靶向表皮生长因子受体变异体III的单克隆抗体CH12调控miR155/GSDME信号轴诱导乳腺癌细胞焦亡的机制

Epidermal growth factor receptor variant III (EGFRvIII) is a unique surface receptor found in tumor cells in recent years. CH12, a monoclonal antibody targeting EGFRvIII, was developed by the team containing the applicant. Our previous studies have shown that the proliferation of breast cancer in vitro and in vivo was inhibited after CH12 treatment, meanwhile, pyroptosis appeared. Pyroptosisis is a newly discovered programmed cell death pathway associated with inflammatory reaction, and apoptosis, necrosis and pyroptosis are commonly known as the three important pathways of cell death. Activation of gasdermin E is one of the important markers of pyroptosis. We found that CH12 treatment could activate GSDME and downregulated the level of oncogene miR-155. Bioinformatics prediction indicated that GSDME was one of the target genes of miR155. These results suggested that CH12 might induce the pyroptosis of breast cancer cells by regulating the miR155/GSDME signal axis. Therefore, we will establish EGFRvIII+ breast cancer model with GSDME overexpression or silencing, and investigate the molecular mechanism of CH12 induced pyroptosis in breast cancer cells at molecular, cellular and animal levels. The results might provide a theoretical basis and new strategies for the treatment of breast cancer.

表皮生长因子受体变异体III (EGFRvIII) 是近年发现的肿瘤特异受体。CH12是申请人参与研发的针对EGFRvIII的单克隆抗体。前期研究发现，CH12可以抑制乳腺癌体外和体内增殖，进一步观察发现CH12处理后细胞发生了焦亡。焦亡(Pyroptosis)是近年发现的一种伴随炎症反应的程序性死亡，与凋亡、自噬共称为细胞死亡的三种重要途径。Gasdermin E（GSDME）活化是细胞焦亡的重要标志。我们发现CH12的处理可以上调GSDME表达，并下调促癌基因miR155，生物信息预测结果显示GSDME是miR-155的靶基因之一。以上提示CH12可能通过调控miR155/GSDME信号轴，诱导乳腺癌细胞的焦亡。因此，本课题拟通过建立GSDME过表达或沉默的EGFRvIII+乳腺癌模型，从分子，细胞和动物水平探讨CH12诱导乳腺癌焦亡的机制，该研究将为乳腺癌治疗提供理论依据和新策略。

表皮生长因子受体 (EGFR) 是肿瘤发生发展的重要驱动基因。EGFRvIII是EGFR重要的变异体之一，可不依赖配体自磷酸化激活，与临床不良预后相关。EGFRvIII仅存在于肿瘤，而在正常组织中不表达，是特异的抗肿瘤治疗靶点。西妥昔单抗是现在临床应用最广泛的靶向EGFR的单抗，CH12是我们实验室自主研发的靶向过表达的EGFR或者EGFRvIII的单抗，在EGFRvIII阳性的小鼠肿瘤模型有很好的抑制效果。焦亡是近年发现的一种伴随炎症反应的程序性死亡，与凋亡、自噬共称为细胞死亡的三种重要途径。诱导肿瘤细胞焦亡，也是逆转耐药的方式之一。.第一项研究显示, 下调miR-155-5p可以增强西妥昔单抗对EGFR过表达三阴乳腺癌的增殖和迁移的抑制作用，促进细胞死亡。GSDMs家族活化是细胞焦亡的重要标志。数据显示，GSDME是miR-155-5p的直接结合靶点。miR-155-5p拮抗剂诱导GSDME裂解成GSDME-N，在细胞膜上聚集并打孔，诱导细胞焦亡。miR-155-5p拮抗剂与西妥昔单抗联合使用，增强了西妥昔单抗在MDA-MB-468-EGFR移植瘤的抗肿瘤作用。因此，西妥昔单抗联合miR-155-5p拮抗剂可能是治疗TNBC的一种新的治疗策略。.第二项研究显示，当NK与肿瘤细胞共培养时，CH12处理后，细胞死亡率显著上升。CH12通过抑制EGFR信号通路，诱导caspase 3的裂解，cleaved caspase3切割GSDME产生GSDME-N，诱导肿瘤细胞焦亡；同时，CH12在体内引发NK细胞的ADCC效应，NK细胞将Granzyme B释放到肿瘤细胞，可以水解GSDME，产生活性GSDME-N，继续诱导肿瘤细胞焦亡；此过程中产生的炎症因子，使免疫细胞处于激活状态，进而在局部持续杀伤肿瘤。.本研究从诱导肿瘤细胞焦亡的角度，为抗体药物的增敏增效提供理论依据和方案选择。