胶质瘤中Wnt/β-catenin通路竞争性募集CBP以推动NKG2D/MICA相关免疫逃逸的新机制

Tumor immunology has become a hotspot of cancer research afresh, while the relationship between immune evasion and signaling pathway has not been studied comprehensively. Explore and develop optimization strategy of glioma immunotherapy. It has been reported that glioma exhibits low level of NKG2D ligand MICA, hence abrogate the immunosurveillance detected by NKG2D on immune cells. Our previous study found that the blockade of Wnt/β-catenin pathway facilitate the expression of MICA and the cytotoxic lysis towards malignant glioma cells, while antagonize CBP could abolish this effect. CBP with histone acetylases is critical for the expression of MICA in epigenetic level via altering nucleosome to 'open'. The aberrant activation of Wnt/β-catenin pathway is the pivotal event in gliomagenesis. β-catenin requires recruiting CBP to act synergistically as a coactivator for efficient activation of transcription, our previous study found that the blockade of Wnt/β-catenin pathway led to increase of the occupancy of CBP on NKG2DL promoter. From this, we propose that Wnt/β-catenin pathway restrain MICA expression and NKG2D/MICA related immunosurveillance through competitively recruiting CBP. Taken together, we will unravel a novel functional model modulating immune evasion, which could be presented as a promising strategy for immunotherapy.

肿瘤免疫研究已重新成为胶质瘤等肿瘤研究领域的热点，而有关肿瘤细胞信号通路与免疫逃逸之间关系的研究尚不全面。现已证实，胶质瘤细胞表面的NKG2D配体MICA处于低表达，从而躲避了免疫细胞上NKG2D的免疫监视。我们前期发现敲低β-catenin能有效提高胶质瘤细胞上MICA的表达，应用CBP的抑制剂后上述作用消失。在表观遗传学水平，CBP能以组氨酸乙酰化作用“解开”染色体构型，使MICA进行转录。Wnt/β-catenin通路的异常表达是胶质瘤发生发展的中心事件，β-catenin须募集CBP作为转录辅助因子以调控下游癌基因表达，我们前期发现敲低β-catenin能有效提高CBP与MICA启动子区的结合。故我们提出Wnt/β-catenin通路通过竞争性募集CBP，在表观遗传学水平抑制MICA的表达，从而实现胶质瘤细胞对免疫攻击作用的逃逸。本课题有望为开发新的胶质瘤免疫治疗手段奠定基础。

NKG2D配体MICA据报道与恶性胶质瘤的免疫监视有关。因为Wnt/β-catenin信号通路是胶质瘤发生中的主要事件，故我们的目标是确定其在恶性胶质瘤MICA/NKG2D相关免疫逃逸中的作用。患者肿瘤标本中MICA与β-catenin的表达由免疫组化进行检测。应用Western blot与流式细胞仪对MICA进行体外检测。应用TOP/FOP荧光试验评价β-catenin转录活性。体外试验中免疫细胞针对多种靶细胞的毒性活性用LDH释放试验确定。Co-IP用于确定CBP与β-catenin的结合情况，染色体ChIP用于确定CBP与MICA的结合情况。截去C端的β-catenin质粒及ICG001用于破坏CBP与β-catenin之间的结合。在胶质瘤标本中，MICA的表达与β-catenin呈负相关，通过回顾性分析，这可以作为对患者预后生存期的预测指标。Wnt/β-catenin信号通路抑制MICA的表达及NK细胞对恶性胶质瘤细胞的毒性作用，破坏CBP与β-catenin的结合将遏止这一效应。对于机制的研究提示β-catenin/CBP复合体的形成减小了CBP在MICA启动子区的占位。并且，在体内试验中，β-catenin与CBP结合的受损将限制MICA的表达及胶质瘤的生长。我们的研究确定了Wnt/β-catenin信号通路在恶性胶质瘤的MICA/NKG2D相关免疫逃逸中的作用。