nuclear envelope rupture and repair during cancer cell ......mar 23, 2016 · cite as: c. m. denais...

Cite as: C. M. Denais et al., Science 10.1126/science.aad7297 (2016).

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First release: 24 March 2016 www.sciencemag.org (Page numbers not final at time of first release) 1

The nuclear envelope (NE), comprised of the inner and outer nuclear membranes, nuclear pore complexes, and the nuclear lamina, presents a physical barrier between the nuclear interior and the cytoplasm that protects the genome from cytoplasmic components and establishes a separate compartment for DNA and RNA synthesis and processing (1). Loss of NE integrity and nuclear pore selectivity have been linked to the normal aging process and a variety of human diseases, including cancer (2). In cancer progression, key steps of tumor cell invasion depend upon deformation of the nucleus into available spaces within the three-dimensional tissue (3–6). Whereas the cytoplasm of migrating cells can penetrate even submicron-sized pores, the deformation of the large and relatively rigid nucleus becomes a rate-limiting factor in migration through pores less than 25 μm2 in cross-section (4, 6–10). We hypothesized that migration through such tight spaces provides a substantial mechanical challenge to the integrity of the nucleus. Thus, we investigated whether cell migration through confining spaces induces NE rupture and compromises DNA integrity, and how cells repair such NE ruptures during interphase.

To model cancer cell invasion with precise control over cell confinement, we designed a microfluidic device contain-ing constrictions with fixed height and varying widths mim-

icking interstitial pore sizes (fig. S1, A and B) (11). We de-tected NE rupture using previously established fluorescent reporters consisting of green or red fluorescent proteins fused to a nuclear localization sequence (NLS-GFP and NLS-RFP, respectively) that rapidly escape into the cytoplasm when NE integrity is lost (12–14). Breast cancer, fibrosar-coma, and human skin fibroblast cells displayed transient loss of NE integrity, coinciding with the nucleus passing through the constrictions (Fig. 1, A and B, fig. S1, C to L; and movie S1). NE rupture was associated with transient influx of fluorescently labeled cytoplasmic proteins into the nucle-us (fig. S2) and could also be detected by accumulation of the fluorescently labeled DNA-binding proteins barrier-to-autointegration factor (BAF) (15) and cyclic GMP-AMP syn-thase (cGAS) (16) at sites of NE rupture (fig. S3).

We then tested whether NE rupture also occurs during cancer cell migration in biological environments. Fibrosar-coma cells and skin fibroblasts exhibited NE rupture during migration in fibrillar collagen matrices (Fig. 1, C and D; fig. S4; and movie S2), with similar kinetics to those recorded in the constriction channels (fig. S1M). NE rupture typically occurred when the minimal nuclear diameter, which closely matches the pore size encountered by the cell (6), dropped to 3 �m (Fig. 1, D and E), thereby linking NE rupture to cell movement through narrow spaces. Accordingly, NE rup-

Nuclear envelope rupture and repair during cancer cell migration Celine M. Denais,1* Rachel M. Gilbert,1* Philipp Isermann,1* Alexandra L. McGregor,1 Mariska te Lindert,2 Bettina Weigelin,2 Patricia M. Davidson,1 Peter Friedl,2,3,4 Katarina Wolf,2 Jan Lammerding1† 1Nancy E. and Peter C. Meinig School of Biomedical Engineering and Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA. 2Department of Cell Biology, Radboud University Medical Center, Nijmegen, The Netherlands. 3Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 4Cancer Genomic Center, The Netherlands (CGC.nl).

*These authors contributed equally to this work.

*Corresponding author. E-mail: [email protected] During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, requiring extensive deformation of the cell and its nucleus. Here, we investigated mammalian tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport-III (ESCRT-III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content, requiring efficient NE and DNA damage repair for cell survival.

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tures were rare (i.e., below 5% per ≈12 hour observation pe-riod) for cells migrating on glass, in low-density collagen matrices, or through 15 × 5 �m2-wide channels, in line with previously reported rates of spontaneous NE rupture in can-cer cell lines (13). However, when matrix pore sizes were reduced to below 5-20 �m2 by increasing collagen concentra-tion and/or by blocking the cells’ ability to cleave collagen fibers and widen pores by addition of a matrix metallopro-tease (MMP) inhibitor, the incidence of NE rupture in-creased 10-fold and more (fig. S5, A and B). Similarly, reducing the pore size of the microfluidic channels to less than 20 �m2 increased NE rupture more than 10-fold (fig. S5, C and D). Irrespective of the experimental model, the inci-dence of NE rupture increased exponentially with decreas-ing pore size (Fig. 1F) and reached over 90% when the nuclear height was confined to 3 �m (fig. S5E). Imaging HT1080 fibrosarcoma cells invading into the collagen-rich mouse dermis in live tumors after orthotopic implantation confirmed that migration-induced NE rupture also occurs in vivo, particularly in individually disseminating cells (Fig. 1, G to J; fig. S6; and movie S3). NE rupture was less prevalent in cells moving as multicellular collective strands (Fig. 1J and fig. S6), which typically follow linear tracks of least re-sistance and undergo less pronounced nuclear deformations (3, 7).

