Replication stress links structural and numerical cancer chromosomal instability

Structural and numerical chromosomal instability are commonly observed together in solid tumours (Supplementary Fig. 1a-c)⁶. This co-occurrence can be experimentally induced via defective mitotic checkpoint function or chromosome attachment to the mitotic spindle, or through pre-mitotic defects affecting chromosome structure, such as faulty DNA repair and replication^6-10. However, the mechanisms underlying CIN in cancer remain unclear.

Colorectal cancers can be broadly classified as CIN+/aneuploid or CIN−/microsatellite unstable³. CIN+ cells displayed an increased frequency of chromosome segregation errors compared to CIN− cells (median = 38% versus 18%, p=0.0025, Mann-Whitney test, Supplementary Fig. 2a)¹¹. To address whether mitotic or pre-mitotic mechanisms are responsible for these segregation errors, we analysed high resolution images of anaphases in a panel of CIN+ CRC cells. The majority of segregation errors consisted of chromosome fragments without centromeres (acentrics, Figure 1a) and anaphase bridges (54-81%, median 70%, Figure 1a,b), indicative of structural chromosome aberrations arising through pre-mitotic defects¹². In contrast, only 10-43% of segregation errors were lagging chromosomes with centromeres (median 20%, Figure 1a,b), suggesting that mitotic dysfunction resulting in improper chromosome attachments⁶ cannot explain the majority of segregation errors in CIN+ CRC cells (Supplementary Fig. 2b). Furthermore, kinetochore distortion of lagging chromosomes (reflecting merotelic attachments⁶) was rare (0-12%, median 8% of segregation errors, Supplementary Fig. 2c). We did not observe differences in mitotic timing, mitotic checkpoint function, sister chromatid cohesion or supernumerary centrioles between CIN+ and CIN− cells, and multipolar spindles were infrequent (0-18%, median 8%, Supplementary Fig. 2d-i). These data suggest that mitotic dysfunction occurs at low frequency in CIN+ CRC cells and that the majority of observed chromosome segregation errors result from structural chromosome aberrations. Accordingly, 22-71% (median 36%) of CIN+ cell metaphases displayed structurally abnormal chromosomes, including acentric chromosomes, dicentric chromosomes and DNA double strand breaks (Figure 1b,c and Supplementary Fig. 2j).

Next we sought a putative cause for these structural chromosome alterations. Activation of the DNA damage response has been observed in both colorectal adenomas and carcinomas^13,14 and is thought to reflect DNA replication stress^13,15,16. Pharmacological induction of replication stress in HCT-116 (CIN−) cells resulted in structural chromosome aberrations and segregation errors, 82% of which were bridges or acentric chromosomes (Supplementary Fig. 3a-f). Importantly, numerical chromosome changes were also induced (Supplementary Fig. 3g,h), demonstrating that replication stress can result in both structural and numerical chromosomal instability¹⁷.

DNA replication stress results in a number of cellular phenotypes including DNA damage in prometaphase^10,15,16, ultra-fine anaphase DNA bridges (UFBs)¹⁶, and 53BP1-positive nuclear bodies in G1 cells^15,18 (Figure 1d and Supplementary Fig. 3i-m). CIN+ cells displayed elevated prometaphase DNA damage (median 74% CIN+ versus 34% CIN− cells with ≥3 γH2AX foci, Figure 1d,e (p=0.033, Mann-Whitney test)) in the absence of increased oxidative DNA damage (Supplementary Fig. 4a-c). γH2AX foci were not confined to telomeres (Supplementary Fig. 4d,e). CIN+ cells also displayed more 53BP1 nuclear bodies in G1 cells (Figure 1d,f, p=0.028, Mann-Whitney test). Consistent with the hypothesis that replication stress may drive chromosome segregation errors in CIN+ cells, UFBs were enriched in anaphases with segregation errors compared to anaphases without segregation errors (Figure 1d,g, p=0.00018, paired t-test).

To assess DNA replication directly, DNA fibre assays were performed for two CIN− and four CIN+ cell lines, to measure progression of individual replication forks, fork stalling and asymmetry between sister replication forks. CIN+ cells displayed significantly slower fork rates relative to CIN− cell lines (0.56-0.83 kb/min compared to 1.11-1.12 kb/min, p<0.05, Figure 1h,i, Supplementary Fig. 5a,b. Additionally, there was evidence of increased replication fork stalling and asymmetric sister fork progression in several CIN+ cell lines (Supplementary Fig. 5c-e), consistent with impaired replication fork progression. These data indicate that elevated replication stress, as demonstrated using established hallmarks and direct measurement of replication fork progression, may contribute substantially to chromosome missegregation in CIN+ CRC cells.

