Chromosomal instability drives metastasis through a cytosolic DNA response

Abstract

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Figures

Extended Data Figure 1. Generation of isogenic tumor models of CIN

a, Weighted genomic instability index (wGII) of brain metastases as a function of the wGII of the matched primary tumor. Red line represents linear regression, n = 79 patients. b, Differences in wGII between metastases and matched primary tumors. RCC, renal cell carcinoma, the “Other” category includes melanoma, sarcoma, ovarian, thyroid, and salivary gland cancers. c, Number of clones (based on single-cell karyotypes) in primary breast tumors (n = 637) or metastases (Mets, n = 131) found in the Mitelman Database, boxes represent median ± interquartile range and bars span the 10th and 90th percentile, significance tested using two-sided Mann Whitney test. d, The number of chromosome aberrations per clone as a function of the total number of chromosomes in a given clone in samples derived from primary breast tumor clones (n = 983) and metastatic clones (n = 186), data points represent average ± SD. e, Percentage of N− or N+ patients as a function of chromosome missegregation frequency (n = 20 patients per condition), significance tested using two-sided Fisher Exact test. f, Immunoblots of cells expressing various GFP-tagged kinesin-13 proteins stained using anti-GFP antibody, β-actin used as a loading control, 2 independent experiments. g, Cellular confluence as a function of time of MDA-MB-231 cells expressing various kinesin-13 proteins or dnMCAK expressing cells depleted of components of the cytosolic DNA-sensing machinery or the noncanonical NF-κB pathway, data points represent mean ± SD, n = 4 independent experiments. h, Left, MCAK and dnMCAK expressing cells stained for microtubules (DM1A), centrosomes (pericentrin) and DNA (DAPI), scale bar 5-μm, 2 independent experiments. Right, Frequency distribution of the number of pericentrin foci per cells. Significance tested using ANOVA. n = 100 cells per condition, 2 independent experiments. i, Cells expressing kinesin-13 proteins stained for microtubules (DM1A), centrosomes (pericentrin) and DNA (DAPI), scale bar 50-μm, 2 independent experiments. Bottom-right, Fluorescence normalized to cell count of MDA-MB0-231 cells expressing kinesin-13 proteins, bars represent mean ± s.e.m., * p < 0.05, two-sided t-test, n = 10 high-power fields encompassing 477–612 cells, 2 independent experiments.

Extended Data Figure 2. Karyotype analysis of human tumor cells

a–b, Immunoblots showing total Rac1 (a) or RhoA (b) levels as well as Rac1 or RhoA that were pulled-down using antibodies that were specific to the GTP-bound form of Rac1 (a) or RhoA (b). Positive and negative controls were total MDA-MB-231 cell lysates supplanted with non-hydrolysable GTP (nhGTP) or GDP, respectively. β-actin was used as a loading control, 2 independent experiments. c–e, Representative karyotypes (DAPI-banding) from parental MDA-MB-231 cells (c), or populations derived from single MCAK (d) or Kif2a (e) expressing cells that were allowed to divide for 30 days. f, The number of non-clonal (present in <25% of the cells in a single clone) structurally abnormal chromosomes in CIN-low or CIN-high MDA-MB-231 cells. ‘Mar’ denotes chromosomes so structurally abnormal that precludes their unambiguous identification by conventional banding, bars represent mean ± SD, n = 140 cells from 7 clonal populations, significance tested using two-way ANOVA test. g, Examples taken from 4 distinct cells belonging to the same clonal population – derived from a single Kif2a-expressing cell – showing convergent translocations involving chromosome 22 with four other chromosomes. h, Deviation from modal chromosome number in single-cell-derived clones grown for 30 days. Four chromosomes were assayed for each clone using centromere-specific probes, *p < 0.05, **p < 0.005 compared to control clone 4, two-sided c2-test, n = 300 cells per clone. Diploid controls were used to determine false positive rate of the centromeric probes.