NE rupture in vitro and in vivo was often accompanied by protrusion of chromatin through the nuclear lamina (Fig. 2, A and B; fig. S7, A to I; and movies S4 and S5). The inci-dence of such “chromatin herniations” increased significant-ly with decreasing pore sizes (Fig. 2C and fig. S7H). In severe cases, small pieces of the nucleus were pinched off from the primary nucleus as cells passed through narrow constrictions (Fig. 2D and movie S6), resulting in an elevat-ed and persistent fraction of cells with fragmented nuclei (Fig. 2E and fig. S7J). Furthermore, cells that had passed through microfluidic constrictions had more nuclear frag-ments positive for �-H2AX, a marker of DNA double-strand breaks (17), than cells that had not yet entered the con-strictions (Fig. 2F), consistent with recent reports that loss of NE integrity in micronuclei can cause DNA damage (14) and chromothripsis (18). Intense �-H2AX staining could also be found at chromatin protrusions (fig. S8A). To confirm that DNA damage was caused by migration-induced nuclear deformation and NE rupture, we performed live-cell imag ing on cells co-expressing NLS-GFP and fluorescently la-beled 53BP1 (RFP-53BP1), another marker of DNA damage (19, 20). NE rupture, and even severe nuclear deformation alone, resulted in rapid formation of new RFP-53BP1 foci as cells squeezed through the constrictions (Fig. 2, G and H, and fig. S8, B to G), consistent with previous reports of in-creased activation of DNA damage response genes after

compression-induced chromatin herniation and NE rupture (21).

To gain further insights into the biophysical processes underlying NE rupture, we analyzed the timing and location of NE rupture in regard to nuclear deformation, local mem-brane curvature, NE composition, and cytoskeletal forces. NE rupture occurred predominantly (≈76%) at the leading edge of the nucleus (Fig. 3, A and B) and was almost always (89.9 ± 2.14%, n = 198 cells) preceded by, or coincided with, the formation of nuclear membrane protrusions (“blebs”) as the nuclei moved through the constrictions (Fig. 1, C and H; Fig. 3, A and C; and fig. S9). Nuclear membrane blebs typi-cally (96.1 ± 1.38%, n = 178 blebs) formed at sites where the nuclear lamina signal, particularly the lamin B1 network, was weak or absent (Fig. 3, D to F, and fig. S10, A and B). These findings indicate that blebs form when segments of the nuclear membrane detach from the nuclear lamina and bulge into the cytoplasm. Depletion of lamins A/C and lam-in B2 significantly increased the likelihood of NE rupture (Fig. 3G and fig. S10C), which together with previous studies (4, 12, 13, 21–23) suggest that lamins are important for stabi-lizing the NE.

Consistent with previous observations (13, 21, 22, 24, 25), the expanding nuclear blebs were devoid of GFP-lamin B1 (Fig. 3, D to F) and nuclear pores (fig. S10D), and initially contained little, or no GFP-lamin A and B2 (fig. S10, A and B). Upon NE rupture the blebs retracted and collapsed (Fig. 1, C and H; Fig. 3, A and C; fig. S9; and fig. S10, E to H), suggesting that hydrostatic pressure was released from the fluid-filled blebs. Although the NE contains pores, recent studies have shown that the NE can provide an effective barrier to support intracellular pressure gradients (26, 27). Nuclear pressurization could arise from actomyosin contrac-tion at the rear of the nucleus that is required to move the nucleus through tight spaces (5, 6), effectively mimicking cellular compression experiments (21, 22). Supporting this idea, treatment with low concentrations of blebbistatin, a myosin II inhibitor, significantly decreased the incidence of nuclear rupture (Fig. 3H) without inhibiting the ability of cells to migrate through larger channels (fig. S10I)

The transient nature of NE rupture suggests that cells can efficiently restore nuclear membrane integrity during interphase. We observed rapid (


