EZH2介导的FGF2对话miRNAs在诱导并维持乳腺癌细胞上皮间质转化及化疗耐受中的作用

The chemoresistance and metastasis are the major obstacles for the successful breast cancer treatment clinically. EMT (epithelial-mesenchymal transition) is expected to clarify the mechanisms for chemresistance and metastasis, and the association between them. We have previously established a 5-Fu resistant breast cancer cell line MCF-7/5-Fu with typical EMT features. We found the conditioned culture media from MCF-7/5-Fu cells enabled chemosensitive and epithelial type breast cancer cells to exhibit EMT phenotype, and found FGF2 mediated Akt/mTOR/EZH2 signal activation perhaps played critical role in the EMT induction and maintaince. In the present program, we will first confirm that FGF2 regulates the activation of Akt/mTOR/EZH2 signal and its biological effects in breast cancer cells. Then, we will explore the cross-talk between FGF2 and miRNAs mediated by the histone methylation in the promoter catalyzed by EZH2, and its biological functions. The third, we will investigate the effects of chemoresistant cells or FGF2 mediated signal pathway on the tumorigenicity, chemosensitivity, invasion-metastasis of epithelial type and chemosensitive breast cancer cells in vivo. Through these researches, we will reveal that chemoresistant cells induce and maintain the EMT via secreted FGF2 in breast cancer, which will provide new clues for the mechanisms study for chemoresistance and metastasis and the intrinsic link between them, and a theoretical basis for the prevention and reversion of chemoresistance and metastasis in breast cancer.

化疗耐受和转移是限制乳腺癌疗效提高的关键原因，EMT有望阐释二者的机制及内在联系。申请者前期建立了呈现EMT表型的乳腺癌耐药细胞株（MCF-7/5-Fu），耐药细胞培养上清能诱导敏感的上皮型乳腺癌细胞发生EMT，筛选发现FGF2活化的Akt/mTOR/EZH2信号可能起重要作用。本项目拟首先应用中和抗体、信号阻断剂及基因干扰技术进一步明确乳腺癌细胞中FGF2对Akt/mTOR/EZH2信号活性的调控及其生物学效应；其次阐述EZH2通过启动子组蛋白甲基化介导FGF2对话miRNAs及其生物学功能；第三将从动物和人体组织水平进一步观察乳腺癌耐药细胞或FGF2介导的信号通路对敏感细胞成瘤能力、化疗敏感性及侵袭转移的影响。本项目的完成将揭示耐药细胞通过分泌FGF2以诱导并维持乳腺癌细胞EMT，将为研究乳腺癌耐药和转移机制及其内在联系提供新思路，并为乳腺癌耐药和转移的早期预防和后期干预提供理论基础。

化疗耐受和转移是导致乳腺癌治疗失败和乳腺癌患者死亡的关键原因，本项目从自分泌和/或旁分泌的微环境维度探讨了TNFα/FGF2-Akt-NF-κB、IL-6-STAT3和Wnt-β-catenin信号在诱导和维持乳腺癌细胞间质表型以介导乳腺癌耐药和转移中的协同作用。我们发现（1）5-Fu体外诱导的乳腺癌多药耐药细胞呈现典型的EMT表型，体内外实验证实了耐药细胞的耐药性和高侵袭转移能力，且伴随乳腺癌干细胞特性的增强；（2）耐药细胞来源的细胞培养液（CM）能诱导亲本细胞发生EMT，且在无CM情况下能维持稳定的耐药、侵袭转移和乳腺癌干细胞样特征等EMT特性；（3）通过细胞因子芯片筛选鉴定耐药细胞培养液中的差异表达细胞因子，并用ELISA进行验证；（4）乳腺癌耐药细胞通过自分泌方式维持TNFα/FGF2-Akt-NF-κB、IL-6-STAT3和Wnt-β-catenin信号活性，调控EMT相关miRNAs表达，NFα/FGF2-Akt-NF-κB、IL-6-STAT3和Wnt-β-catenin信号协同维持耐药细胞的EMT表型；（5）乳腺癌耐药细胞可通过旁分泌方式创建细胞外微环境活化NFα/FGF2-Akt-NF-κB、IL-6-STAT3和Wnt-β-catenin信号以触发亲本细胞的EMT进程，并诱导亲本细胞获得稳定的EMT表型；（6）临床上化疗能诱导乳腺癌患者血清中TNFα、FGF2、IL-6和Wnt4蛋白水平升高，TNFα/FGF2-Akt-NF-κB、IL-6-STAT3和Wnt-β-catenin信号活化与乳腺癌转移密切相关。本项目的研究发现将为乳腺癌的化疗耐受机制研究提供新线索，并为开发以TNFα、FGF2 、IL-6和Wnt4细胞因子组合检测试剂盒以追踪乳腺癌治疗疗效，以及为开发以肿瘤微环境触发的信号通路为靶的新治疗策略以提高乳腺癌治疗效果提供理论依据。基于本项目资助，已发表SCI学术论文4篇（包括Clinical Cancer Research已在线发表1篇），核心部分内容正在撰写成文；申请并已被受理专利3项；参加在国内举办的国际学术会议3次；协助培养毕业硕士研究生2名。