Myosin-mediated Force Inhibits Collective Cancer Cell Migration Via FAK-Rho-ROCK Signal Pathway

Breast cancer has been one of the biggest killers of women due to its susceptibility and high metastasis.During breast cancer progression,the surrounding normal tissues could be invaded through infiltrative growth.Not only the process involves the migration of single cells,but there are also collective cells which are oriented in the same direction at the same speed.Tumor cells migration is regulated by many signaling pathway.There are research findings that FAK-Rho-ROCK signal pathway is upregulate obviously in various malignant tumors.Meanwhile,highly active FAK can protect cells from cell apoptosis under low adhesion conditions and regulate cell adhesion to ECM in the progress of single cell migration.However,the activation of Rho-Rock signal pathway can increase cell contractility and allow cells to migrate at a higher speed.The collective cells migration remains unclear.Recent work has been shown that inhibited by the activity of myosin and reduce cell contractility.It not only will not slow down the speed migration,but also enhance the migration ability in the collective cell migration.Therefore,in this research,it is time to investigate how the contractile force produced by myosin affects the tumor cells migration,and how the FAK-Rho-ROCK signal pathway responds to change intracellular contractile force.In this research,we choose three stably expressing breast cancer cells MCF-7 which is collective cells migration characteristics with different myosin activities using lentiviral transfection were successfully constructed.Western Blot experiment verified that the stable transfected cell lines have been successfully constructed and used it for other experiments.In the meantime,we use time-lapse photography to investigate the wound healing process.Meanwhile,it was found that the increase in myosin activity not only did not increase the migration speed of collective cells,but also reduced the migration speed of collective cells,while the inactivated myosin caused the opposite result.Next,we explored the three factors that affect collective cells migration: cell polarity,cell contractility and adhesion.Furthermore,it is also found that although activated myosin increases cell contractility,it affects the distribution of force within the collective cells.After that,the directionality of migration is weakened.The directional polarization is lost.The adhesion of cells to matrix is greatly increased.Therefore,these three factors lead to a decrease in the speed of collective cells migration.FAK is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration,proliferation and survival.It can affect the intracellular force by adjusting Rho-ROCK pathway.As a mechanically sensitive molecule,it can also respond to mechanical signals in the cells.Thence,we further probed how changes in myosin activity affect the FAK-Rho-ROCK signal pathway.Western Blot experiment and immunofluorescence experiments can be proved that high cell contractility can lead to the activation of FAK,and then the degree of phosphorylation of FAK at the Y397 is enhanced.On the other hand,optogenetic experiment was used to found that inhibit the activity of myosin resulting in a decrease FAK in the cells.Based on the information above,the decrease in FAK activity is due to the decrease in cell contractility.Next,the molecular conformation of FAK was changed due to the high contractility which leads to the phosphorylation of FAK at the Y397 by using fluorescence resonance energy transfer experiment and other testing methods.Then through immunofluorescence,optogenetic inhibitors and Western Blot experiments,it was found that the activation of FAK can activated the downstream Rho-Rock signal pathway.Finally,after adding p FAKY397 inhibitor to collective cell with high myosin activity using by wound healing assay.These results show that the migration speed was restores.In conclusion,it has found in this research that activated myosin changes the molecular structure of FAK through high cell contractility which leads to the activation of FAK,which in turn activates the Rho-ROCK pathway to further strengthen intracellular contractility and form a positive feedback loop.In this research,we clarified that myosin is not only located at the end of the FAK-Rho-ROCK signal pathway,but also can affect its upstream signaling pathway through mechanical mechanical signals.It provides a new research direction for probing the mechanism of breast cancer cell collective migration.