Extended Data Figure 3. CIN promotes formation and maintenance of metastasis

a, Chromosome missegregation in H2030 and 4T1 cells expressing kinesin-13 proteins. Bars represent mean ± SD, n = 150 cells, 3 independent experiments, significance tested using two-sided t-test. b, Left, Normalized photon flux over time of whole animals injected with MDA-MB-231 cells expressing kinesin-13 proteins Bars represent mean ± s.e.m. n = 8 (MCAK), 7 (Kif2b), 5 (Control), 4 (Kif2a), and 9 (dnMCAK) mice per group, 3 independent experiments. Right, Mice injected with MDA-MB-231 cells expressing dnMCAK (above) or Kif2b (below) where disease burden was tracked using BLI, 3 independent experiments. c, Photon flux (p/s) of whole animals imaged 5 weeks after intracardiac injection with control or MCAK expressing H2030 cells. Bars represent the mean, significance tested using two-sided Mann Whitney test, n = 10 mice in the MCAK group and 5 mice in the control group. d, Left, Representative BLI images from two independent experiments of mice orthotopically transplanted with MDA-MB-231 cells before (Day 33) and after (Day 90) tumor excision. Metastasis can be detected in the mouse transplanted with dnMCAK expressing cells at day 90. Middle, Total flux (p/s) emitted from primary tumors 52 days after transplantation. Bars represent mean ± SD, n = 5 (CIN-low) and 14 (CIN-high) mice, p = 0.13, two-sided Mann Whitney test. Right, Distant metastasis-free survival (DMFS) of mice orthotopically transplanted with MDA-MB-231 cells with various levels of CIN. n = 15 (CIN-low) and 29 (CIN-high) mice, pairwise significance tested with two-sided log-rank test. e, Tumor volume at 8 days (top) and survival (bottom) of mice transplanted with murine 4T1 cells in the mammary fat pad. Bars represent median ± interquartile range, pairwise significance tested with two-sided t-test (top) and two-sided log-rank test (bottom). n = 20 (CIN-low) and 30 (CIN-high) mice. f, Top, immunoblots of MCAK or MCAK and mad2 overexpressing MDA-MB-231 cells stained for mad2 using anti-mad2 antibody with a-tubulin used as a loading control, 3 independent experiments. Bottom, Percentage of anaphase cells exhibiting evidence of chromosome missegregation in MCAK or MCAK and mad2 overexpressing cells, bars represent mean ± SD, n = 150 cells, 3 experiments, significance tested using two-sided t-test. g, Top, immunoblots of dnMCAK or dnMCAK and Lamin B2 overexpressing MDA-MB-231 cells stained for Lamin B2 using anti-Lamin B2 antibody with β-actin used as a loading control, 2 experiments. Bottom, Percentage of anaphase cells exhibiting evidence of chromosome missegregation in dnMCAK or dnMCAK and Lamin B2 overexpressing cells, bars represent mean ± SD, n = 150 cells, 3 experiments, significance tested using two-sided t-test. h, Photon flux (p/s) of whole animals after intracardiac (left) or tail vein (right) injection with dnMCAK or dnMCAK and Lamin B2 expressing MDA-MB-231 cells. Bars represent the median, significance tested using two-sided Mann Whitney test, n = 9 (dnMCAK), 15 (dnMCAK and Lamin B2) mice in the intracardiac injection cohort and 5 mice per group in the tail vein injection cohort.

Extended Data Figure 4. Transcriptional consequences of CIN in cancer cells

a–b, Principle component analysis (PCA) (left) and unsupervised clustering (right) of 5 MDA-MB-231 cell lines expressing different kinesin-13 proteins based on bulk RNA expression data. b–e, Gene set enrichment analysis (GSEA) results showing HALLMARK gene sets that are highly enriched in CIN-high (control, Kif2a, and dnMCAK) compared with CIN-low cells (MCAK and Kif2b) cells (b–c) or STING-depleted cells (e), or after comparing metastases with primary tumors (d), significance tested using one-sided Weighted Smirnov-Kolmogorov test corrected for multiple tests. f, Heatmap of consensus chromosomal karyotypes of cells derived from primary tumors and metastases showing selective increase in chromosome 1 copy number in metastases compared with primary tumors.

