低氧诱导的氧化型ATM调节乳腺癌CAFs能量代谢重塑的机理及其促乳腺癌侵袭转移研究

Invasion and metastasis of cancer cells are the leading causes resulting in the death of breast cancer patients. The cancer-associated fibroblasts (CAFs) promote the invasion and metastasis of breast cancer cells. Our previous work has found that the aberrant activity of oxidized ATM is independent of DNA double strands breaks and involved in the hypoxia-induced energy metabolism reprogramming (EMR) in CAFs. However, its potential mechanisms and roles in the invasion and metastasis of breast cancer remain unknown. Based on our previous work, we will mimic the hypoxia status in breast cancer microenvironment to reveal the molecular mechanisms (including the target proteins and their associated signaling pathways) of oxidized ATM regulating the EMR of breast CAFs by employing phosphorylated proteomics technology, bioinformatics method and site-directed mutagenesis technology. To further understand its role of CAF EMR mediated by oxidized ATM, the characteristics of CAFs, and the invasion and metastasis of breast cancer cells in the cross-talking with CAFs will be studied by using siRNA and specific chemical inhibitors to EMR-related key targets or signaling pathways. Our results will disclose a novel mechanism of CAF EMR, and provide the novel strategies or find the new molecular targets to prevent and treat the metastasis of breast cancer.

癌细胞侵袭转移是引起乳癌患者死亡的主因，癌相关成纤维细胞(CAF)促进乳癌侵袭转移。前期工作发现：乳癌CAF存在非DNA双链断裂依赖的ATM异常活化、能量代谢变化，但氧化型ATM对低氧诱导的CAFs能量代谢重塑机制和促乳癌侵袭影响不明。本项目拟在前期研究基础上，以CAF和NF为模型，模拟体内乳癌微环境低氧状态，运用磷酸化蛋白质组学技术、生物信息学手段、定点突变技术挖掘氧化型ATM下游能量代谢有关的靶蛋白及其信号途径，以揭示氧化型ATM调控CAFs EMR的内在分子机制；以siRNA沉默、小分子抑制剂干预关键靶蛋白和信号通路，探寻氧化型ATM介导的CAFs EMR对CAFs生物学特性、乳癌侵袭转移的潜在影响，为寻找乳腺癌转移防治新靶点提供新思路和可靠的实验依据，研究结果具有重要理论意义和潜在的临床价值。

经过4年来努力，在课题组全体人员的艰辛劳动与有力配合下，圆满完成了本课题的研究内容，取得了丰硕的成果，主要成果总结如下：1. 揭示乳腺肿瘤微环境基质细胞CAF中存在DNA损伤非依赖的ATM（即oxidized ATM）活化。活化的ATM激活ERK、PI3K/AKT，信号调控c-Myc、cyclinD1促进并维持CAF增殖。2. Oxidized ATM特异磷酸化CAF中相关蛋白。经磷酸化组学鉴定到333个oxidized ATM靶蛋白。oxidized ATM特异磷酸化靶蛋白Cortactin的Serine113位点，增强与Arp2/3的亲和力而形成复合物，使得运动相关骨架蛋白actin有序组装，促乳腺肿瘤细胞侵袭与转移。3. CAF中异常表达IL32，特异识别并高亲和整合素ITGB3，使其发生磷酸化活化，激活下游P38/MAPK信号，促EMT相关蛋白表达，进而促进癌细胞侵袭与转移。4. 揭示乳腺肿瘤癌前病变非典型增生组织中存在活化的成纤维细胞（AHFs）。AHF中miR-200b/c低表达，miR-200b/c靶向IKKb活化NF-kB信号，上调PAI-1，促进癌变的乳腺上皮细胞极性变化。这一研究首次揭示肿瘤微环境在乳腺癌起始与早期发展中的重要作用。5.待发表的研究工作中，我们揭示oxidized ATM特异磷酸化GLUT1、BNIP3等，GLUT1磷酸化发生膜转位，促进CAF糖酵解与乳酸产生，激活癌细胞的TGFb信号和线粒体活性，促癌细胞侵袭转移。BNIP3的磷酸化增强CAF自噬，促外泌体GPR64聚集，活化癌细胞NF-kB信号促转移。此外，另2项工作揭示oxidized ATM调控糖代谢，通过柠檬酸积累促TNBC侵袭；或通过Ac-CoA促CSC富集与干性维持。已发表SCI收录论文5篇，待发表SCI论文4篇。已培养博士研究生3名、硕士研究生6名。