Chromosomal instability drives metastasis through a cytosolic DNA response

In the paper

“Chromosomally unstable cells exhibit evidence of chromosome missegregation during anaphase3,4”

What the sources say

• Supported

  The Results and discussion section reports lagging chromatids during anaphase in chromosomally unstable cell lines, identifying them as the most common mitotic defect observed. It also reports more lagging chromatids per anaphase in unstable than in stable cells. Page numbers are not provided in our copy. (LLM, conf=0.94)

  cites [3] Thompson, S. L. & Compton, D. A. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–672 (2008) source
• Partly supported

  The Results section reports lagging chromosomes and occasional sister-chromatid missegregation during anaphase in PtK1 cells. It does not establish that the cells studied were chromosomally unstable, so the claim’s population qualifier is not supported by this paper. (LLM, conf=0.84)

  cites [4] Cimini, D. et al. Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J. Cell Biol. 153, 517–527 (2001) sourceExperimental — accuracy not yet measured

In the paper

“we overexpressed Kif2a, a third member of the kinesin-13 proteins that lacks kinetochore and centromere localization domains13, and observed no effect on CIN despite exhibiting microtubule-depolymerizing activity on interphase microtubules”

What the sources say

• Partly supported

  The citation is attached to the description of Kif2A, not to the citing authors’ own overexpression and CIN results. Section 3.1 identifies Kif2A as a kinesin-13 found mainly at spindle poles, but also reports its association with centromeres; the copy does not establish that it lacks kinetochore and centromere localization domains. The cited review does not report the stated overexpression experiment. (LLM, conf=0.75)

  cites [13] Ems-McClung, S. C. & Walczak, C. E. Kinesin-13s in mitosis: key players in the spatial and temporal organization of spindle microtubules. Semin. Cell Dev. Biol. 21, 276–282 (2010) sourceExperimental — accuracy not yet measured

In the paper

“Destabilization of microtubule attachments to chromosomes at the kinetochores, through overexpression of the non-motile microtubule depolymerizing kinesin-13 proteins, Kif2b or MCAK/Kif2c, directly suppresses CIN in otherwise chromosomally unstable cells5–7.”

What the sources say

• Partly supported

  In the main text (page unavailable), GFP–Kif2b overexpression reduced karyotypic heterogeneity in two CIN+ cell lines, supporting suppression of CIN. The paper also reports earlier results for kinesin-13 overexpression in two other CIN+ lines, but our copy does not identify MCAK/Kif2c or establish its effect separately. (LLM, conf=0.88)

  cites [5] Bakhoum, S. F. et al. The mitotic origin of chromosomal instability. Curr. Biol. 24, R148–R149 (2014) sourceExperimental — accuracy not yet measured
• Supported

  In Results, “Extra-nuclear DNA damage in irradiated mitotic cells” (page unavailable), the paper reports that Kif2b overexpression reduced chromosome segregation errors in chromosomally unstable U251 cells by destabilizing kinetochore–microtubule attachments. In Results, “w-CIN influences viability of irradiated mitotic cells” (page unavailable), it also identifies MCAK overexpression as suppressing whole-chromosome CIN, albeit in a slightly different manner. (LLM, conf=0.92)

  cites [6] Bakhoum, S. F. et al. Numerical chromosomal instability mediates susceptibility to radiation treatment. Nat. Commun. 6, 5990 (2015) source
• Supported

  The ‘Suppression of chromosomal instability’ section reports that GFP-Kif2b or GFP-MCAK overexpression reduces chromosome mis-segregation and suppresses CIN in the unstable U2OS and MCF-7 cell lines. It attributes this effect to increased kinetochore–microtubule turnover that releases inappropriate attachments. (LLM, conf=0.95)

  cites [7] Bakhoum, S. F., Thompson, S. L., Manning, A. L. & Compton, D. A. Genome stability is ensured by temporal control of kinetochore–microtubule dynamics. Nat. Cell Biol. 11, 27–35 (2009) source

In the paper

“whereas overexpression of a dominant negative MCAK mutant12 (dnMCAK) led to a modest increase in chromosome missegregation in MDA-MB-231 cells.”

What the sources say

• Supported

  The citation is attached to the MCAK mutant, rather than to the citing paper’s result in MDA-MB-231 cells. In Results and discussion (Fig. 2; page not given), the source identifies and uses GFP-hypir-MCAK, a motor-domain mutant inactive in microtubule depolymerization. It does not report the chromosome-missegregation result. (LLM, conf=0.86)

  cites [12] Moore, A. T. et al. MCAK associates with the tips of polymerizing microtubules. J. Cell Biol. 169, 391–397 (2005) source

In the paper

“Cells overexpressing Kif2b or MCAK continue to propagate abnormal aneuploid karyotypes albeit in a stable manner7.”