Extended Data Figure 5. Prognostic impact of the CIN signature

a, Volcano plot showing differentially expressed genes between CIN-high and CIN-low MDA-MB-231 cells. Red data points denote genes subsequently used for determining the CIN signature. b–e, Enrichment plots for all differentially expressed genes (a) or those on Chromosome 1 (d–e). Circos plot (c) showing genomic location (outer circle), log2-fold expression of genes significantly differentially expressed in metastases compared to primary tumors (middle circles), and the log10(p value, inner circle) for genomic amplifications (red) or deletions (blue) in metastases relative to primary tumors. n = 2 (CIN-low), 3 (CIN-high), 11 (primary tumors), 28 (metastases). Significance tested using two-sided Wald test (a), one-sided Weighted Smirnov-Kolmogorov test (b, d, e), and one-sided hypergeometric test (c) all corrected for multiple testing. f–g, DMFS of breast cancer patients stratified by lymph node status, grade, and receptor status, from a meta-analysis (f, n = 664 patients) or a validation cohort (g, n = 171 patients) divided based on their average expression of the CIN gene expression. Significance tested using two-sided log-rank test.

Extended Data Figure 6. Single-cell sequencing and population detection

a, The cellular composition of every subpopulation presented Figure 4b. b, Violin plots showing expression probability density of key metastasis and invasion genes in a subpopulation of cells (n = 1273 cells) enriched for EMT and CIN genes (subpopulation ‘M’) compared with the remaining subpopulations (n = 5548 cells) that were identified using graph-based unsupervised K-nearest neighbor embedding. c, Representative low-power field images (left) and numbers (right) of MDA-MB-231 cells which invaded through a collagen membrane within 18 hours of culture. Bars represent mean ± SD, Significance tested using two-sided Mann Whitney test, n = 10 high-power fields, 2 independent experiments. d, Representative images of MDA-MB-231 cells expressing MCAK or dnMCAK stained for β-actin, Vimentin, and DNA scale bar 50-μm, n = 2 independent experiments. e, Single-cell correlation plots between CIN signature genes, canonical NF-κB and type I interferon target genes, n = 6,821 cells. e, Representative phase-contrast images of a wound-healing assay of cells MCAK, MCAK+mad2 or dnMCAK expressing MDA-MB-231 cells and MCAK cells treated with cGAMP, scale bar 800-μm, 4 experiments.

Extended Data Figure 7. CIN promotes in vitro invasion and migration

a, Left, representative phase contrast images of MDA-MB-231 cells in the wound area, 36-hours after wound creation, 4 experiments. Right, length-to-width ratio of cells expressing different kinesin-13 proteins. Bars span the interquartile range, n = 100 cells, 2 independent experiments, significance tested using two-sided Mann Whitney test. b, Representative MDA-MB-231 cells stained for β-catenin (anti-β-catenin antibody) or DNA (DAPI). Changes in β-catenin are seen upon alteration of CIN; enriched at cell-to-cell junctions in MCAK expressing cells but cytoplasmic and nuclear in dnMCAK expressing cells, scale bar 30-μm, 2 experiments. c, Above, Phase-contrast images of a wound-healing assay of cells expressing kinesin-13 proteins, scale bar 800-μm, 2 experiments. Bottom, Wound area (normalized to the 0h time point) 24h and 45h after wound creation, bars represent mean ± SD, n = 4 experiments, significance tested using two-sided t-test. d, Above, Low-power field images of MDA-MB-231 cells that have migrated through a polycarbonate membrane containing 8-μm pores within 18 hours of culture. Below, Normalized O.D. of cells scraped from the bottom of the membrane, bars represent mean ± s.e.m., significance tested using two-sided t-test, n = 3 experiments. e–f, Left, Number of MDA-MB-231 cells that have successfully invaded through a collagen basement membrane 24 hours after plating. Bars represent mean ± SD, n = 20 high power fields from 2 independent experiments, significance tested using two-sided Mann-Whitney test. Right, representative images from high-power fields, 2 independent experiments. g, i, Representative time-lapse fluorescence and phase-contract image sequences of control cells expressing NLS-GFP undergoing unconfined migration (g) or going through 1×5 μm2 constrictions (i). Scale bar 20-μm. Arrows in (g) indicate cytoplasmic NLS-GFP. Arrows in (i) indicate formation of nuclear protrusion and subsequently fragment during confined migration, 3 independent experiments. h, j, The probability of primary nuclear rupture during unconfined conditions (h, top) or after migration through 1×5 μm2 wide constrictions (j, top). The number of cells migrating through more than one 1-μm wide constrictions (j, bottom) and the duration of nuclear rupture (h, bottom) as measured by the length of time in which NLS-GFP signal is observed in the cytosol. Bars represent mean ± s.e.m., n = 3 independent experiments (except for unconfined rupture probability – 2 independent experiments) encompassing 390–665 (h) and 150–336 (j) cells observed during unconfined and confined migration, respectively, significance tested using two-sided t-test.

