沉默STAT3增强SNX-2112诱导食管癌干细胞样细胞凋亡的作用机制研究

Many studies indicate that cancer stem cells (CSCs) play an important part in treating patients. Heat shock protein 90 is a potential anti-tumor target. Our applicants have been engaged in the study of esophageal cancer stem cells. In the phase of my doctor study we found that the esophageal cancer stem-like cells (ECSLCs) were isolated using serum-free suspension media to form spheres. The ECSLCs were characterized by qPCR and flow cytometry and these analysis demonstrated that Nanog, Sox2, Bmi1 and STAT3 were over-expressed in the ECSLCs. And slience of STAT3 inhibited the proliferation of ECSLCs. Moreover, Our former studies demonstrated that Hsp90 inhibitor SNX-2112 has strong anti-tumtor capacity, such as hepatocellular carcinoma, melanoma and leukemia cells. Base on these studies, we are going to study synergistic efficiacy of SNX-2112 with slience of STAT3. We intend to sudy whether SNX-2112 with slience of STAT3 synergistically induce apoptosis of ECSLCs. In addition, it will be confirmed whether SNX-2112 with slience of STAT3 combination have synergistic anti-tumor effect. These studies will provide strong evidence for the esophageal cancer therapeutic potential of the combination of SNX-2112 and slience of STAT3.

研究表明，肿瘤干细胞是肿瘤研究的重点。热休克蛋白90是潜在的抗肿瘤靶点。申请人博士阶段一直从事食管癌干细胞的靶向研究，前期博士阶段研究发现：①采用无血清培养的方法从食管癌细胞获得食管癌干细胞样的细胞；②经分析，该食管癌干细胞样细胞高表达Nanog、Sox2、Bmi1、STAT3等干性因子；③慢病毒沉默STAT3抑制该肿瘤干细胞样细胞增殖；④前期研究基础表明， Hsp90抑制剂SNX-2112具有良好的抗肿瘤能力，如肝癌、黑色素瘤、白血病等肿瘤细胞生长的能力。基于此，本课题拟以STAT3与SNX-2112的药效为切入点，我们推测，①沉默STAT3协同增强SNX-2112抑制食管癌干细胞样细胞增殖；②在小鼠体内实验，二者联合作用同样具有抗食管癌干细胞样细胞生长的效果。本课题将为阐明沉默STAT3增强SNX-2112抑制食管癌干细胞样细胞增殖提供依据，也为食管癌的临床治疗提供指导方向与治疗策略。

食管癌是世界第七大肿瘤致死性疾病，其类型主要包括鳞癌与腺癌。本项目研究沉默STAT3后Hsp90的小分子抑制剂SNX-2112对食管癌干细胞样细胞的增殖抑制影响。测序与差异化表达分析发现STAT3与p-STAT3（Tyr705）在人食管癌临床标本高表达，western blot发现肿瘤组织STAT3表达显著高于癌旁组织。沉默STAT3后SNX-2112抑制食管癌干细胞样细胞的存活率(20.4±3.3)%，降低Bcl2、ABCB1与ABCG2表达，提高Bax表达，降低食管癌干细胞样细胞克隆形成能力(25.3±5.6)%，阻滞其细胞周期在G2/M期，提高其凋亡水平(70.5±5.6)%，降低Hsp90客户蛋白MAPK信号通路蛋白（p-P38,p-JNK,p-ErK）表达。小鼠体内实验亦证明此效果，两者联合应用降低小鼠瘤重(1.3-1.8)倍、抑制瘤体积增长（2-2.5）倍，并发现小鼠体重没有明显变化；瘤组织免疫组化结果表明Hsp90的客户蛋白Akt（2.2倍）/ErK（2.5倍）水平降低，Tunel实验显示细胞凋亡显著提高（25.0±5.9）%。如果提高STAT3表达则出现相反的结果。过表达STAT3显著降低SNX-2112对食管癌干细胞增殖抑制效果，如提高Hsp90客户蛋白表达（p-P38,p-JNK,p-ErK），降低细胞凋亡比例（40.0±8.4）%，提高ABCB1与ABCG2表达水平（分别是0.5与0.6倍）。这些研究表明，沉默STAT3有助于提高Hsp90的抑制剂SNX-2112抑制食管癌干细胞样细胞的增殖抑制效果。这些研究为食管癌的靶向治疗奠定了基础。