LGR5调控乳腺癌干细胞的休眠作用与机制研究

Tumour dormancy has been recognized as a clinical phenomenon in numerous types of cancer for many years because the disseminated stem cells can enter dormancy and become refractory to targeted or conventional therapies. But regulation of the switch from quiescent dormancy to active regrowth is poorly understood, this poses a tremendous challenge to the researchers. Applicant had proved that LGR5 significantly Tumour dormancy has been recognized as a clinical phenomenon in numerous types of cancer for many years because the disseminated stem cells can enter dormancy and become refractory to targeted or conventional therapies. But regulation of the switch from quiescent dormancy to active regrowth is poorly understood, this poses a tremendous challenge to the researchers. Applicant had proved that LGR5 significantly associated with recurrence and poor outcome in breast cancer, LGR5 promoted cell mobility, tumour formation, and epithelial-mesenchymal transition by activating Wnt/β -catenin signaling (Stem cells, 2015). In addition, LGR5 was specificity expressed in the active proliferation stem cells when study in well builded stem cell lines (patent NO. 201510330647.5), and LGR5 overexpression in breast cancer predicted short disease-free survival period. Thus, We put forward the scientific hypothesis: LGR5 is required for dormant tumour stem cell reactivation and boosted tumour recurrence. This project aims to study the potential roles of LGR5 in breast cancer stem cell dormancy and reactivation in noninvasive imaging models; use the microarray gene chips and experimental studies of mechanisms, and provide an experimental basis for the development of breast cancer and targeted therapy.

研究证实在多种类型肿瘤细胞中发现一群能够休眠和早期播散的细胞，这类细胞能幸免于肿瘤的常规药物治疗和靶向治疗，称为休眠干细胞。它们是怎样维持休眠又是如何被“唤醒”？探讨这一过程的分子机制是目前研究肿瘤复发与转移的热点。申请者前期构建稳定传代的乳腺癌干细胞株（中国专利，201510330647.5），证实LGR5与乳腺癌患者临床预后相关，过表达LGR5可促进乳腺癌干细胞的侵袭与转移（Stem Cells，2015），进一步研究发现LGR5在代谢活跃的乳腺癌干细胞中表达显著增加，且LGR5高表达的乳腺癌患者更早出现复发。由此我们提出科学假说：LGR5可能参与加速乳腺癌复发转移及起始休眠干细胞增殖的调控过程。本项目拟采用体内动物成像技术研究LGR5调控乳腺癌干细胞休眠作用；通过基因芯片筛查和一系列实验阐明LGR5调控乳腺癌干细胞休眠的分子机制，为乳腺癌发病机制提供新的理论依据和治疗靶点。

乳腺癌治疗的难题是治疗多年后发生复发和转移。能够通过休眠状态逃脱放化疗及靶向治疗，从而引起复发转移的这群细胞，在化疗药物作用后，从分裂旺盛到进入休眠状态再到被重新激活，了解这个过程的肿瘤机制是防治肿瘤复发的关键，许多科学家也提出可以利用这一机制维持肿瘤细胞的休眠状态以延长肿瘤复发和抑制转移。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的表达与肿瘤干细胞的增殖与休眠状态密切相关。在本研究中，我们采用了流式分选，将已经培养成功的人源性的干细胞系，包括分离来自乳腺癌患者的原代肿瘤干细胞以及分离自乳腺癌细胞系的肿瘤干细胞，通过流式技术结合LGR5MACS特异性磁珠抗体，分选为LGR+/LGR5-两种进行后续的功能及表型检测，以探索干细胞休眠现象在肿瘤耐药及复发进程中的作用机制。结果提示：LGR5+细胞群的确具有更强的增殖能力，LGR5-乳腺癌干细胞群虽增殖缓慢，但具有长期增殖潜能；5FU与治疗后的肿瘤中LGR5-的干细胞群比例增加，证明LGR5-干细胞是造成肿瘤耐药的细胞群；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探索调控LGR5-乳腺癌干细胞休眠的基因；TGFBR1在乳腺癌干细胞休眠状态调控中具有重要作用；TGFβ信号可抑制乳腺癌干细胞对化疗敏感性。因此我们也在此项目中进一步在细胞系中研究了LGR5蛋白表达对HER2基因表达及肿瘤恶性度的影响和作用，并进一步研究了解这种机制是与微环境互作过程中的一种表现，还是其基因型决定的属性。