三维基质粘弹性影响乳腺癌细胞凋亡和自噬交互作用的力学生物学机制研究

Apoptosis and autophagy are both cellular degradation pathways essential for organismal homeostasis. Therefore, both autophagy and apoptosis have been implicated in protecting organisms against a variety of diseases, especially cancer. Extracellular matrix (ECM) viscoelasticity, an important tumor microenvironmental factor of ECM mechanics, has attracted much attention in recent years. A better understanding of the cross-talking between autophagy and apoptosis under various ECM viscoelasticity and its mechano-mechanisms will probably be an important area in the cancer biomechanics and caner biology. Our previous studies have demonstrated that the interaction of caveolin-1 (cav-1) and integrin β1 is related to malignant progression and metastasis of human breast cancer cells (BBA-Mol Cell Res, 2017, 1864: 12-22; Oncotarget, 2016, 7: 16227-16247). However, the mechanobiological roles of cav-1/integrin β1 interaction in various ECM viscoelasticity of human breast cancer cells under 3D cell culture condition and its mechanotransduction or regulatory mechanisms are still not well understood and remain to be elucidated. In this project, we will firstly investigate the effects of various ECM viscoelasticity in 3D cell culture condition on the cav-1 expression, distribution, activation, and the cav-1-dependent integrin β1 trafficking by combining immunofluorescence cytochemistry, western blot, co-immunoprecipitation, real time fluorescence quantitative PCR, flow cytometry, and living cell microscopy. Then we further explore how the various ECM viscoelasticity in 3D cell culture condition regulates the mechanosensitive complex of cav-1/integrin β1 interaction and its triggered mechanotransduction pathways. The complexity and underlying mechanisms of the interactions between the apoptotic and autophagic machinery will be intensively studied. Through the above-mentioned experiments, we want to elucidate the relationship between ECM viscoelasticity-induced apoptosis and autophagy and the mechanobiological pathways of cav-1/integrin β1 interaction in 3D cancer cell culture condition. This research proposal will provide insight into the mechanobiology of cancer progression, and identify novel therapeutic targets for cancer prevention or auxiliary diagnosis in the future.

胞外基质粘弹性作为基质力学微环境一个重要物理特性备受关注，阐明三维基质粘弹性如何影响肿瘤细胞凋亡与自噬交互作用的力学生物学机制有重要意义。我们前期研究发现小窝蛋白-1(cav-1)/整合素β1相互作用与乳腺癌细胞恶性发展和转移潜能密切相关，但胞外基质粘弹性变化与肿瘤细胞凋亡和自噬交互作用的力学调控机制仍不清楚。该项目采用三维细胞培养方法联合免疫细胞化学、免疫共沉淀、流式细胞术及活细胞显微技术等分析手段，研究不同胞外基质粘弹性对肿瘤细胞cav-1表达、分布、活化以及cav-1依赖的整合素β1转运规律，重点探讨不同三维基质粘弹性如何调控cav-1/整合素β1相互作用及所引发的信号转导途径, 深入分析胞外基质粘弹性对力敏感复合物cav-1/整合素β1形成及其信号转导与肿瘤细胞凋亡/自噬交互作用调控途径之间的内在关系, 挖掘乳腺癌发生发展的生物力学基础和分子调控机制，为肿瘤治疗提供潜在作用靶点。

肿瘤发生发展过程通常伴随着胞外基质（ECM）的异常交联与沉积，进而导致ECM重塑和刚度增加，肿瘤力学微环境在肿瘤的发生发展中扮演着重要角色。然而，基质刚度如何影响肿瘤细胞凋亡和自噬交互作用及其力学生物机制仍知之甚少。该项目以乳腺癌细胞为研究对象，重点探讨基质刚度对乳腺癌细胞凋亡和自噬交互作用的力学-生物学耦合机制。主要研究结果如下：（1）深入研究了基质刚度变化对肿瘤细胞凋亡与自噬的影响规律，阐明了软基底诱导肿瘤细胞发生凋亡和保护性自噬的分子机制。研究发现基底刚度显著影响肿瘤细胞生长，而软基底（10 kPa）上细胞活力明显降低，细胞凋亡水平增强，自噬小体和自噬溶酶体数量增加，且抑制自噬能显著增加软基底诱导的细胞凋亡。而ROS/JNK通路活化促进了Bax易位到线粒体膜上和细胞色素C释放，引发线粒体途径的细胞凋亡。同时，Beclin-1与Bcl-2相互作用减弱，游离Beclin-1与Vps34/Atg14组成自噬起始复合物促进保护性自噬发生。（2）揭示了基质刚度影响肿瘤细胞线粒体动力学的变化规律及其力学生物机制。发现软基底诱导DRP1募集到线粒体上，通过S616位点磷酸化促进线粒体分裂，导致细胞线粒体片段化程度增加和线粒体功能异常。DRP1介导的Parkin和PINK1易位到线粒体膜上，促进线粒体自噬发生，并导致IP3R钙通道开放，显著增加内质网钙释放和线粒体钙摄取，且线粒体分裂和自噬受到内织网-线粒体（ER-Mito）钙转运的调节，发现了软基底通过ER-Mito钙转运诱导肿瘤细胞发生DRP1介导的线粒体分裂和自噬。相关研究成果发表在Nat Commun、Biomaterials、Cell Mol Immunol、Free Radical Biology and Medicine等国际重要学术期刊上。通过该项目的研究，阐明了基质刚度通过ROS/JNK通路和ER-Mito钙转运共同调节乳腺癌细胞的凋亡-自噬交互作用，揭示了软基底通过ROS/JNK通路诱导线粒体途径的细胞凋亡和保护性自噬，以及通过ER-Mito钙转运调控线粒体动力学，深化了基质力学影响肿瘤恶性演进的力学信号转导过程与机制，为肿瘤发生发展及其治疗提供参考信息和实验证据，圆满完成了该项目的主要研究任务，达到了预期研究目标。

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