CDK2AP1在乳腺癌中抑制癌变发生的分子信号通路和临床意义

Breast cancer is the most serious threat to women's health among malignant tumors. In the preliminary research, through the histomorphological study and in vitro, in vivo research, we find CDK2AP1 play an important role in inhibiting the proliferation of breast cancer. As a cell cycle regulator, CDK2AP1 is involved in the development of breast cancer. The dysfunction of the CDK2AP1 is one of the key factors for the progression of breast cancer, On the basis of the previous research, we find that CDK2AP1 directly controls CDK2, but the function and its sub-cellular localization may also relevant. This project intends to through Pathway array combining research methods and ChiP – qPCR to clarify the relationship between CDK2AP1 associated molecular signaling pathways and the inhibition course of breast cancer. This project also intends to research the relationship between the nucleolus orientation of CDK2AP1 and functions of assotiated promoters, and study the relationship between the CDK2AP1 molecular mechanism and its clinical pathological feature. Our aim is to add the theoretical foundation for the development of the clinical diagnosis of breast cancer as molecular markers and the possible application of biological treatment that is a potential therapeutic candidate to suppress tumorigenicity in breast cancer.

乳腺癌是严重威胁女性健康的最常见恶性肿瘤。申请者在前期工作中通过组织形态学和体外、体内实验等研究，证实CDK2AP1在抑制乳腺癌恶性增殖中发挥重要作用，其功能是通过调控细胞周期实现的，CDK2AP1的功能缺失正是导致乳腺癌恶变的关键因素之一。在前期研究基础上，申请者发现在乳腺癌中CDK2AP1直接调控CDK2，并存在不同亚细胞定位。本项目拟通过Pathway array结合ChiP-qPCR等研究方法，阐明CDK2AP1在乳腺癌中抑制癌变发生的分子信号通路，研究不同亚细胞定位与相关信号分子功能的联系，并通过临床样本分析CDK2AP1的表达、定位与病理特征和临床预后的相关性，为开发其作为临床分子诊断标记物和可能的生物治疗应用奠定理论基础。

细胞周期蛋白依赖性激酶2关联蛋白1（cyclin-dependent kinase 2-associated protein 1，CDK2AP1）是一个重要的生长抑制因子，在人类正常组织中普遍表达。本研究的重点是其在乳腺癌中的作用及其机制。利用酵母双杂交筛选CDK2AP1的相互作用蛋白，并用pull down验证发现，与肿瘤侵袭转移密切相关的组织蛋白酶L亚型（Cathepsin L1，CTSL）可能是与CDK2AP1相互作用的分子。应用免疫组化检测临床标本，发现CDK2AP1高表达与转移和HER2高表达相关。CDK2AP1核定位表达与年龄和PR表达水平相关，但与转移关系并不密切。通过蛋白芯片比较CDK2AP1敲减组和过表达组信号通路蛋白的表达变化，发现CDK2AP1表达改变和STAT3通路的激活相关；过表达CDK2AP1的细胞中p-STAT3增加。对乳腺癌患者有转移且CDK2AP1阳性表达，和无转移且CDK2AP1阴性表达的活检新鲜临床病理样本进行lncRNA芯片筛选，发现lncRNA-ATB（又称为lncRNA-AL589182.3）、lncRNA-DC、DANCR等显著升高。与相应的空载体组细胞相比，经慢病毒载体过表达或敲减CDK2AP1基因的MCF-7细胞和MDA-MB-231细胞，发现lncRNA-ATB的表达与CDK2AP1的表达水平相关，而lncRNA-DC、DANCR的表达与CDK2AP1的变化不相关。并在组织水平上发现lncRNA-ATB及炎性因子（Il-6、TGFβ）在CDK2AP1阳性表达的乳腺癌病理样本中的mRNA水平显著升高。过表达乳腺癌细胞MDA-MB-231细胞的lncRNA-ATB后的显著引起p-STAT3水平增加，并检测到炎性因子(IL-6、TGF-β、TNF-α)的mRNA水平显著增高。通过细胞功能学实验证明敲减lncRNA-ATB对乳腺癌细胞侵袭的能力影响不大。说明lncRNA-ATB影响乳腺癌的转移不是通过该基因的直接作用，而是通过调节炎性因子的表达及分泌间接实现的。拟在此基础上，深入研究lncRNA-ATB调节乳腺癌细胞炎症因子产生的信号通路，从而促进乳腺癌细胞转移的机制。