基于肿瘤干细胞免疫微环境TAMs/CXCL-1通路探讨消癖颗粒预防乳腺癌的分子机制和物质基础

The disorder of immune microenvironment is a critical mechanism regulating the origination and function of cancer stem cells (CSCs). Traditional Chinese Medicine (TCM) has unique priority in breast cancer prevention and treatment via regulating immunity. Identification of key immune signalings of CSCs and development of natural targeting phytochemicals will provide novel strategies for breast cancer prevention with high efficiency and safety. Previous clinical trials demonstrated that a TCM formula XIAOPI could block or reverse the progress of severe mammary atypical hyperplasia, a well known precancerous lesion. However, its underlying bioactive compounds and molecular mechanisms are still remained largely unknown. Our pilot study revealed that XIAOPI formula could significantly prevent tumor occurrence and growth of breast cancer on MMTV-PyMT transgenic mice, accompanying with a sharp reduction of CSCs. However, in vitro experiments indicated that XIAOPI formula did not exhibit similar bioactivities as in vivo findings, suggesting that the in vivo microenvironment might be a critical step in mediating the bioactivity of XIAOPI formula. Therefore, we utilized cytokine array to reveal the in vivo effects of XIAOPI formula and found that it could significantly inhibit the CXCL-1 expression of tumor associated macrophages (TAMs). Based on above findings, we hypothesized that XIAOPI formula might prevent breast cancer via inhibiting TAMs to express CXCL-1 cytokine, a critical signaling regulating the self-renewal and multi-differentiation abilities of CSCs. Taken together, our study will comprehensively explore the significance of CXCL-1 in regulating breast CSCs and the bioactive phytochemicals in XIAOPI formula by utilizing multiple techniques including clinical specimen detection, three-dimensional CSCs culture, in vitro and in vivo gene modification and bioactivity-guided fractionation, etc. The study will provide novel strategies and powerful tools for molecular elucidation of breast cancer occurrence and developing new preventional drugs from TCM.

免疫微环境是诱导肿瘤干细胞发生与功能调控的重要机制，中医药在调节免疫防治乳腺癌领域具有独特优势，鉴定肿瘤干细胞免疫调节关键信号及开发天然靶向制剂将对乳腺癌预防提供安全有效的崭新手段。临床试验证实消癖颗粒对乳腺中到重度不典型增生具有阻断及逆转作用，但其药效物质基础与分子机制尚不清楚。我们前期研究发现消癖颗粒可有效预防乳腺癌自发转基因小鼠肿瘤发生与生长，降低体内乳腺癌干细胞群落数量，但其体外无显著作用。细胞因子芯片实验证实消癖颗粒体内外均可显著降低肿瘤巨噬表达分泌的CXCL-1水平，由此我们设想消癖颗粒可通过调节CXCL-1信号抑制乳腺癌干细胞自我更新及多向分化能力，从而阻断乳腺癌前病变。本项目拟通过临床标本检测、干细胞三维培养、体内外基因重组及生物活性导向分离等技术探讨CXCL-1对乳腺癌干细胞的调节机制及消癖颗粒的物质基础。本研究的开展将为乳腺癌发生机制及预防药物开发提供新思路和新工具。

本研究拟探讨消癖颗粒调节TAMs/CXCL1通路预防乳腺癌的分子机制和物质基础。我们首先通过细胞因子芯片发现CXCL1是TAMs分泌含量最高的细胞因子，并与乳腺癌细胞上皮间质化过程密切相关，分子机制研究表明TAMs/CXCL1可通过NF-κB/SOX4途径促进乳腺癌细胞转移。体内研究表明CXCL1在TAMs中敲除后乳腺癌原位生长和肺转移显著抑制，同时CXCL1与乳腺癌患者总生存期和无复发生存期显著相关；在此基础上，网络药理分析显示消癖颗粒主要靶点为响细胞外环境，结合细胞因子芯片和生物学功能实验发现TAMs/CXCL1通路在介导消癖颗粒抑制乳腺癌发生发展过程中发挥主要作用，同时消癖颗粒可通过TAMs/CXCL1通路抑制乳腺癌干细胞干性；血清代谢物质谱分析确定消癖颗粒入血成分有63种，其中丹参酮I和II类化合物为主要入血成分，网络药理学结合KEGG分析显示消癖颗粒主要影响体内胆汁酸代谢途径，可通过上调CYP7A1加速胆汁酸的合成和分泌。在此基础上，我们构建了CXCL1双荧光素酶报告基因体系，采用生物活性追踪分离方法确定宝霍苷I是消癖颗粒中对CXCL1抑制活性最强的化合物，并采用体内外实验验证了其对乳腺癌生长、转移及肿瘤干细胞的抑制作用，提示其可作为消癖颗粒的生物活性质控标准之一。通过该项目的实施，共发表14篇通讯作者SCI收录文章，进行国际交流2次，发表会议论文2篇，培养3人次省级人才项目，培养广州中医药大写优秀博士后2名，申请专利2项，发表英文论著1部，申请科技成果登记1项，新药转化1项。研究的开展为乳腺癌预防及肿瘤干细胞调控提供了新的分子靶点，同时为乳腺癌防治药物开发提供了新的药物。