similar function in interphase NE repair, we generated GFP-fusion constructs of the ESCRT-III subunit CHMP4B, in-volved in recruiting other ESCRT-III proteins and facilitat-ing membrane scission, and the ESCRT-III–associated AAA-ATPase VPS4B, which is required for disassembly and recy-cling of ESCRT-III proteins (30). Upon NE rupture induced by confined migration or laser ablation, CHMP4B-GFP and VPS4B-GFP rapidly (≤2 min) formed transient foci at the site of nuclear membrane damage (Fig. 4, A to D; fig. S12; fig. S13, A to C; and movie S7). Super-resolution microscopy confirmed recruitment of endogenous ESCRT-III proteins to sites of NE rupture into complexes ≤ 160 nm in size (Figs. 4E and fig. S13, D to H). Recruitment of the ESCRT-III ma-chinery was independent of microtubules (fig. S14). Deple-tion of the ESCRT-III subunit CHMP2A, CHMP7, or ectopic expression of a dominant-negative VPS4B mutant (GFP-VPS4BE235Q) that prevents ESCRT-III subunit recycling, sig-nificantly increased the time required for nucleo-cytoplasmic re-compartmentalization (Figs. 4, F and G, and fig. S13, I to N), indicating a crucial role of ESCRT-III pro-teins in restoring nuclear membrane integrity. To assess the functional relevance of NE repair, we quantified cell viabil-ity after NE rupture. Under normal condition, the vast ma-jority (>90%) of cells survived even repeated NE rupture (Fig. 1A 4H and fig. S4A). Inhibiting either ESCRT-III medi-ated NE repair or DNA damage repair pathways alone did not reduce cell viability, but inhibition of both NE and DNA repair substantially increased cell death after NE rupture (Fig. 4H).

Taken together, our studies demonstrate that cell migra-tion through confining spaces, as frequently encountered during cancer cell invasion, can challenge the integrity of the NE and DNA content, which could promote DNA dam-age, aneuploidy and genomic rearrangements, and—in the absence of efficient repair—cell death (31). We propose a biophysical model in which cytoskeletal-generated nuclear pressure results in the formation and eventual rupture of nuclear membrane blebs at sites of high membrane curva-ture and weakness in the underlying nuclear lamina (fig. S15). These events could be particularly prominent in cells with reduced levels of lamins, whose expression is deregu-lated in many cancers and often correlates with negative outcomes (32, 33). While NE rupture, and resulting genomic instability, may promote cancer progression, it may also represent a particular weakness of metastatic cancer cells and an opportunity to develop novel anti-metastatic drugs by specifically targeting these cells, for example, by blocking NE repair and inhibiting DNA damage repair.

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ACKNOWLEDGMENTS

The authors thank G. Lahav, A. Loewer, M. Zwerger, M. Smolka, S. Emr, N. Buchkovich, B. Burke, H. Worman, S. Young, L. Fong, D. Discher, J. Broers, J. Goedhart, H. Yu and W. Zipfel for cells, reagents, and helpful discussion. The authors acknowledge E. Wagena for mouse surgery and cell transfection, C. Paus for generation of in vitro collagen data, and K. Zhang, E. Ghazoul, D. Huang, G. Fedorchak, G.-J. Bakker and F. Ahmadpour for help with image analysis. The authors are grateful to M. Piel and colleagues for sharing their unpublished data and manuscript. This work was supported by awards from the National Institutes of Health (NIH) (R01 HL082792 and R01 NS059348), the Department of Defense Breast Cancer Research Program (BC102152, BC BC150580), the National Science Foundation (NSF) (CBET-1254846), the National Cancer Institute through the Cornell Center on the Microenvironment and Metastasis (U54 CA143876), and an NSF Graduate Research Fellowship to A.L.M (DGE-1144153). This work was further supported by the Netherlands Science Organization (NWO-VIDI 917.10.364 to K.W. and NWO-VICI 918.11.626 to P.F.) and the Cancer Genomics Center, Netherlands. This work was performed in part at the Cornell NanoScale Facility, which is supported by the NSF (Grant ECCS-15420819). Imaging was performed in part through the Cornell University Biotechnology Resource Center, which is supported by NYSTEM (CO29155), NIH (S10OD018516), and NSF (1428922), and by the Radboud UMC Microscopic Imaging Center and the preclinical animal imaging center (PRIME). Additional experimental details and data are available in the supplementary materials.

SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aad6xxxx/DC1 Materials and Methods Figures S1 to S15 Movies captions S1 to S7 References (34–43) Movies S1 to S7 26 October 2015; accepted 25 February 2016 Published online 24 March 2016 10.1126/science.aad7297

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Nuclear envelope rupture and repair during cancer cell migration

Davidson, Peter Friedl, Katarina Wolf and Jan LammerdingCeline M. Denais, Rachel M. Gilbert, Philipp Isermann, Alexandra L. McGregor, Mariska te Lindert, Bettina Weigelin, Patricia M.

published online March 24, 2016

ARTICLE TOOLS http://science.sciencemag.org/content/early/2016/03/23/science.aad7297

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2016/03/23/science.aad7297.DC1

CONTENTRELATED

http://stke.sciencemag.org/content/sigtrans/11/537/eaar5401.fullhttp://stke.sciencemag.org/content/sigtrans/7/321/ec100.abstracthttp://science.sciencemag.org/content/sci/352/6283/359.fullhttp://science.sciencemag.org/content/sci/352/6283/295.full

REFERENCES

http://science.sciencemag.org/content/early/2016/03/23/science.aad7297#BIBLThis article cites 43 articles, 14 of which you can access for free

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Copyright © 2016, American Association for the Advancement of Science

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nuclear envelope rupture and repair during cancer cell ......mar 23, 2016 · cite as: c. m. denais...

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