Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

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Life sciences study design
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Sample size
No statistical tests were performed to pre-determine sample size. Sample sizes for the spontaneous metastasis assays (10 mice per experimental condition) were selected due to more than 20 years previous experience demonstrating that this is the minimum number of animals necessary to achieve statistically significant and reproducible results (

Replication
Experiments have been repeated at least duplicate or triplicates. For studies involving mice (metastasis assay), experiment was performed once for each orthopic transplantation models to limit the use of animals. We have used 3 different immunocompetent orthotopic transplantation model of metastasis and 2 different mice background to validate the robustness of key results in different biological systems.
All the attempts at replication of experimental conditions were successful.
Randomization For animal studies, mice were randomly chosen into each experimental groups. For all other samples used in this study, including cultured cells, were allocated randomly to each condition.

Blinding
Investigators were not blinded during allocating animal experiment because the investigators needed to know what cell type they will be injecting and they had to culture and process the cells for mouse injection by themselves. Assessment of immunohistochemistry result was performed by independent researcher in the blinded fashion. In many applications (Mass spectrometry analysis, Accessible chromatin analysis, ChIA-PET seq, RNA-seq, ChIP-seq, high-resolution microscopy, DNA fluoresence in situ hybridization etc), acquisition of the data was performed by expert technicians without the knowledge of the experimental groups in a blinded fashion.

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Authentication
Cell lines were not authenticated in our laboratory.

Mycoplasma contamination
We have extensively checked our cell lines for Mycoplasma contamination using Mycoalert Mycoplasma detection kit (Lonza) and found negative.

Commonly misidentified lines (See ICLAC register)
None of the cell lines used in this study were found in the database of misidentified cell lines

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals~6 weeks old, female BALB/cJ (000651) and FVB/NJ (001800) mice were purchased from The Jackson Laboratory. Mice were housed in 12 hours dark/12 hours light cycle, ambient temperature and humidity condition.

Wild animals
The study did not involve any wild animal.

Field-collected samples
The study did not involve field-collected samples.

Ethics oversight
Usage of animals described in this study was performed under the animal study protocol LCBG-004 approved by the National Cancer Institute (NCI) at Bethesda Animal Use and Care Committee.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
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Methodology
Replicates ChIP-seq was performed on one replicate per condition. ChIP enrichment was normalized to negative control (IgG).

Sequencing depth
All ChIP-seq experiments were performed in single-end read, 75bp. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Circulating tumor cell (CTC) analysis: 100,000 6DT1 cells with or without Nup210 knockdown were injected into the fourth mammary fat pad of FVB/NJ mice. Ten mice were used in each group and three mice were kept uninjected for use as healthy controls. One month after injection, mice were anesthetized with avertin injection. Through cardiac puncture, 600-1000 ul blood per mouse was collected in 50 ul of 0.5 M EDTA solution. An equal volume of blood was taken for red blood cell lysis using ACK lysis buffer. 100 ul of the peripheral blood lymphocyte (PBL) fraction was subjected to fixation with 2% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS containing 0.1% Triton X-100. Cells were vortexed briefly and kept at room temperature for 30 min. 0.5% BSA in PBS was added and cells were pelleted by centrifugation. Cells were then resuspended in ice-cold 50% methanol in PBS and incubated for 10 min on ice. 150,000 fixed cells were stained for CD45, a pan-lymphocyte marker, and pan-keratin, a tumor cell marker. Before staining with antibodies, cells were incubated with FcR Blocking Reagent (1:10 dilution; Miltenyi) for 10 min at 4 degree C. Cells were then stained with APC-conjugated CD45 (1:25 dilution; Miltenyi) and Alexa Fluor 488-conjugated pankeratin (1:25 dilution, Cell Signaling Technology) antibodies for 10 min at 4 degree C. After washing with MACS buffer (PBS, 0.5% BSA, and 2 mM EDTA), cells were incubated with 1 ug/ml Hoechst 33342 (Thermo Fisher Scientific) for 5 min. Cells were then washed again with MACS buffer and resuspended in 200 ul buffer for analysis using a BD FACSCanto II flow cytometer. A CTC (CD45-/Cytokeratin+) gate was created based on the staining pattern of 6DT1 tumor cells in culture and primary tumor cells derived from 6DT1-injected mice.
Cell cycle analysis: Cell cycle analysis was performed using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) and FxCycle Violet Stain (Thermo Fisher Scientific) according to manufacturer's instructions. Briefly, 4T1 cells were seeded onto 15 cm tissue culture dishes at a seeding density of 3x106 cells/dish. 24 h later, cells were pulsed with Click-iT EdU (5-ethynyl-2´deoxyuridine) for 1 h. After harvesting the cells through trypsinization, cells were fixed with Click-iT fixative and permeabilized with saponin-based permeabilization agent. The Click-iT reaction was then performed for 30 min at room temperature. Cells were then washed with wash buffer and stained for DNA content analysis with FxCycle Violet, a DNA-selective dye. Finally, cell cycle analysis was performed using a BD FACSCanto II flow cytometer (BD Bioscience).

Instrument
BD FACSCanto II flow cytometer was used for data acquisition Software BD FACSDiva software was used for data collection and FlowJo V10 was used for data analysis Cell population abundance For flow cytometry analysis, peripheral blood lymphocyte was isolated from each mouse blood. 20,000 to 100,000 cells were captured during each session/sample.

Gating strategy
After excluding dead cells and cell clumps, individual cell types were identified based on the following markers: Circulating tumor cells: CD45 negative, pan-cytokeratin positive Peripheral blood lymphocytes: CD45 positive Sample acquisition gates were created for CTCs based on the staining pattern of 6DT1 cells lines in culture which is provided in figure 8.
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