What the sources say

• Supported

  The ‘Suppression of chromosomal instability’ section reports that U2OS clones overexpressing GFP-Kif2b or GFP-MCAK retained their aneuploid modal chromosome numbers while showing reduced deviations from those numbers. Those reductions persisted for 50 generations, supporting stable propagation of abnormal karyotypes. (LLM, conf=0.93)

  cites [7] Bakhoum, S. F., Thompson, S. L., Manning, A. L. & Compton, D. A. Genome stability is ensured by temporal control of kinetochore–microtubule dynamics. Nat. Cell Biol. 11, 27–35 (2009) source

In the paper

“Chromosomal instability (CIN) correlates with tumor metastasis1,2”

What the sources say

• Partly supported

  The Results passage (page not available in our copy) links a high proportion of subclonal copy-number alterations to a higher risk of recurrence or death in patients with non–small-cell lung cancer. The Discussion describes copy-number heterogeneity in relation to chromosomal instability, but neither passage establishes a correlation with metastasis specifically. (LLM, conf=0.84)

  cites [1] Jamal-Hanjani, M. et al. Tracking the evolution of non-small-cell lung cancer. N. Engl. J. Med. 376, 2109–2121 (2017) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] Turajlic, S. & Swanton, C. Metastasis as an evolutionary process. Science 352, 169–175 (2016) source

In the paper

“We found a striking localization of cGAS to approximately half of all micronuclei as previously observed22,23.”

What the sources say

• Partly supported

  The section labeled Abstract (page not provided) reports that cGAS localizes to micronuclei, and Extended Data Figure 4 describes measuring cGAS-positive micronuclei. However, the passages do not report that approximately half of all micronuclei are cGAS-positive. The reported majority concerns micronucleated daughter cells in a live-imaging experiment, not all micronuclei. (LLM, conf=0.85)

  cites [22] Harding, S. M. et al. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466–470 (2017) sourceExperimental — accuracy not yet measured
• Partly supported

  The paper reports that cGAS localizes to micronuclei, but its quantified result is 83.3 ± 1.4% GFP-cGAS-positive micronuclei in RNase H2-deficient mouse fibroblasts (Abstract, Fig. 1d), not approximately half. It also describes localization in other settings without establishing a proportion for all micronuclei. (LLM, conf=0.88)

  cites [23] Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017) sourceExperimental — accuracy not yet measured

In the paper

“Impeding micronuclear rupture through Lamin B2 overexpression20 significantly diminished the relative fraction of cGAS+ micronuclei (Fig. 6a–b).”

What the sources say

• Supported

  The Results section (“NE collapse is triggered by lamin disorganization”) reports significantly more intact micronuclei after Lamin B2 overexpression and concludes that increased lamin B can prevent micronuclear disruption. Citation 20 is attached to that intervention; the decrease in cGAS-positive micronuclei is presented as the citing paper’s own Figure 6a–b result. (LLM, conf=0.92)

  cites [20] Hatch, E. M., Fischer, A. H., Deerinck, T. J. & Hetzer, M. W. Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell 154, 47–60 (2013) source

In the paper

“The top 23 differentially expressed genes in CIN-high cells (referred to as CIN signature) predicted distant-metastasis-free survival (DMFS) in a meta-analysis16 as well as a validation cohort17 of patients with breast cancer, irrespective of tumor subtype, grade, or lymph node status”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  cites [16] Györffy, B. et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res. Treat. 123, 725–731 (2010) sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Methods describe predictors using 33 and 27 probe sets for early relapse, not a 23-gene CIN signature. The Results report distant-relapse-free survival in an independent validation cohort, but our copy does not say that the CIN signature was tested there or that it predicted survival irrespective of subtype, grade, or lymph-node status. Searches for the CIN signature and those validation claims found no supporting passage; page numbers were unavailable. (LLM, conf=0.76)

  cites [17] Hatzis, C. et al. A genomic predictor of response and survival following taxane–anthracycline chemotherapy for invasive breast cancer. J. Am. Med. Assoc. 305, 1873–1881 (2011) sourceExperimental — accuracy not yet measured

In the paper

“These two mutually exclusive states, which were recently observed in a pan-cancer genomic analysis of metastatic tumors41, likely account for the reversibility in chromosome missegregation rates seen in primary tumors and metastases”

What the sources say

• Supported

  The citation is attached to the observation of two mutually exclusive states, not to the citing authors’ proposed explanation for reversible chromosome missegregation rates. In “Transcriptional signatures of metastatic disease,” the source identifies two metastatic-tumor subtypes and reports mutual exclusivity between their proliferative and EMT gene sets. (LLM, conf=0.93)

  cites [41] Robinson, D. R. et al. Integrative clinical genomics of metastatic cancer. Nature 548, 297–303 (2017) source

In the paper

“The emergence, and subsequent tolerance, of CIN represents an important bottleneck during tumor evolution38–40.”

