Nuclear softening is essential for protease-independent migration
Introduction
The composition and organization of the extracellular matrix (ECM)—which provides structural and functional support to cells within tissues—is altered in diseased contexts such as fibrosis and cancer [1]. Both these diseases are characterized by increased deposition of the fibrillar ECM protein collagen and its crosslinking [2]. These alterations effectively reduce the pore size of the ECM network and provide increased steric hindrance. Cancer cells invade through these dense interstitial matrices using mesenchymal mode of invasion wherein cells exhibit elongated morphologies, adhere to the ECM, and secrete proteases including matrix-metalloproteinases (MMPs) for degrading the ECM fibers and generating free space for migration [3,4]. Mesenchymal to Amoeboid transition, MAT—first demonstrated by Friedl and co-workers in MDA-MB-231 breast cancer cells and HT-1080 fibrosarcoma cells—refers to the phenotypic switch from protease dependent to protease independent mode of migration when pericellular proteolysis is abrogated [[5], [6], [7], [8]]. Amoeboidal migration, also observed in dictostelium and leukocytes, can be sustained by multiple molecular mechanisms including actomyosin contractility-dependent squeezing, bleb-based protrusion and actin polymerization-dependent gliding [9,10]. Recent studies also suggest that confinement and low adhesivity can induce mesenchymal cells to adopt an amoeboidal mode of migration [11]. Intriguingly, other microenvironmental cues such as hypoxia and TGFβ have also been shown to induce a phenotypic switch in cancer cells to an amoeboidal state [12]. These signals also induce distinct phenotypic effects on fibroblasts in a stiffness-dependent manner [13,14].
For sustaining protease-independent amoeboidal migration, given that the ECM cannot be degraded, it is likely that cells alter their own physical properties, namely their stiffness, for squeezing through pores in the matrix. Indeed, cell softening has been linked with higher invasiveness in a range of different cancer cell lines and patient-derived cells [15] and has also been proposed as a biomarker of metastatic potential [16]. Recently we demonstrated that in MDA-MB-231 and HT-1080 cells, both of which exhibit MAT, inhibition of MMP proteolytic activity induces cell softening. However, this softening response is absent in MCF-7 cells, which are less aggressive raising the possibility that cell softening might be an adaptive response utilized by highly invasive cancer cells.
Though cell softening may help in amoeboidal migration, it may not be sufficient for sustaining amoeboidal invasion through dense interstitial matrices where nuclear squeezing represents the rate limiting factor. Seminal work by Friedl and co-workers demonstrated that the nucleus cannot be deformed below 10% of its uncompressed size [17]. Loss of lamin A/C, the major nuclear envelope protein dictating physical properties of the nucleus [18]—observed in multiple cancers [19]—has been shown to promote cell migration under confinement through increased nuclear deformability [20]. Nuclear translocation through confined geometries is mediated by myosin II motors which deform the nucleus through a combination of pulling from the front and squeezing from the rear [21]. Given that actomyosin contractility is partly inhibited in MDA-MB-231 and HT-1080 cells upon inhibition of MMP proteolytic activity [22,23], it remains unclear how nuclear deformation is achieved in these cells.
In this study, we document biophysical alterations in cell and nuclear properties that accompany drug-induced MAT, and demonstrate their importance in sustaining amoeboidal migration in highly invasive MDA-MB-231 and HT-1080 cells. We show that inhibition of MMP proteolytic activity perturbs both cytoskeletal and nuclear organization. Specifically, treatment with the broad-spectrum inhibition GM6001 leads to nuclear softening via phosphorylation of nuclear lamin A/C. By stiffening the nucleus through inhibition lamin A/C phosphorylation, we show that nuclear softening is necessary for migration through sub-nuclear size transwell pores. We further show that nuclear softening is not sufficient. Instead, in addition to nuclear softening, baseline contractility and peri-nuclear actin are required for mediating nuclear translocation through pre-existing paths.