Extended Data Figure 8. CIN generates micronuclei and cytosolic DNA

a–b, Percentage of micronuclei in samples depicted in Figure 3c–d: injected cells (blue), first-passage cells derived from primary tumors (green), or metastases (orange denotes spontaneous metastases arising from primary tumors, red denotes metastases obtained from direct intracardiac implantation). Bars represent mean ± s.e.m., n = 10 high-power fields encompassing 500–1500 cells/sample, 3 independent experiments, * p < 0.05 and denotes samples with higher missegregation rates than the injected lines, # p < 0.05 and denotes samples with lower missegregation rates than the injected lines, ** p < 0.05 and it denotes significant differences between metastases and matched primary tumors from the same animals, two-tailed t-test. c, Correlation between the percentage of cells exhibiting evidence of chromosome missegregation and percentage of micronuclei in all injected cell lines as well as cells derived from primary tumors and metastases. Data points represent mean ± s.e.m., n = 44 samples. d–f, Representative images of cells stained for DNA (DAPI), cytosolic single-stranded DNA (ssDNA) (d), Dnase2 (RFP reporter) (e), or cytosolic double-stranded DNA (dsDNA) (f), scale bar 20-μm, arrows in e denote Dnase2 expressing cell, 2 independent experiments. g, Representative images of dnMCAK expressing cells treated with ssDnase or dsDnase for 10 min. after selective plasma membrane permeabilization (using 0.02% saponin) stained for DNA (DAPI) and cytosolic dsDNA, scale bar 20-μm, one experiment. h, Representative images of dnMCAK expressing cells stained for mitochondria (using anti-CoxIV antibody), DNA (DAPI) or for cytosolic DNA (using anti-dsDNA antibody), scale bar 20-μm, 2 independent experiments. i, Immunoblots of lysates from cells expressing different kinesin-13 proteins, control or STING shRNA, β-actin used as a loading control. j, Normalized ratio of phosphorylated p52-to-p100 (left) and p100-to-total p100 (right) protein levels. Bars represent mean ± s.e.m., n = 5 independent experiments.

Extended Data Figure 9. Alternative response to cytosolic DNA in cancer cells

a–d, Representative images of MDA-MB-231 cells stained for DNA (DAPI), and for p65 (a), IRF3 (b), or RelB (c–d). Images were individually contrast-enhanced to emphasize nuclear versus cytosolic localization of p65, IRF3, and RelB. For quantitative comparisons of the identical images, please refer to Supplementary Figure 3. Arrows (c–d) point to RelB-positive nuclei, scale bars, 20-μm, 3 independent experiments. e, Immunoblots of fractionated lysates. a-tubulin and Lamin B2 were used as loading controls for the cytoplasmic and nuclear fractions, respectively, 3 independent experiments. f, h, Interferon-β levels in conditioned media from DLD-1 cells (f) MDA-MB-231 or HEK293 cells with and without cGAMP addition (h). Bars represent mean ± s.e.m. n = 3 experiments, significance tested using one-sided Mann-Whitney test. g, i, Relative levels of interferon responsive genes obtained by RT-qPCR, DLD-1 cells (g) normalized to untreated conditions or MDA-MB-231 cells (i) normalized to control cells. Bars represent mean ± SD n = 3 experiments, significance tested using 2-sided t-test. j, Immunoblots of lysates of dnMCAK expressing cells that also co-express control shRNA or shRNAs targeting components of cytosolic DNA sensing or the noncanonical NF-κB pathway. shRNA hairpins are numbered in ascending order based on the efficiency of the protein knockdown, 2 independent experiments.

