小窝蛋白-1在肿瘤细胞抗失巢凋亡力学信号转导中的功能与调控机制研究

Hematogenous metastasis is the mainly leading cause of death in cancer patients, which is a multistep process including dissociation of cancer cells from primary sites, survival in the vascular system, and proliferation in distant target organs. The acquisition of anoikis resistance (anti-anoikis) for metastatic cancer cells is required for hematogenous metastasis. Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body. Currently, anoikis resistance becomes a hot topic in cancer research. A better understanding of the underlying molecular and cellular mechanisms is crucial for the development of effective treatment for metastatic cancer. Our previous studies demonstrated that caveolin-1 (cav-1) is related to cell survive and migration of human breast cancer cells (MDA-MB-231), and low shear stress up-regulated cav-1 expression. However, the mechanobiological roles of cav-1 in anti-anoikis of human breast cancer cells under shear flow conditions and its mechanotransduction or regulatory mechanisms are not well understood and remain to be elucidated. In this project, we will firstly investigate the effects of low shear stress on the expression, distribution, internalization and activation of cav-1 by suspenssion cultured shear stress loading apparatus (Couette rotation system) combining immunofluorescence cytochemistry, real time fluorescence quantitative PCR and western blot. Then we further explore how the low shear stress regulates cav-1 function and its triggered mechanotransduction pathways. The affinity of beta(1) integrin, FAK/beta(1) integrin interaction, and the expression of anti-apoptosis related genes will be also investigated after MDA-MB-231 cells exposure to low shear stress. Through the above-mentioned experiments, we want to elucidate the relationship between of cav-1 and anti-anoikis, and the mechanobiological roles and regulatory mechanisms of cav-1 in anti-anoikis of cancer cells under shear flow conditions. The detailed contribution of cav-1 and shear stress to anti-anoikis of human breast cancer cells, and their internal relationships will be illuminated through this proposed project investigation. These studies will provide insight into the biology of cancer metastasis and identify novel therapeutic targets for prevention of cancer dissemination.

肿瘤细胞获得抗失巢凋亡能力是其发生转移的先决条件之一，阐明肿瘤细胞抗失巢凋亡分子机制有重要意义。我们前期研究结果发现小窝蛋白-1(cav-1)与乳腺癌细胞的生存和迁移能力密切相关，且切应力作用可以上调cav-1表达，但切应力条件下cav-1在肿瘤细胞抗失巢凋亡力学信号转导中的具体功能与调控机制仍不清楚。该项目采用悬浮培养切应力加载技术联合免疫细胞化学、免疫印迹及定量PCR等分析方法，研究切应力作用对肿瘤细胞膜上小窝形成及cav-1表达、分布、内化和活化的影响规律，重点探讨切应力如何调控cav-1功能改变及所引发的信号转导途径、整合素beta(1)亲和性、及抗细胞凋亡相关基因表达水平，揭示cav-1在肿瘤细胞抗失巢凋亡力学信号转导中的功能与影响机制，阐明切应力和cav-1与细胞抗失巢凋亡的内在关系和贡献大小，从而开辟遏制肿瘤细胞抗失巢凋亡能力的新途径，为防治肿瘤转移和抗癌药物开发提供新靶点。

循环肿瘤细胞在通过血液或淋巴系统转移时，需要获得抗失巢的能力才能生存下来。然而，肿瘤细胞如何在流体切应力环境中如何获得抗失巢凋亡能力及其调控机制仍知之甚少。该项目以乳腺癌细胞为研究对象，重点探讨肿瘤力学微环境对肿瘤细胞转移影响的力学-生物学耦合机制。主要研究结果如下：（1）深入研究了悬浮培养条件下小窝蛋白-1(Cav-1)在乳腺癌细胞抗失巢凋亡的力学生物学功能，阐明了切应力作用影响肿瘤细胞抗失巢凋亡的分子机制。研究发现切应力通过上调Cav-1表达，提高了肿瘤细胞抗失巢凋亡能力。细胞通过解离Cav-1/Fas复合物、抑制死亡复合体(DISC)的形成和Caspase-8活性，调节外源凋亡途径抑制细胞凋亡；通过增加integrin β1/FAK介导的细胞聚团、抑制t-BID介导的内外源途径间交互作用、提高Bcl-2水平和恢复线粒体功能来调节内源凋亡途径，抑制肿瘤细胞失巢凋亡。（2）深入探讨了切应力条件下肿瘤细胞侵袭伪足形成的力学调控机制。发现切应力通过活化Cav-1引发PI3K/Akt/mTOR信号通路去上调MT1-MMP的表达，并通过Cdc42和Rac1来调节MT1-MMP向侵袭伪足部位的胞内转运与聚集，加速细胞外基质的降解，从而促进了肿瘤细胞的迁移与转移。（3）揭示了Cav-1介导切应力引起的肿瘤细胞骨架重排和细胞粘附的变化规律及其生物力学机制。切应力可以通过Cav-1调控ROCK/p-MLC通路影响细胞骨架重排，也可通过FAK/Src信号通路调控黏着斑的动态变化，两者协同作用促进了肿瘤细胞运动。相关研究成果发表在Nat Commun、J Cell Physiol、Cell Mol Immunol、BBA-Mol Cell Res等国际重要学术期刊上。通过该项目的研究，阐明了切应力作用可以通过抑制Cav-1依赖内外源细胞凋亡途径提高了肿瘤细胞抗失巢凋亡的能力，揭示了肿瘤力学环境对肿瘤进程的调节作用，深化了切应力调控肿瘤细胞血源性转移的力学信号转导过程与机制，尤其是肿瘤细胞粘附、运动和转移的力学生物规律，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。