中心体调控因子Nek2A在三阴性乳腺癌中与化疗药物敏感性关系的研究

Nek2A is an important centrosome associated kinase, our previous study found that Nek2A high expressed in breast invasive carcinoma compared with normal tissues, triple-negative breast cancer（TNBC） was significantly higher than that of luminal type. And TNBC patients with high expression Nek2A pathological complete response rate is low, prone to drug resistance. TNBC with poor prognosis, lack of effective means of targeted therapy and endocrine therapy, is difficult in clinical treatment. This study intends to study sensitivity of Nek2A with chemotherapy drugs in triple negative breast cancer cell lines; in vivo tumorigenicity (the chemotherapy group, siRNA interference group and their combination group were implanted in nude mice, observed in situ tumor and distant metastasis and tumor HE staining was observed response to chemotherapy), in TNBC patients samples (detect Nek2A expression in TNBC tumor samples, analyze the relationship between its expression and response to chemotherapy to analyze the relationship between the prognosis of patients). And explore molecular mechanism of them. This study in-depth investigate whether Nek2A can affect chemosensitivity in triple negative breast cancer and study of the mechanism. Thus opening up new avenues to enhance chemosensitivity and reverse chemoresistance, providing clues for triple negative breast cancer targeted therapy.

Nek2A是一种重要的中心体相关激酶，我们前期研究发现：Nek2A在乳腺浸润性癌表达增高，并且三阴性乳腺癌（TNBC）中表达明显高于腺腔型乳腺癌；并发现Nek2A高表达TNBC患者病理完全缓解率较低，易出现化疗耐药。TNBC预后差，缺乏有效的个体化治疗手段，化疗耐药的TNBC患者是临床治疗难点。本课题拟从TNBC细胞系、体内裸鼠成瘤实验（观察化疗药物组、siRNA干扰组及二者联合组各组细胞原位成瘤及远处转移能力及化疗反应）、新辅助化疗TNBC患者肿瘤标本（分析Nek2A表达与化疗反应的关系及与患者预后的关系）、探讨相关分子机制多个层面进行Nek2A与化疗药物敏感性的研究。本项目将深入研究Nek2A对TNBC化疗药物敏感性的影响及相关机制，从而探寻增强化疗敏感性甚至逆转化疗耐药的新途径，为三阴性乳腺癌个体化治疗提供线索。

Nek2A是一种重要的中心体相关激酶，研究发现Nek2A在乳腺浸润性癌表达增高，并且三阴性乳腺癌（TNBC）中表达明显高于腺腔型乳腺癌。TNBC预后差，缺乏有效的个体化治疗手段，化疗耐药是TNBC患者临床治疗难点。本课题在三阴性乳腺癌细胞系、裸鼠及新辅助化疗TNBC患者肿瘤组织标本中研究。发现TNBC细胞系中Nek2A异常高表达，增殖异常增高，并且γ-tubulin（中心体）异常增多，α-tubulin（细胞骨架蛋白）表达异常。Nek2A-siRNA转染三阴性乳腺癌细胞系后，与转染对照-siRNA组相比，细胞增殖减慢、Ki67增殖指数表达降低，并且γ-tubulin（中心体）异常表达比例降低。流式细胞术检测细胞周期显示细胞周期分布无差异、划痕实验检测细胞迁移运动未有明显差异，行CCK8实验检测细胞IC50发现，降低Nek2A表达可以增强紫杉醇及顺铂化疗药物敏感性，而对长春瑞滨化疗药物的敏感性改变未见统计学差异。在三阴性乳腺癌细胞系中行ICC（免疫细胞化学方法）检测发现β-catenin、CDC20、Ki-67及ABCC1与Nek2表达相关。裸鼠成瘤实验发现，Nek2A-siRNA联合TAX及DDP化疗药物组裸鼠成瘤肿物最小，对瘤体及裸鼠重要脏器进行取材、固定、切片、HE染色发现：空白对照组可见1只裸鼠发生肺及肝两处内脏转移，1只裸鼠发生皮下多发转移。从我院行新辅助化疗≥4周期后进行手术的TNBC患者临床病例中随机抽取100例，并评估方法化疗反应，0级的14例，Ⅰ级的36例，Ⅱ级的38例，Ⅲ级的12例。并统计分析Nek2A的表达与患者新辅助化疗后病理完全缓解率的关系，结果发现：Nek2A高表达患者与Nek2A低表达患者相比较，病理完全缓解率较低，易出现化疗耐药。Nek2A异常增高可能与三阴性乳腺癌化疗耐药相关。降低Nek2A表达可以增强紫杉醇及顺铂化疗药物敏感性，并发现β-catenin、CDC20及ABCC1与Nek2表达相关。其可能通过影响Wnt通路、ABC转运体等发挥作用。为三阴性乳腺癌个体化治疗提供线索。