Extended Data Figure 10. Effect of cytosolic DNA sensing pathways on prognosis

a, Distant metastasis-free survival (DMFS), Relapse-free survival (RFS) and Progression-free survival (PFS) of breast and lung cancer patients, respectively stratified based on their expression of NF-κB and interferon pathways, significance tested using two-sided log-rank test. b, Disease-specific survival of mice injected with dnMCAK expressing MDA-MB-231 cells co-expressing either control shRNA, STING shRNA, NFKB2 shRNA, or RelB shRNA n = 35, 16, 19, and 20 mice in the control, STING shRNA, NFKB2 shRNA, and RelB shRNA groups respectively, significance tested using two-sided log-rank test. c, Number of MDA-MB-231 cells expressing shRNA targeting genes belonging to the DNA sensing or noncanonical NF-κB pathways that invaded through a collagen membrane within 24 hours of culture. Bars represent mean ± SD, ** p < 0.0001, two-sided Mann Whitney test, n = 20 high-power fields, 2 independent experiments. d, Number of different normal tissues (vascular, neuronal, or soft tissues) invaded by orthotopically transplanted tumors. Bars represent mean ± s.e.m., *p < 0.05, two-tailed t-test, n = 13 tumors (CIN-high), 20 tumors (noncanonical NF-κB depleted), 19 tumors (cGAS-STING depleted). e, Oncoprints showing genomic alterations in STING (TMEM173) and cGAS (MB21D1) in breast and lung cancers from the TCGA database.

Figure 1. Human metastases enrich for CIN

a, wGII of matched primary tumors (P) and brain metastases (M), n = 79 patients. b–c, Karyotype probability density (b) and chromosomal aberrations (c) in 983 primary tumor and 186 metastatic breast cancer clones. d, Images of a head and neck squamous cell carcinoma cells undergoing anaphase. Arrows point to chromosome missegregation, scale bar 5-μm. Right, Chromosome missegregation in tumors from patients with (N+, n = 22 patients) or without (N-, n = 18 patients) clinically detectable lymph node metastases. Boxes represent median ± interquartile range, confidence intervals denote 10th–90th percentile (a, c–d), significance tested using two-sided Wilcoxon matched-pairs signed rank test (a) and two-sided Mann Whitney test (b–d).

Figure 2. CIN is a driver of metastasis

a, Anaphase cells stained for centromeres and DNA, scale bar 5-μm. b, Chromosome missegregation in cells expressing kinesin-13 proteins. Bars represent mean ± SD, n = 150 cells. c, Whole animal bioluminescence (BLI) 7 weeks after intracardiac injection of MDA-MB-231 cells. Bars represent the median, n = 12 (MCAK+Mad2), 20 (MCAK), 7 (Kif2b), 9 (control), 9 (Kif2a), 8 (dnMCAK) mice. d, Ex-vivo BLI of organs with metastases from MDA-MB-231 cells expressing dnMCAK. e, Disease-specific survival of mice injected with CIN-high (n = 33) or CIN-low (n = 20) MDA-MB-231. Significance tested using two-sided t-test (b), two-sided Mann Whitney test (c), and two-sided log-rank test (e) n = 3 (a–b) and 5 (d) independent experiments. Throughout the manuscript, pairwise comparisons between individual CIN-low and CIN-high conditions are smaller than the stated p value.

