore test the following hypotheses: 1) TRAIL- resistant Rh28 and Rh41 can be sensitized to TRAIL by cycloheximide, actinomycin-D or IFN-gamma, and 2) specific chemotherapeutic agents can be identified that elicit synergistic interactions with TRAIL in TRAIL-sensitive RMS cell lines. Thirdly, we will test the hypothesis that TRAIL induces antitumor responses in human RMS xenografts when administered alone, or in combination with chemotherapeutic agents based upon data derived in tissue culture. The long-term objectives of the proposal are to develop highly effective therapy for metastatic disease in pediatric RMS from: 1) understanding specific signaling pathways involved in the regulation of cell death and apoptosis, 2) the identification of new molecular targets, and 3) developing new therapeutic strategies based upon specific molecular characteristics.