HE4与ANXA2相互作用调节肿瘤上皮间质转化介导卵巢癌耐药的机制研究

HE4 is a novel ovarian cancer marker with highest sensitivity and specificity. Although it could be a candidate to predict chemoresistance of ovarian cancer, the mechanism mediated by HE4 is still poorly understood. Our previously researches discovered that: 1) HE4 could interact with Annexin A2 (ANXA2), and their high expression levels facilitate the chemoresistance; 2) The expression level of EMT-related molecules changed in accordance with the expression of HE4 and ANXA2; 3) Imbalance of intracellular calcium could promote EMT; Intracellular calcium could be regulated by the spatial conformation change of ANXA2 after interaction with other proteins; HE4 could influence the expression of calcium channel protein TRPC6; 4) Both of HE4 and ANXA2 exist modification of fucosylation, and the glycosylation strengthened the function of HE4; 5) HE4 could influence the expression of MAPK and TGF-βsignaling pathway. Our hypothesis: The interaction of HE4 and ANXA2 could influence the spatial conformation of ANXA2, resulting in the increasing of intracellular calcium, to activate signal pathways and promote EMT and chemoresistance, furthermore, these effects could be enhanced by fucosylation of HE4 and ANXA2. The antibody specific to the binding site could interrupt the contribution of the complex to chemoresistance. This project aims to elucidate the mechanisms of ovarian chemoresistance mediated by the interaction of HE4 and ANXA2.

HE4是目前敏感性及特异性最高的新型卵巢癌标志物，可作为化疗耐药的预测因子，但其与卵巢癌耐药机制研究目前几乎空白。我们发现：1.HE4与ANXA2为互作蛋白，二者高表达促进化疗耐药；2. HE4及ANXA2的表达影响上皮间质转化（EMT）相关分子变化；3.细胞内Ca2+稳态失衡促进EMT发生，ANXA2与互作蛋白结合后发生空间构象改变，影响细胞内Ca2+的调节；HE4上调钙通道蛋白TRPC6的表达；4.HE4/ANXA2均存在岩藻糖基化修饰，糖基化修饰增强HE4的功能；5.HE4影响MAPK及TGF-β通路相关分子表达。推测：HE4与ANXA2结合，影响ANXA2空间构象，增加细胞内Ca2+浓度，激活信号通路，影响细胞EMT，促进化疗耐药，糖基化修饰增强上述作用。针对HE4与ANXA2结合位点的抗体能阻断该复合体对耐药的影响。本项目拟阐明HE4与ANXA2相互作用介导卵巢癌化疗耐药的机制。

人附睾蛋白4(HE4)是目前敏感性及特异性高的卵巢癌标志物之一，但目前其相关机制研究极少且粗浅。本研究为前期国家自然科学基金研究的后续，目的探究HE4与膜连蛋白A2（ANXA2）相互作用调节肿瘤上皮间质转化（EMT）介导卵巢癌耐药的机制。首次证明：①HE4与ANXA2互作关系，互作区域为ANXA2氨基端232-301位氨基酸；②HE4与ANXA2均存在糖基化修饰，Lewis y为优势糖链，Lewis y修饰增强二者及互作蛋白功能，增强癌细胞EMT及耐药；③HE4通过ANXA2通过p-MEK/p-ERK/twist和p-TAK/p-P38/twist途径促进癌细胞EMT。上述现象也存在于卵巢癌以外的其他癌症。④HE4通过ANXA2作为“桥梁”连接S100家族、CD147、MMPs、P-gp等，与ANXA2-MMP2形成蛋白复合体、通过ANXA2调控MMP2促进卵巢癌细胞侵袭转移，通过PI3K信号通路促进卵巢癌细胞增殖、EMT和自噬而影响耐药；通过ANXA2抑制hsa-miR-129-5p的表达，促进P-gp的表达介导卵巢癌细胞对阿霉素的耐药；通过与S100A相互作用调控细胞Ca2+通道蛋白TRPC6的表达。⑤HE4通过促进ZNF703的核易位，促进ZNF703与PEA15增强子区域结合最终促进PEA15的转录，从而促进癌细胞的恶性生物学行为，包括侵袭转能力移及EMT水平等。⑥克隆针对ANXA2氨基端232-249位氨基酸制备单克隆抗体，证明抗体可识别卵巢癌组织及细胞中内源性ANXA2和外源性ANXA2重组蛋白，具有较好的敏感性及特异性，抗体在体内外具有抑制卵巢癌细胞增殖、侵袭和迁移等能力。除此之外，还研究了其他HE4结合蛋白及下游蛋白等对卵巢癌发生发展的影响及机制，以及ANXA家族其他成员在卵巢癌发生、发展及耐药中的作用。研究为预测卵巢癌耐药、寻找卵巢癌治疗靶点提供实验依据。