LGR5调控乳腺癌干细胞HER2异质性诱导耐药的作用机制研究

The clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Recently, studies considered that the equilibrium between HER2+ and HER2− cells within a heterogeneous tumour population is driven by spontaneous interconversion between these phenotypes. However, what induce this transition that ledding tumor drug resistance is unknown. We proved that the stem marker LGR5 induced EMT and maintained stemness by Wnt pathway in several breast cancer stem cell lines (stem cells, 2015); and we found in our advanced study that the LGR5+ stem cells expressed high level of HER2, the cells became reproliferative and LGR5/HER2 expression upregulated after drug withdrew. Thus, we put forward the scientific hypothesis: LGR5 regulated HER2 heterogeneity of breast stem cells and induced drug resistance through Wnt dependent EMT. This project aims to study the potential roles of LGR5 in breast cancer acquired drug resistance. We designed to use flow cytometry, CRISPR-Cas9-lentivirus to studies whether LGR5 could regulate HER2 expression and the state of cancer stem cell, inducing drug resistance. In conclusion, this project will study the role of cancer stem cell and provide an experimental basis for overcoming clinical drug resistance.

HER2异质性是HER2基因扩增型乳腺癌靶向治疗耐药的重要机制。最新研究发现肿瘤细胞可自发性或诱导性在HER2+/HER2-状态间转换。然而，HER2表达转变导致耐药这一过程又是何种机制介导？申请者前期分离纯化了多株可稳定传代的乳腺癌干细胞，证实LGR5通过激活Wnt通路诱导EMT发生从而维持肿瘤干性（Stem cells, 2015）；进一步研究发现LGR5+细胞中HER2高表达，经药物诱导后LGR5/ HER2在耐药细胞中表达下降，撤除药物后细胞恢复增殖，LGR5/ HER2表达上调。据此我们提出假设：LGR5通过Wnt通路介导EMT，调控乳腺癌干细胞HER2表达改变诱导耐药发生。本项目拟采用流式分选、CRISPR-Cas9-慢病毒等工具研究LGR5能否通过调控HER2表达及干细胞功能状态转变诱导耐药发生，以期发现肿瘤干细胞参与的获得性耐药机制，为克服临床耐药提供新的实验依据。

尽管对于早期乳腺癌的治疗可以采用多种手段进行综合治疗，并取得了非常好的治疗效果，但目前对于进展期的乳腺癌患者仍然没有非常有效的治疗方案。复发和转移是进展期患者面临的问题，而在细胞层面上认为是由于乳腺癌肿瘤中的一群肿瘤干细胞导致了治疗失败。因而了解这群细胞的特征及调控机制是防治肿瘤复发的关键。本课题按时按质完成了原计划的研究目标，我们根据前期对LGR5在干细胞的调控作用，以及总结了多个最新的国际前沿研究进展发现，LGR5的表达与化疗以及靶向治疗耐药密切相关。在本研究中，我们采用了流式分选乳腺癌干细胞，通过流式技术结合LGR5/MACS特异性磁珠抗体，分选为LGR5+/LGR5-两种进行后续的功能及表型检测，以探索LGR5对特殊干细胞群的标记作用及这类细胞的功能特征和调控机制。结果提示：LGR5的确可以精确标记间质型乳腺癌干细胞，并且相比较ALDH1对小鼠模型的肺转移灶具有更特异的标记功能； LGR5可增加HER2+乳腺癌细胞的干性，LGR5高表达可降低HER2+乳腺癌干细胞的干性并降低其对HER2+靶向药物的敏感性；基因差异性表达分析发现LGR5高表达细胞群中ID3被激活，且具有维持此类细胞的干性功能；功能研究发现ID3能通过促进和维持LGR5下游的Wnt信号，进而维持肿瘤干性。