第8节 Ewing肉瘤的基础研究进展
Ewing肉瘤是一种低分化、高度恶性的小圆形细胞肿瘤。James Ewing于1921年首次报道了一种以圆形细胞为主要成分的肉瘤，当时称作“弥散性骨内皮瘤”，后来命名为Ewing肉瘤。1976年，Nesbitt和Vidone描述神经上皮瘤为原始神经外胚层肿瘤（primitive neurotodermal tumour, PNET）。1979年Askin描述了一种胸肺部起源的小圆细胞肿瘤，后来称Askin瘤。由于Ewing肉瘤、PNET、Askin瘤有着同样的病理学特征，并且大都存在t（11；22）（q24；q12） 染色体异位，表达EWS-FLI1融合蛋白，所以上述肿瘤同为Ewing肉瘤家族肿瘤。
一、 Ewing肉瘤的组织起源
关于Ewing肉瘤的细胞起源至今尚无定论，早期根据其细胞形态和胞质稀少的特点归类为内皮细胞来源。1971年时根据其超微结构特征类似骨髓细胞，将其归类于髓源性肿瘤。目前，关于Ewing肉瘤组织起源的假说主要集中在神经嵴细胞和间充质干细胞。关于起源于神经嵴细胞的依据在于：Ewing肉瘤细胞表达烯醇酶、S-100蛋白等神经细胞特有的标志物，在电镜下可以观察到Ewing肉瘤细胞中存在神经内分泌颗粒，而且Ewing肉瘤细胞的基因表达谱与神经嵴细胞十分接近；而有趣的是，在横纹肌肉瘤细胞中表达EWS-FLI1蛋白可促使该细胞表达神经细胞的标志物，并诱导该细胞的表型向Ewing肉瘤转化。也有许多证据表明Ewing肉瘤细胞可能来源于间充质干细胞。在骨髓间充质干细胞中表达Ewing肉瘤细胞特有的EWS-FLI1蛋白，可以抑制干细胞向成骨细胞或脂肪细胞分化；在小鼠骨髓间充质干细胞中表达EWS-FLI1蛋白后接种裸鼠，可形成Ewing肉瘤样的肿瘤；同时，在Ewing肉瘤细胞中敲除EWS—FLI1后其基因表达谱近似间充质干细胞。所以，EWS-FLI1融合蛋白已被视为Ewing肉瘤发生和发展的核心因素。与其争议Ewing肉瘤的组织来源，许多学者更倾向于依据EWS-FLI1来界定Ewing 肉瘤。
二、 Ewing肉瘤特征性的染色体异位
染色体易位是恶性肿瘤的常见现象，Ewing肉瘤是典型的以染色体易位为重要特征的恶性肿瘤。目前，在Ewing肉瘤中已发现多种染色体易位方式，其中约85%为t（11；22）（q24；q12）易位。这种易位使第11号染色体上的FLI1基因易位到第22号染色体，并转录正常组织不存在的融合蛋白EWS-FLI1。这种融合蛋白保留了EWS蛋白5’端有富含谷氨酰胺的转录活性区，以及FLI1蛋白3’端的DNA结合序列，形成了具有强转录活性的蛋白结构。EWS-FLI1蛋白通过结合GGAA/T序列启动靶基因的转录，造成多种基因异常表达。其余的易位方式包括t（21；22）（q22；q12）易位，形成融合基因EWS-EGR，约占ESFT染色体易位的5%；t（7；22）（p22；q12）易位，形成融合基因EWS—ETV1；t（17；22）（q12；q12）易位，形成融合基因EWS-E1AF；t（2；22）（q33；q12）易位，形成融合基因EWS-FEV；t（16；21）（p11；q21）易位，形成融合基因FUS-EGR；t（2；16）（q35；p11）易位，形成融合基因FUS-FEV。上述易位所形成的融合基因中，仅EWS-FLI1与Ewing肉瘤的形成和发展紧密相关，既可以作为一个特异性分子参考诊断，又可以作为潜在的分子靶点，探索治疗策略。
三、 Ewing肉瘤的分子治疗