Section snippets
Inhibition of MMP catalytic activity perturbs actomyosin organization
In addition to matrix degradation, an increasing body of work has illustrated the importance of MMPs in regulating various aspects of cell behaviour [24]. For example, both in astrocytes [25] and in cardiac fibroblasts [26], MMP-2 has been shown to regulate cell proliferation by perturbing focal adhesions. Recently, we showed that MMP proteolytic activity regulates the phenotype of invasive cancer cells by modulating the localization and activation of integrins [23]. Interestingly, MMP
Discussion
In summary, our results reveal that inhibition of MMP proteolytic activity leads to cell softening via reduction in pMLC levels and nuclear softening via increased phosphorylation of lamin A/C. Nuclear softening is essential for sustaining protease-independent migration through pre-existing paths smaller than nuclear dimensions, but not through dense interstitial matrices with randomly aligned fibers (Fig. 7). In addition to nuclear softening, baseline contractility and perinuclear actin are
Cell culture and reagents
MDA-MB-231 breast cancer cells and HT-1080 fibrosarcoma cells were obtained from National Center for Cell Science (NCCS) (Pune, India), and cultured in DMEM (high glucose, Invitrogen) containing 10% fetal bovine serum (FBS, Hi-media). Cells were maintained in 25 cm2 tissue culture flask (SPL) at 37oC at 5% CO2 humidified atmosphere and passaged when 80–90% confluent using 0.25% trypsin-EDTA (Hi-media). While GM6001 (Tocris, Biochemicals Cat. # 2983, 10 μM) was used as a broad spectrum MMP
Acknowledgements
Authors acknowledge financial support from Department of Biotechnology (Govt. of India) (Grant # BT/PR12705/BRB/10/1361/2014). AD was supported by fellowships from Department of Biotechnology (Govt. of India). Authors would also like to thank IRCC, IIT Bombay for providing Bio-AFM, Confocal microscopy and Cryo-FEG SEM facilities.
References (69)
- et al.
Extracellular matrix: the driving force of mammalian diseases
Matrix Biol.
(2018) Decoding fibrosis: mechanisms and translational aspects
Matrix Biol.
(2018)- et al.
Regulation of matrix biology by matrix metalloproteinases
Curr. Opin. Cell Biol.
(2004) Breast cancer cells alter the dynamics of stromal fibronectin-collagen interactions
Matrix Biol.
(2017)- et al.
Mechanical modes of ‘amoeboid’ cell migration
Curr. Opin. Cell Biol.
(2009) Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells
Cell
(2015)Hypoxia induces a HIF-1-dependent transition from collective-to-amoeboid dissemination in epithelial cancer cells
Curr. Biol.
(2017)- et al.
Targeting TGF-β signaling for the treatment of fibrosis
Matrix Biol.
(2018) Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts
Matrix Biol.
(2018)- et al.
A combination of actin treadmilling and cross-linking drives contraction of random actomyosin arrays
Biophys. J.
(2015)
A quantitative analysis of contractility in active cytoskeletal protein networks
Biophys. J.
MMP secretion rate and inter-invadopodia spacing collectively govern cancer invasiveness
Biophys. J.
Matrix elasticity regulates lamin-A, C phosphorylation and turnover with feedback to actomyosin
Curr. Biol.
Lamin A and lamin C but not lamin B1 regulate nuclear mechanics
J. Biol. Chem.
Dynamic interplay between the collagen scaffold and tumor evolution
Curr. Opin. Cell Biol.
Prioritising guidance cues: directional migration induced by substratum contours and electrical gradients is controlled by a rho/cdc42 switch
Dev. Biol.
Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization
Biophys. J.
Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility
Biochim. Biophys. Acta-Mol. Cell Res.
Fascin regulates nuclear movement and deformation in migrating cells
Dev. Cell
Matrix metalloproteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics
Biochim. Biophys. Acta-Mol. Cell Res.
Serglycin promotes breast cancer cell aggressiveness: induction of epithelial to mesenchymal transition, proteolytic activity and IL-8 signaling
Matrix Biol.
MT1-MMP-dependent control of skeletal stem cell commitment via a β1-integrin/YAP/TAZ signaling axis
Dev. Cell
Migration in confined 3D environments is determined by a combination of adhesiveness, nuclear volume, contractility, and cell stiffness
Biophys. J.
Adaptive force transmission in amoeboid cell migration
Nat. Cell Biol.
Compensation mechanism in tumor cell migration: mesenchymal–amoeboid transition after blocking of pericellular proteolysis
J. Cell Biol.
Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells
Cell Commun. Signal
The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells
Cell. Mol. Life Sci.
Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement
J. Leukoc. Biol.
Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines
Cancer Res.
Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells
PLoS One
Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force
J. Cell Biol.
Nuclear shape, mechanics, and mechanotransduction
Circ. Res.
The clinicopathological significance of lamin A/C, lamin B1 and lamin B receptor mRNA expression in human breast cancer
Cell. Mol. Biol. Lett.
Design of a microfluidic device to quantify dynamic intra-nuclear deformation during cell migration through confining environments
Integr. Biol.
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