What the sources say

• Partly supported

  The Results (no page supplied) show that chromosome gains and transiently induced karyotypic abnormalities impair proliferation and transformation, consistent with a barrier to tumor development. But the authors explicitly avoided continuous CIN in those experiments; the paper does not establish that the emergence and subsequent tolerance of CIN itself is a bottleneck during tumor evolution. (LLM, conf=0.86)

  cites [38] Sheltzer, J. M. et al. Single-chromosome gains commonly function as tumor suppressors. Cancer Cell 31, 240–255 (2017) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [39] Weaver, B. A. A., Silk, A. D., Montagna, C., Verdier-Pinard, P. & Cleveland, D. W. Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 11, 25–36 (2007) source
• Supported

  The Discussion describes early Mad2-induced instability as detrimental: many cells die, while a small fraction overcomes arrest and Mad2 expression persists in tumors. It explicitly describes the mouse models as combining chromosome missegregation with negative selection and tolerance, supporting the claimed bottleneck in tumor evolution. (LLM, conf=0.88)

  cites [40] Rowald, K. et al. Negative selection and chromosome instability induced by Mad2 overexpression delay breast cancer but facilitate oncogene-independent outgrowth. Cell Reports 15, 2679–2691 (2016) source

In the paper

“It also leads us to suggest that CIN drives the subset of human metastases characterized by EMT and inflammation41.”

What the sources say

• Supported

  The ‘Transcriptional signatures of metastatic disease’ section identifies an EMT-like subtype of metastatic tumors associated with inflammation signatures. The statement that CIN drives this subtype is framed as the citing authors’ own suggestion, not a finding attributed to the source. (LLM, conf=0.91)

  cites [41] Robinson, D. R. et al. Integrative clinical genomics of metastatic cancer. Nature 548, 297–303 (2017) source

In the paper

“Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol.”

In the paper

“The number of chromosomal aberrations was highest in tumor samples with karyotypes ranging between the diploid and tetraploid (4n) range (Fig. 1b–c and Extended Data Fig. 1c–d).”

In the paper

“and had twice as many structural or numerical chromosomal aberrations per clone.”

In the paper

“Overexpression of Kif2b or MCAK suppressed chromosome missegregation”

In the paper

“histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma11 revealed a significant association between anaphase chromosome missegregation and the incidence of lymph node metastasis”

What the sources say

• ?Not checked — no full text of the sourcecites [11] Chung, C. H. et al. Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression. Cancer Cell 5, 489–500 (2004) source

In the paper

“Metastases exhibited increased wGII compared to primary tumors (Fig. 1a, Extended Data Fig. 1a–b).”

In the paper

“karyotype analysis of primary breast tumors and metastases archived in the Mitelman Database of chromosomal translocations10 revealed a predilection for near-diploid (2n) karyotypes in primary tumors.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [10] Mitelman, F., Johansson, B. & Mertens, F. Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancerhttps://cgap.nci.nih.gov/Chromosomes/Mitelman (2017)

In the paper

“Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models”

In the paper

“Conversely, metastases were enriched for cells with near-triploid (3n) karyotypes”

In the paper

“whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner.”

In the paper

“Furthermore, the ability of Kif2b or MCAK overexpression to suppress invasion in vitro was dependent on the cell cycle, as the addition of thymidine after transient transfection of either proteins abrogated this phenotype”

In the paper

“RNA-seq of primary tumor-derived and metastasis-derived cells revealed pathways that were shared among metastases and CIN-high cells.”

In the paper

“Similarly, RNA-seq data from primary breast cancer in the TCGA database demonstrated increased expression of CIN-responsive NC-NF-κB genes in tumors with higher CIN signature genes (Fig. 6e)”

In the paper

“Unsupervised graph-based clustering, based on all expressed genes, identified 12 phenotypically distinct subpopulations.”

In the paper

“Bulk RNA sequencing (RNA-seq) of CIN-low and CIN-high MDA-MB-231 cells revealed 1,584 differentially expressed genes.”

In the paper

“There was a strong positive correlation between inflammation-related, CIN signature, and EMT genes in the scRNA-seq and bulk RNA-seq data”

In the paper

“One subpopulation was defined by increased expression of genes involved in EMT and metastasis (referred to as subpopulation ‘M’) and was concomitantly enriched for CIN signature genes.”

In the paper

“This subpopulation comprised 45% of dnMCAK expressing cells compared to 6% of the CIN-low cells (Fig. 4b and Extended Data Fig. 6a–b).”

