GFRα1b调控肝癌发生发展的作用和免疫逃逸机制研究

Tumorigenesis and progression are always accompanied the fighting with the immune system, tumor microenvirment escapes the attacks of host immune system by regulating certain molecular expression. GFRα1 is the receptor of neurotrophic factor GDNF, and GFRα1b was found to be expressed predominantly in the fetal and adult liver, but it’ function and significant remain elusive. Our previous studies showed that GFRα1b was down-regulated abundantly at mRNA level in hepatocellular carcinoma (HCC) compared to the matched non-neoplastic tissue in nearly 90% of HCC patients. And GFRα1b expression level in HCC is strongly negatively correlates with the survival of HCC patients. In DEN-induced HCC mice model, we also found that the expression of GFRα1b in nomal liver significently higer than in HCC tissue both at mRNA and protein level. And accompanying thedown-regulated expression of GFRα1 in tumor tissues, there are abundant immune cells infiltration. These results suggested that the down-regulated expression of GFRα1b maybe contribute to HCC progression and immune escape. Here, based on the previous studies, we want to further investigate whether condition knockdown of GFRα1b expression in liver could affect HCC generation? And whether over-expression or silence of GFRα1b expression in HCC cells could affect HCC development and metastasis in vitro and in vivo? And demonstrate the mechanisms that GFRα1 regulate HCC generation and progression via evading the immune system recognization and attacks. Finally providing a novel strategy and target for HCC immunotherapy.

肿瘤的发生发展始终伴随着与免疫系统的博弈，肿瘤通过调控某些分子的表达营造适宜的微环境而逃避免疫系统的识别和攻击。GFRα1是神经营养因子GDNF的受体，有GFRα1a/1b两种剪切体，GFRα1b优势表达于肝脏。我们前期2个临床中心318例肝癌样本的研究发现，近90%患者癌组织GFRα1b的表达显著低于癌旁组织，且癌组织GFRα1b的表达与肝癌患者的生存率呈显著负相关。DEN诱导的小鼠肝癌显示，癌组织中GFRα1b表达水平较正常肝组织显著降低，且伴随大量免疫细胞的浸润。这些结果提示GFRα1b在肝脏表达的降低可能与肝癌的发生发展及免疫逃逸显著相关。本研究旨在上述研究的基础上，利用基因敲除小鼠，基因沉默，双光子显微镜及小动物成像等平台，阐明GFRα1b表达的降低或缺失与肝癌发生发展的相关性，明确GFRα1b调控肝癌发生发展逃避机体免疫系统识别和攻击的机制，为肝癌的免疫治疗提供新的策略和靶标。

大量研究表明神经营养因子及其受体在肿瘤的进展、转移、化疗抵抗等中扮演重要角色。胶质细胞系源性神经营养因子(glial cell line derived neurotrophic factor，GDNF)家族成员（GDNF, neurturin, artemin, artemin和 persephin）在肿瘤进展中的作用也逐渐得到关注和广泛研究。我们前期的研究也发现，GDNF家族成员artemin的血清含量在肝癌患者中较健康人、慢性肝炎及肝硬化患者显著增高，且与肝癌患者的生存期显著相关；体内给予重组artemin蛋白显著促进小鼠肿瘤的生长，给予阻断性抗体或者敲减其受体GFRα3的表达显著抑制肿瘤的生长。本课题在此基础上，进一步研究发现，GDNF家族另一成员GDNF受体GFRa1在正常肝脏组织高表达，而来自两个不同肝癌临床中心的肝癌组织和癌旁组织样本的荧光定量结果显示，有84%以上肝癌患者的癌组织中GFRα1的mRNA表达低于相应癌旁组织；且GFRα1的低表达与肝癌患者的生存率显著相关；肝癌组织芯片免疫组化结果也显示，有65.5%肝癌患者肝癌组织中GFRα1蛋白的表达显著低于相应癌旁及正常肝组织，且GFRα1 蛋白的低表达与肝癌患者的生存率显著相关。在DEN诱导的小鼠肝癌模型中，GFRα1基因敲除能够显著增加肝癌发生率和促进肝癌的进展。肝癌细胞系体内外实验发现，沉默GFRα1基因的表达能够显著促进肿瘤细胞的侵袭和转移，促进肿瘤的生长和转移，缩短荷瘤小鼠的生存期；而过表达GFRα1基因，则能够显著抑制肿瘤细胞的增殖能力，加细肿瘤胞的粘附能力，抑制肿瘤的生长和转移，延长荷瘤小鼠的生存期。此外，肝脏特异性的GFRα1过表达病毒载体（AAV8-GFRα1）瘤内注射治疗，能够显著抑制小鼠肝癌的生长，延长荷瘤小鼠生存期。这些结果提示，GFRα1可能是肝癌的候选预后生物标志物和潜在的治疗靶点。