以往Ewing肉瘤的主要治疗方式是手术加放疗、化疗，对于尚未发生转移的患者，治疗后5年生存率可以达到70%左右。然而，有20%～25%的患者在明确诊断时就已经发生远处转移。对于肿瘤复发或远处转移的患者，传统治疗方式效果不明显，所以总体生存率仍不乐观。为探索更为有效的治疗方式，学者们把目光集中在分子靶向治疗上。
目前研究显示，Ewing肉瘤的基因组突变率在所有恶性肿瘤中是最低的，然而特异性的染色体易位产生了Ewing肉瘤标志性的融合蛋白EWS-FLI1。EWS-FLI1蛋白仅在Ewing肉瘤细胞中表达，并且与Ewing肉瘤的恶性生物学行为紧密相关，这使得EWS-FLI1成为十分理想的靶点。自1992年EWS-FLI1被鉴定后，许多研究证明EWS-FLI1对于保持肿瘤恶性特征有至关重要的作用。使用小干扰RNA（siRNA）干扰EWSFLI1表达，可以明显抑制Ewing肉瘤细胞增殖、侵袭以及相关肿瘤学表型。然而由于siRNA药代动力学上的缺陷，致其在目前阶段尚无法应用于临床。Toretsky等利用噬菌体文库筛选能与EWSFLI1结合的多肽，发现EWS-FLI1可与RNA解旋酶A的末端解螺旋结构域结合，随后通过等离子共振技术在3 000个化合物中筛选可以与EWSFLI1匹配的化合物，最后鉴定出YK-4-279可以结合EWS-FLI1并抑制其与RNA解旋酶A解离。进一步研究发现，YK-4-279通过阻断EWS-FLI1与RNA解旋酶A的结合，明显抑制了EWS-FLI1的转录活性，并对Ewing肉瘤细胞产生细胞毒性作用。目前YK-4-279药物（TK216）已经进入临床试验阶段，并表现出良好的临床效果。
基因表达谱和组织芯片分析发现，赖氨酸去甲基化酶1（lysine-specific demethylase 1，LSD1）在Ewing肉瘤中高表达，且通过对比发现Ewing肉瘤中LSD1的表达程度在36种恶性肿瘤中排第二；同时，研究发现LSD1可参与调控EWS-FLI1的转录活性。因此，LSD1也可作为Ewing肉瘤的理想靶点。在Ewing肉瘤细胞培养基中加入LSD1抑制剂HCI-2509可逆转EWS-FLI1所介导的基因表达变化，这已预示着良好的临床前景。目前，有3个LSD1抑制剂-苯环丙胺、GSK-2879552、ORY-100，在急性髓细胞白血病的研究中已进入临床试验阶段。
聚腺苷二磷酸-核糖聚合酶（poly ADP-ribose polymerase, PARP）超家族是一组多功能酶，对于单链DNA修复具有重要作用。PARP1是该家族的重要成员之一，在Ewing肉瘤中PARP1表达升高，抑制PARP1活性可导致EWS-FLI1蛋白表达降低。奥拉帕尼（olaparib）是PARP1的抑制剂，Ewing肉瘤细胞对奥拉帕尼的敏感性要优于其他恶性肿瘤。同时，前临床试验发现奥拉帕尼联合放疗可增强放疗对DNA的损伤。目前，奥拉帕尼对于Ewing肉瘤的治疗已进入Ⅱ期临床试验。
在以Ewing肉瘤特异性分子为靶点探索靶向药物的同时，一些恶性肿瘤所共有的靶点分子也在Ewing肉瘤中开展探索，如针对IGF-1受体（IGF-1 receptor, IGF1R）、mTOR、血小板衍生生长因子受体α （platelet-derived growth factor receptor α，PDGFR α）、EGFR、VEGF受体（VEGF receptor, VEGFR）等靶点的药物，在Ewing肉瘤的研究中也取得了较好的效果。
随着分子生物学技术的不断发展，对Ewing肉瘤恶性生物学行为内在机制的了解不断加深，对于Ewing肉瘤的靶向治疗一定会取得令人瞩目的成效。
（周旺 李磊 姜丛 李珍惜
王辉 万宗淼 陈天睿）
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