Figure 3. Opposing roles for CIN in primary tumors and metastases

a, Experimental schema. b–d, Chromosome missegregation in injected cells (blue), cells derived from primary tumors (green), metastases (met) arising spontaneously (orange) or after intracardiac injection (red). ST, soft tissue. Bars represent mean ± SD, n = 150 cells, 3 independent experiments, *p < 0.05, #p < 0.05, **p < 0.05 denote samples with higher or lower chromosome missegregation than the injected lines, and spontaneous metastases with higher missegregation than matched primary tumors, respectively. Significance tested using two-sided t-test (b–d).

Figure 4. CIN enriches for mesenchymal cell traits

a, Heatmap showing expression of EMT genes in 6,821 MCAK, Kif2b, and dnMCAK expressing MDA-MB-231 cells. b, Above, t-stochastic neighbor embedding (tSNE) projection of all cells in a. Below, Heatmap of normalized enrichment score (NES) for pathways with an FDRq < 0.05 based on GSEA using one-sided Weighted Smirnov-Kolmogorov test on subpopulations identified using Phenograph. Boxes outline populations with mutually exclusive transcriptional profiles.

Figure 5. CIN generates cytosolic DNA

a, Heatmap showing gene-gene correlations in 6,821 cells and HALLMARK gene sets significantly enriched in Module 2, one-sided Weighted Smirnov-Kolmogorov test. b, Top right, normalized expression level of key gene signatures in 6,821 MCAK, Kif2b, and dnMCAK expressing MDA-MB-231 cells. Bottom left, Correlation plots for key gene signatures. c, A primary nucleus and a micronucleus stained for centromeres and DNA, scale bar 5-μm. d–e, Percentage of micronuclei cells expressing various kinesin-13 proteins (d) or in cells derived from 10 primary tumors and 28 metastases (e). n = 20 high-power fields per sample (d) or average values derived from 10 high-power fields per sample (e). f, MCAK and dnMCAK expressing MDA-MB-231 cells stained using DAPI and anti-dsDNA antibody, scale bar 20-μm. g, Cytosolic-to-nuclear DNA ratios in CIN-high (n = 5) and CIN-low (n = 4) MDA-MB-231 (n = 5) and H2030 (n = 4) cells. h, Left, DLD-1 cells stained for DNA and hybridized to chromosome-specific FISH probes, scale bar 5-μm. Right, Percentage of cells containing micronuclei or small cytosolic DNA fragments (n >500 cells per condition). i, MDA-MB-231 cells stained using DAPI, anti-dsDNA antibody, or mCherry-Lamin B2 (arrow), scale bar 10-μm. Bars represent median ± inter-quartile range (d–e), mean ± SD (g), mean ± sem (h). n = 6 (c–d), 3 (e–f, h), 1 (g) and 2 (i) independent experiments. Significance tested using two-sided Mann-Whitney test (d–e) or two-sided t-test (g–h), * p <0.05.

Figure 6. Metastasis from a cytosolic DNA response

a, MDA-MB-231 cells stained for DNA and cGAS, scale bar 5-μm. b, Percentage of micronuclei with (cGAS+) or without (cGAS-) cGAS localization, n = 200 cells. c, Cells stained for DNA and STING, scale bar 20-μm. d, Percentage of MDA-MB-231 cells with nuclear RelB, n = 150 cells. e, Average z-normalized expression of CIN-responsive noncanonical NF-κB target genes in breast cancer patients with low (<30th percentile, n = 330) or high (>30th percentile, n = 332) CIN gene expression signature. f–g, Photon flux (p/s) of whole animals after intracardiac injection with MDA-MB-231 cells expressing control shRNA, STING shRNA (f), RelB shRNA (g) or NFKB2 shRNA (g), n = 9, 7, and 9 mice for the control, STING shRNA1, and STING shRNA2 groups, respectively (f), n = 22, 10, 10, 10, and 9 mice for the control, RelB shRNA1, RelB shRNA2, NFKB2 shRNA1, and NFKB2 shRNA2 groups, respectively (g). Bars represent mean ± SD (b, d), median ± interquartile range with bars spanning 10th–90th percentile (e), median (f–g), significance tested using two-sided t-test (b, d), two-sided Mann Whitney test (e–g), n = 4 (a–b), and 3 (c–d) independent experiments.