ROCK And NMII Isoforms Differentially Modulate Breast Cancer Cell Migration By Mechanosensing The Substrate Stiffness

Breast cancer is one of the most prevalent cancers in women and its incidence showed a significant younger trend,with metastasis being the main reason for the poor prognosis of most patients.And the progression of breast cancer is usually accompanied by abnormal deposition,cross-linking,remodeling,and increased tissue stiffness of extracellular matrix(ECM).As one of the most important mechanical signals in tumor microenvironment(TME),substrate stiffness has a profound impact on the distal metastasis of tumor cells and the formation of secondary lesions.However,previous work has mainly focused on the influence of biochemical signals in TME on the malignant behavior of tumor cells.In recent years,an increasing number of studies have revealed that the mechanical features of ECM also play an extremely important role in tumor initiation and progression.As a complex disease regulated by multiple genes,cancer involves numerous cellular molecules and signaling pathways,thus it is essential to explore the mechanisms of tumorigenesis and metastasis,which can help the screening and identification of molecular targets for anti-cancer drugs.Therefore,this dissertation not only explores the effect of substrate stiffness on the motility behavior and mechanical properties of breast cancer cells,but also provides an in-depth investigation of the underlying molecular mechanisms.In this dissertation,based on the ECM mechanical characteristics in tumor development,we used a highly metastatic breast cancer cell line MDA-MB-231 as the primary subject and prepared cell culture substrates with elastic modulus values of 10 k Pa(soft),38 k Pa(stiff),and 57 k Pa(rigid)by polyacrylamide(PAA)gels,which were used to respectively simulate benign,malignant,and tissue stiffness during bone metastasis.We successfully constructed cell culture models comparable to tissue stiffness at different progress stages of breast cancer in vitro,and glass was used as a control substrate for cell culture in traditional mode.The results showed that stiff substrates induced migration phenotype formation through integrin β1-FAK-mediated mechanotransduction pathways.Subcellular structure observations suggested that stiff substrates could drive membrane protrusion at the cell leading edge by promoting cytoskeletal reorganization,which in turn led to the formation of the large broad lamellipodium with numerous longer filopodia,thereby regulated focal adhesions maturation and traction force polarized distribution.Wound-healing assay,transwell assay,and time-lapse microscopy exhibited that cells on the stiff substrate had a stronger migration and invasion abilities,and the velocity,Euclidean distance,accumulated distance,and directionality were significantly higher than other groups.We further demonstrated that substrate stiffness regulates the biomechanical behavior of breast cancer cells via the Rho A/ROCK signaling pathway,and ROCK isoforms were differentially involved in this process.Specifically,ROCK1 and ROCK2 were distributed in specific cytoskeletal structures,namely ROCK1 was mainly colocalized with F-Actin in actin bundles along the dorsal side of cells,whereas ROCK2 was more frequently co-localized with F-Actin at the edge of cell membrane protrusions.Molecular mechanism studies demonstrated that ROCK1 facilitated the generation of traction force by regulating the phosphorylation of MRLC,whereas ROCK2 contributed to cytoskeletal assembly by regulating the phosphorylation of cofilin.These results suggested that substrate stiffness jointly modulated breast cancer cell motility through two signaling pathways,Rho A/ROCK1/p-MRLC and Rho A/ROCK2/p-Cofilin,respectively.The above results showed that substrate stiffness can enhance tumor cell motility by affecting the mechanical responsiveness of the cytoskeleton.Accordingly,we investigated the distribution,function,and mechanism of nonmuscle myosin II(NMII),a motor protein that provided contractile stress for cytoskeletal remodeling,in migrating cells based on the results of Chapter 2.The findings revealed that the stiff substrate favored the localization and activation of NMIIA at the cell leading edge to promote actin polymerization,lamellipodia formation,and FAs dynamics by regulating the phosphorylation of its heavy chain terminus at the S1916 site.At the same time,the stiff substrate also contributed to the aggregation of NMIIB in the perinuclear and posterior region to promote traction force generation and polarized distribution by regulating the phosphorylation of its heavy chain terminus at the S1935 site.Molecular mechanism studies showed that the phosphorylation of NMIIA was regulated by the Rac1/PAK1 signaling pathway and dominated in substrate stiffness-mediated cell migration;while the phosphorylation of NMIIB was directly related to PKCζ activity.These results suggested that substrate stiffness modulated the phosphorylation of NMII heavy chains in an isoform-specific manner,thereby augmenting the directional migration of breast cancer cells.In summary,substrate stiffness enhanced breast cancer cell migration and invasion through integrin β1-FAK-mediated mechanotransduction pathways and regulating cytoskeletal reorganization in conjunction with ROCK and NMII isoforms to induce migration phenotype formation.This dissertation elucidated the process and regulatory mechanism by which substrate stiffness regulated breast cancer cell motility,providing new targets and inspiration for clinical research and cancer therapy.