槲皮素-3-甲酯抑制乳腺癌上皮-间质转化及肿瘤干细胞形成及其机制的研究

Breast cancer is a leading cause of cancer-related death in women. Although progress has been made in the early detection of cancer and in improvements of cancer therapies, the ability to provide long-term survival has been limited. Increasing evidence suggests that biologically unique population of cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) exist in breast cancer and may be responsible for tumor initiation, progression, metastasis, and relapse. EMT is a transient process occurring during carcinogenesis, characterized by phenotypic and molecular alterations, resulting in increased invasive and metastatic capabilities of cancer cells and drug resistance. Moreover, EMT is associated with increased enrichment of CSCs in breast tissues. Breast CSCs are characterized by the expression of several stem cell-specific genes, including nanog. Among these, Polycomb Repressive Complexes (PRCs) play a crucial role. Polycomb genes are organized in multimeric complexes that mediate specific histone post-translational modifications and gene silencing PRC2 mediates histone H3 lysine 27 trimethylation, thereby silencing lineage-specific genes and maintaining stem cell pluripotency. Enhancer of Zeste Homolog 2 (EZH2) is a critical component of the PRC2, which is involved in gene silencing and histone H3 lysine 27 methylation. High EZH2 level is strongly associated with aggressive and metastatic breast cancer. In breast cancer cells, EZH2 is over-expressed in the CD44+/24- fraction, and mediates E-cadherin gene silencing during EMT. Hence, the killing of drug-resistant EMT-type cells (CSCs) and the potential for targeting EZH2 could be the novel and important therapeutics. Our previous study showed quercetin-3-methyl ether (Q3ME) was able to produce a significant inhibition of cell growth in lapatinib-sensitive and -resistant breast cancer cells, which was associated with changes in the level of factors that regulated cell cycle G2/M progression and apoptosis including cyclin B1, p-Cdc25c (Ser216), Chk1, caspase 3, caspase 7, and PARP. However, the direct target of Q3ME is still unclear. Reverse docking study indicates EZH2 could be a potential target of Q3ME. Based on these, it is necessary to study whether Q3ME inhibits EMT and CSCs in breast cancer by targeting EZH2.

乳腺癌是妇女最常见的恶性肿瘤之一。EMT和CSCs参与乳腺癌的发生、发展、复发和转移等过程，并EMT能促进CSCs的形成。EZH2在乳腺癌组织中过表达，能诱发EMT，促进CSCs的形成。我们的研究表明槲皮素-3-甲酯（Q3ME）在乳腺癌细胞中显著抑制拉帕替尼敏感和耐药细胞株的增殖和克隆形成，诱导细胞周期阻滞，促进凋亡；但尚未找到该药直接作用的蛋白靶点。在此基础之上，利用超级计算机模拟技术发现，Q3ME有可能是EZH2的抑制剂，通过抑制EZH2 阻断乳腺癌EMT，杀死CSCs。本研究以乳腺癌患者来源的细胞株为研究模型，利用细胞生物学、分子生物学和干细胞实验技术，拟深入观察Q3ME对乳腺癌细胞EMT和CSCs形成及特性的影响，并探讨该化合物是否通过抑制EZH2这个靶点来阻断EMT和CSCs形成，从而有效发挥抗癌作用，减少乳腺癌复发和转移，为治疗癌症研发新药，达到延长病患生存时间的目的。

乳腺癌是妇女最常见的恶性肿瘤之一。上皮间质转化（epithelial-mesenchymal transition，EMT）和肿瘤干细胞（cancer stem cells，CSCs）参与乳腺癌的发生、发展、复发和转移等过程，并EMT能促进CSCs的形成。前期研究结果表明槲皮素-3-甲酯（Q3ME）能显著抑制拉帕替尼敏感和耐药乳腺癌细胞的增殖和克隆形成，诱导细胞周期阻滞，促进凋亡；但尚未完全明了该天然化合物的抗癌作用机制。本项目以乳腺癌细胞MCF-7和MDA-MB-231作为研究模型，主要深入研究Q3ME对乳腺癌细胞EMT和CSCs形成的影响，并探讨该化合物阻断EMT和CSCs形成过程的分子作用机制。关于表型研究的实验结果：1. Wound healing assay和Transwell assay结果显示Q3ME显著抑制乳腺癌细胞的侵袭和转移；2. Western-blot结果显示Q3ME促进乳腺癌细胞E-cadherin的表达，抑制Vimentin和MMP-2的表达，表明该化合物有效阻抑EMT过程；3. Mammosphere assay结果显示Q3ME能明显抑制乳腺癌细胞成球，同时Western-blot结果显示Q3ME抑制SOX2和Nanog的表达，说明Q3ME能抑制CSCs的形成。关于分子机制研究的实验结果：1.Q3ME处理乳腺癌细胞后，发现EZH2的表达无明显差异，但EZH2下游基因Tri-Methyl-Histone H3 (Lys27)表达下降，提示EZH2可能是Q3ME的靶点之一；2.Q3ME能抑制Notch1、Notch3、PI3K和Akt的表达，说明该化合物通过抑制Notch-PI3K-Akt信号通路发挥生物学功能，Noth可能也是Q3ME的靶点之一。本研究初步证实Q3ME通过抑制EZH2和Notch信号通路来阻断EMT和CSCs的形成，从而有效发挥抗癌作用，抑制乳腺癌的侵袭和转移，为进一步研发Q3ME为抗癌新药提供了理论基础和实验依据。