EpCAM调控Wnt/β-catenin通路促进卵巢癌细胞成球产生耐药的机制研究

Chemoresistance is one of the main cause of unfavorable prognosis of epithelial ovarian canecer. Directed by the multi-cellular chemoresistance theory, spheroids are more resistant to chemotherapy and therefore are considered to be ideal models for chemoresistance research due to the difference of monolayer cells and three-dimensional solid tumors. We design the research in cancerous spheroids and select cell adhesion molecules as target genes which play an important role in maintenance of both structures and functions of spheroids. Our previous study demonstrated that EpCAM was related to the chemoresistance of primitive ovarian cancerous spheroids and the expression by IHC was significantly correlated with the chemoresistance and prognosis. It was concluded that EpCAM was a key factor inducing spheroids formation and chemoresistance. Chip and EpCAM knock down experiments demonstrated that Wnt/β-catenin pathway was regulated by EpCAM. This subject is designed to make further identification of the relationship between EpCAM expression and spheroids forming and chemoresistance and explore possible mechanisms. The efficiency of target antibody treatment will also be evaluated. It is meant to clarify the mechanism of EpCAM inducing chemoresistance and explore a new marker to guide chemoresistance prediction and treatment.

化疗耐药是上皮性卵巢癌预后不良的主要原因之一。“多细胞耐药”理论认为三维培养的细胞耐药性增强，单层贴壁培养细胞不能模拟实体肿瘤的三维结构，利用球形培养细胞研究化疗耐药更合理。目前研究显示粘附分子在球形体细胞的立体结构形成及功能维持中均发挥重要作用，但其对卵巢癌细胞成球及耐药的影响所知甚少。我们前期发现粘附分子EpCAM与原代上皮性卵巢癌细胞成球及化疗耐药关系密切，且其组织表达和化疗耐药及预后显著相关，提示EpCAM是调控卵巢癌细胞成球及化疗耐药的关键分子；芯片及EpCAM敲降实验提示Wnt/β-catenin通路受 EpCAM调控。本课题拟运用分子与细胞生物学手段研究EpCAM对卵巢癌细胞细胞成球及化疗耐药的影响，揭示其作用机制，并评估EpCAM靶向抗体用于逆转化疗耐药的可行性，结果有助于深入理解卵巢癌耐药的机制，拓展其在临床耐药预测及逆转化疗耐药中的应用。

化疗耐药是上皮性卵巢癌预后不良的主要原因之一。“多细胞耐药”理论认为利用三维球形培养细胞的进行化疗耐药研究较单层贴壁细胞更合理。我们通过测序筛选在原代球形培养细胞中高表达的细胞粘附分子EpCAM，组织表达和卵巢癌化疗耐药及预后相关。本研究对EpCAM对于肿瘤细胞的成球及化疗耐药能力进行验证并对其可能的作用机制进行初步的探索。研究发现EpCAM过表达后的 SKOV3及HEY细胞悬浮培养成球能力增加，化疗耐药试验显示对于顺铂化疗药物的耐药性有增加趋势，进一步机制研究发现SKOV3及HEY 细胞在EpCAM过表达后Wnt/β-catenin通路相关基因β-catenin mRNA水平均呈下降趋势，而蛋白水平在HEY 细胞及SKOV3细胞株均无显著变化；流式检测细胞周期和凋亡细胞比率发现EpCAM过表达后SKOV3细胞株细胞凋亡受到抑制，而HEY 细胞凋亡比率反而增加，两株细胞EpCAM过表达后G2期细胞比例增加而G1期比例减少。本课题的完成为卵巢癌耐药相关研究提供新的研究靶点，同时三维球形培养为后续机制及治疗研究提供更贴合实际的模型。