In the paper

“Clustering of single cells using EMT genes successfully classified most cells based on their CIN-status”

In the paper

“Quantification of dsDNA levels after subcellular fractionation revealed a four-fold reduction in cytosolic DNA in CIN-low compared to CIN-high cells (Fig. 5g).”

In the paper

“Accordingly, it reduced metastasis after intracardiac or tail vein injections (Fig. 5i and Extended Data Fig. 3g–h).”

In the paper

“Furthermore, CIN-high cells exhibited increased levels and perinuclear localization of STING congruent with pathway activation (Fig. 6c).”

In the paper

“mice harboring CIN-high cells rapidly succumbed to metastatic disease with a median survival of 70 days.”

In the paper

“whereas cells derived from most primary tumors had significantly lower rates of CIN (Fig. 3b–d).”

In the paper

“Conversely, the addition of cGAMP increased invasion and migration of CIN-low cells (Fig. 6f–g, Extended Data Figs. 6f, 9j, and 10b-d).”

In the paper

“and higher expression of key regulators of the noncanonical NF-κB pathway or its CIN responsive target genes was associated with shorter DMFS and disease-free survival in breast and lung cancers.”

In the paper

“Whole-genome sequencing of subcellular fractions at 30x coverage confirmed the genomic origin of cytosolic DNA (not shown).”

In the paper

“Similar results were obtained after injection of lung adenocarcinoma H2030 cells (Fig. 2c–e and Extended Data Fig. 3b–c).”

In the paper

“Many metastases from CIN-low cells waxed-and-waned and, at times, spontaneously resolved”

In the paper

“In line with the scRNA-seq data, CIN-high cells exhibited increased migratory and invasive behavior in vitro”

In the paper

“Whole-chromosome FISH probes targeting the Y chromosome or an independent autosome (chr15) revealed selective incorporation of the Y chromosome into micronuclei two days following chromosome missegregation induced by doxycycline and auxin (Dox/IAA) treatment.”

In the paper

“Bulk RNA-seq data revealed a number of noncanonical NF-κB target genes that were upregulated in response to CIN (CIN-responsive NC-NF-κB genes).”

In the paper

“and revealed a fraction of cells (primarily CIN-high) that was highly enriched in mesenchymal markers (Fig. 4a).”

In the paper

“Suppression of CIN reduced both numerical and structural karyotypic heterogeneity in single-cell derived clones as supported by the presence of fewer chromosomes exhibiting non-clonal structural abnormalities and decreased numerical chromosome heterogeneity in CIN-low cells”

In the paper

“Metastasis-related and epithelial-to-mesenchymal (EMT) gene sets were relatively enriched in CIN-high cells.”

In the paper

“Regardless of the CIN status of the injected cells, the majority of metastases enriched for higher rates of chromosome missegregation”

In the paper

“Expectedly, Mad2 overexpression rescued invasion and migration of MCAK expressing cells.”

In the paper

“Interestingly, there was no evidence for robust activation of downstream canonical NF-κB or type I interferon signaling in CIN-high cells as evidenced by the lack of a significant increase in p65 or IRF3 phosphorylation, absence of p65 or IRF3 nuclear translocation, undetectable levels of interferon-β, and failure to induce ISGs”

In the paper

“we assessed the nuclear localization of RelB, the binding partner of p52, and observed increased nuclear localization in CIN-high cells.”

In the paper

“when CIN-high cells (Fig. 3d, dnMCAK, blue bars) were injected in the mammary fat pad, chromosome missegregation rates decreased in the primary tumors (green bars) before increasing once more in metastases spontaneously arising within the same animal (orange bars).”

In the paper

“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.”

In the paper

“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)”

In the paper

“Metastasis can be detected in the mouse transplanted with dnMCAK expressing cells at day 90.”

In the paper

“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.”

In the paper

“By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumor cells co-opt chronic activation of innate immune pathways to spread to distant organs.”

In the paper

“Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA.”

In the paper

“Thus chromosome 1 loss is a recurrent event during primary tumor growth in this model (Extended Data Figs. 4c–f and 5a–e).”

In the paper

“We found that CIN induces a transcriptional shift from a proliferative and highly metabolic state, ideally suited for primary tumor growth, to a mesenchymal state associated with upregulation of inflammatory pathways”

In the paper

“We found that STING and downstream noncanonical NF-κB activity are mediators of metastasis in a tumor cell-autonomous fashion”

In the paper

“Effect of cytosolic DNA sensing pathways on prognosis”

In the paper

“Prognostic impact of the CIN signature”

In the paper

“Alternative response to cytosolic DNA in cancer cells”

In the paper

“Transcriptional consequences of CIN in cancer cells”