Nesprin-1/2 Regulate Nuclear Deformation During Tumor Cells Pore Migration

The spread and metastasis of breast cancer cells is one of the main reasons for treatment failure.In the process of cancer cell invasion and establishment of distant metastatic tumors,cells must migrate through the constricted environment of cell junctions,extracellular matrix and basement membrane.The nucleus is the largest and hardest organelle,so it can resist huge changes in shape.Therefore,the deformability of the nucleus is a limiting factor for migration through confine environment.Nesprin-1/2provide the mechanical connection between the nucleus and cytoskeleton,studies have found that they regulate the movement,positioning and deformation of nucleus.However,the specific mechanisms of Nesprin-1/2 regulating tumor cell nucleus deformation are unclear.In addition,accumulation of non-muscle myosin IIb at the rear of the cell as the nucleus is squeezed through a constriction indicates actomyosin contractility pushes the nucleus forward.However,the hour-glass shape of fibroblast nuclei deforming through microfluidic constrictions and the patterns of strain in the nuclear interior suggests a pulling force from the front of the nucleus as the origin of nuclear motion.Therefore,this paper mainly explores the molecular mechanisms that Nesprin-1/2 affect migration by regulating nucleus deformation and force transmission in breast cancer.We explored the migration of cells in a narrow environment by using 3 μm pore transwell chamber,and found that Nesprin-1/2 knockdown significantly reduced the ability of migration.Confocal microscopy and atomic force microscopy were used to evaluate the deformability of nucleus,and it was found that Nesprin-1/2 knockdown reduced the stiffness of nuclear and made it easier to deform.Nuclear stiffness is related to the expression level of lamins.In order to further explore the mechanisms of nuclear deformation,we used Western Blot and immunofluorescence technology to detect the expression level of Lamin A/C,and found that Nesprin-1/2 knockdown leads to Lamin A/C expression decrease.However,we found that Lamin A/C did not change at the mRNA level through q-PCR experiments.In order to investigate whether Lamin A/C protein has been degraded,we used the Caspase-6 inhibitor Z-VEID-FMK,the autophagy inhibitor 3-methyladenine(3-MA),or the broad-spectrum proteasome inhibitor MG132 to treat the experiment cells,Nesprin-1/2 deficiency increases Lamin A/C degradation through a proteasomal pathway.To further confirm this,we examined the levels of lamin A/C ubiquitination in Nesprin-1/2 knockdown and control cells by immunoprecipitation and Western Blot experiments,Nesprin-1/2 knockdown increased the ubiquitination of lamin A/C.On the other hand,we used inhibitors to treat experimental cells,and found that Nesprin-1/2 depletion results in the activation of cyclin-dependent kinase 5(CDK5),which causes the phosphorylation of lamin A/C(mainly at S392 site)and its subsequent translocation to the cytoplasm for degradation.Finally,in order to explore the mechanisms of Nesprin-1/2 transferring force to the nucleus,we observed the distribution of F-actin and p-MLC in the process of pore migration through immunofluorescence experiments,and found that Nesprin-1/2knockdown led to the disappearance of the nuclear tail thrust,unable to push the nucleus forward.In summary,our study found that Nesprin-1/2 regulate the deformation of the nuclear by modulating expression of Lamin A/C and the distribution of cytoskeletal force to affect cell pore migration,providing a new research direction for breast cancer metastasis mechanism.