自噬相关基因Beclin1在HER2阳性乳腺癌曲妥珠单抗耐药中的作用及表达调控机制研究

Trastuzumab is a target drug recommended by NCCN guidelines for treating patients with HER2-positive breast cancer. However, the majority of patients with HER2-positive breast cancer do not respond to trastuzumab in the clinic. Therefore, there is an urgent need to elucidate the mechanism of resistance to trastuzumab. The present research shows that the resistance to trastuzumab involves the participation of a variety of mechanisms, and the latest studies suggest that autophagy is closely related to resistance. In our previous work, we identified a new autophagy-related gene Beclin1 regulating the resistance to trastuzumab in trastuzumab resistant breast cancer cells and demonstrated that knockdown of Beclin1 could increase the sensitivity of trastuzumab-sensitive and -resistant breast cancer cells to trastuzumab by CCK-8 and colony formation assay in vitro. In the present study, we will further analyze whether knockdown of Beclin1 could enhance trastuzumab induced breast cancer cell cycle arrest, apoptosis, and inhibition of tumor growth in nude mice in vivo. What’s more, we will elucidate the mechanism responsible for differently expression of Beclin1 in resistant breast cancer cells and the relationship between the expression of Beclin1 and therapy resistance of breast cancer patients treated with trastuzumab to reveal new expression regulation mechanism of Beclin1 and investigate the possibility of Beclin1 as a new biomarker to predict trastuzumab treatment efficacy. Our research will provide new ideas for treating breast cancer.

曲妥珠单抗是乳腺癌临床治疗指南对于HER2阳性患者的推荐靶向药物，但临床应用发现大多数HER2高表达的乳腺癌患者对其不产生反应，因此阐明其耐药机制是临床治疗的迫切需求。目前研究显示曲妥珠单抗耐药涉及多种机制的参与，最新研究提示自噬与耐药密切相关。在前期工作中，我们在曲妥珠单抗耐药的乳腺癌细胞中筛选出新的调控曲妥珠单抗耐药的自噬相关基因Beclin1，并通过CCK-8和平板克隆形成实验证实在体外敲低Beclin1可以增强曲妥珠单抗敏感和耐药的乳腺癌细胞对曲妥珠单抗的敏感性。课题将在此基础上观察敲低Beclin1是否增强曲妥珠单抗诱导的乳腺癌细胞周期阻滞、凋亡以及裸鼠体内生长的抑制，并通过对Beclin1在耐药细胞中表达变化的机制以及与乳腺癌患者曲妥珠单抗耐药关系的深入研究揭示Beclin1表达调控的新机制和探讨其作为新的预测曲妥珠单抗疗效的生物标志物的可能性，为乳腺癌的治疗提供新的思路。

曲妥珠单抗是乳腺癌临床治疗指南对于HER2阳性患者的推荐靶向药物，但临床应用发现大多数HER2高表达的乳腺癌患者对曲妥珠单抗不产生反应，因此阐明其耐药机制是临床治疗的迫切需求。目前研究显示乳腺癌曲妥珠单抗耐药涉及多种机制的参与，最新研究提示自噬与曲妥珠单抗耐药相关，但具体机制并不清楚。在本项目中，我们首先通过自噬PCR array在曲妥珠单抗耐药的乳腺癌细胞中筛选出新的可能参与调控曲妥珠单抗耐药的自噬相关基因Beclin1，接着通过CCK-8法和克隆形成实验证实在体外敲低Beclin1可以明显增强曲妥珠单抗对敏感和耐药的乳腺癌细胞的生长抑制，通过流式细胞术分析发现敲低Beclin1可以增强曲妥珠单抗对乳腺癌细胞周期进程的抑制，通过裸鼠成瘤实验发现敲低Beclin1可以增强曲妥珠单抗对乳腺癌细胞体内生长的抑制；机制分析显示敲低Beclin1能够增强曲妥珠单抗诱导的细胞周期抑制蛋白p27的表达，增强曲妥珠单抗对MAPK和PI3K/AKT信号通路的抑制；对Beclin1表达升高的机制研究发现在曲妥珠单抗耐药的乳腺癌细胞中高表达的转录因子c-Jun能够更多地结合Beclin1启动子，促进Beclin1转录；最后通过检索GEO数据库发现低表达Beclin1或c-Jun的乳腺癌患者可以从曲妥珠单抗治疗中获益，而高表达的患者不能。本项目首次揭示了c-Jun-Beclin1通路在乳腺癌曲妥珠单抗耐药中的作用和机制，同时发现高表达的Beclin1和c-Jun与乳腺癌患者曲妥珠单抗耐药相关。本项目的开展进一步揭示了曲妥珠单抗的耐药机制，并且Beclin1和c-Jun可能成为新的预测曲妥珠单抗疗效的生物标志物，为曲妥珠单抗在HER2阳性乳腺癌中更有效的应用奠定基础，对于提高乳腺癌患者的预后以及避免不必要的经济付出都具有极其重要的意义。