Elsevier

Matrix Biology

Volume 82, September 2019, Pages 4-19
Matrix Biology

Nuclear softening is essential for protease-independent migration

https://doi.org/10.1016/j.matbio.2019.01.001Get rights and content

Highlights

  • Inhibition of MMP activity induces loss of stress fibers and loss of cell contractility.

  • Inhibition of MMP activity induces nuclear softening through phosphorylation of Lamin A/C.

  • Nuclear softening is essential for sustaining protease-independent migration through pre-existing paths.

  • Baseline peri-nuclear actomyosin contractility is required for mediating pore entry of soft nuclei for transmigration.

  • Peri-nuclear actin is required for maintaining nuclear deformation during pore migration.

Abstract

During amoeboidal migration, cancer cells migrate in a protease-independent manner by squeezing through pre-existing gaps in the extracellular matrix (ECM). However, the extent to which cells alter their physical properties in order to sustain this mode of migration remains unclear. Here, we address this question by documenting biophysical changes in the properties of highly invasive MDA-MB-231 and HT-1080 cells upon inhibition of pericellular proteolysis. Remarkably, treatment with the broad spectrum MMP inhibitor GM6001 not only induces cell rounding and loss of actomyosin contractility, but also induces nuclear softening via increased phosphorylation of the nuclear membrane protein lamin A/C. Though nuclear softening is necessary for sustaining migration through sub-nuclear sized transwell pores, it is not sufficient. In addition, baseline levels of contractility mediating pore entry and peri-nuclear actin inside the pores mediating pore migration are also required. Taken together, our results suggest that protease-independent migration through sub-nuclear sized pre-existing tracks is enabled by deformation of a softened nucleus by contractility and the peri-nuclear actin network.

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.

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