We sought a genetic basis for the elevated replication stress and chromosome segregation errors in CIN+ cells. Examining whole-exome sequencing data in colorectal tumours from The Cancer Genome Atlas (TCGA)¹⁹, and mutation status for 64 genes in CRC cell lines (COSMIC), revealed that the only gene mutated at significantly higher frequencies in CIN+ tumours or cell lines was TP53, which is also mutated in 13-33% of CIN− tumours and cell lines (Supplementary Fig. 6a,b). Mutations in TP53 are thought to be permissive for CIN rather than causative²⁰. These data suggest that whilst mutations in known oncogenes or tumour suppressors may contribute to replication stress in cancer cells^14,21, these are unlikely to exclusively account for the elevated replication stress in CIN+ CRC cells. We therefore addressed whether additional genetic aberrations may contribute to CIN.

We hypothesised that regions of consistent somatic copy number loss in CIN+ CRC might encode CIN-suppressor genes, loss of which could contribute to the induction of chromosome missegregation. To identify CIN-specific regions of loss, comparative genomic hybridisation data (CGH) was analysed for a cohort of 26 aneuploid colorectal tumours and 20 CIN+ cell lines. Chromosome 18q was most frequently subject to copy number loss, observed in 88% of aneuploid tumours and 80% of CIN+ cell lines, (Figure 2a,b and Supplementary Table 1) consistent with published studies in CRC and other tumour types^22-24. Copy number losses in CIN+ tumours and cell lines were highly concordant (p<0.001, Fisher’s exact test, Supplementary Table 2). Next we examined the temporal sequence and consequences of 18q copy number loss in tumours. In a cohort of 28 adenomas (preinvasive tumour) with carcinoma in the same specimen (Figure 2c), 18q loss of heterozygosity (LOH - indicative of copy number loss) was observed in 21/28 (75%) carcinomas, compared to 10/28 (35.7%) adenomas, implicating 18q loss in the adenoma-carcinoma transition. 18q LOH was significantly associated with aneuploidy in both adenomas and carcinomas (Figure 2d, Supplementary Fig. 6c).

To identify candidate CIN-suppressor genes encoded within regions of recurrent copy number loss, HCT-116 cells were transfected with pools of four siRNAs targeting the most frequently lost genes, present at ≤1 copy in at least 30% of CIN+ cell lines (94 genes encoded on chromosome 18q, Supplementary Table 3). After 48 hours anaphase segregation errors were quantified (Supplementary Fig. 7a). Importantly, this approach would identify both mitotic and pre-mitotic defects. SiRNA pools inducing segregation errors to ≥3 standard deviations above the frequency in control-transfected cells were assessed in validation assays. All sequences targeting PIGN, RKHD2 and ZNF516 induced segregation errors and efficiently depleted mRNA levels (Supplementary Fig. 7b-d). Additional independent siRNA sequences targeting each of the three genes also induced segregation errors (Supplementary Fig. 7e) and these genes were prioritised for further analysis, although we cannot exclude a contribution of other genes encoded on 18q to CIN. Of note, RKHD2 is the only protein-coding gene located between two genes implicated in CRC carcinogenesis, DCC and SMAD4. Copy number loss of PIGN, RKHD2 or ZNF516 is observed in 85% of the 20 CIN+ cell lines and 84% of aneuploid tumours (n=103) in the independent TCGA cohort¹⁹, with loss of all three genes in 70% of CIN+ cell lines and 79% of aneuploid tumours (Supplementary Table 4). Importantly, reduced copy number correlated significantly with reduced mRNA expression for all three genes both in the TCGA tumour cohort and CRC cell lines (Figure 2e, Supplementary Fig. 8a and Supplementary Table 5).

We next ensured off-target siRNA effects reported against MAD2 (mitotic checkpoint protein) and RAD51 (DNA repair protein), were not causing chromosome missegregation (Supplementary Fig. 8b-e)²⁵. Two RKHD2 sequences partially depleted MAD2 protein, and were excluded from further analysis. Additionally, expression of exogenous RKHD2-GFP rescued segregation errors induced upon silencing endogenous RKHD2 using a 3′ UTR-targeted siRNA, and expression of siRNA-resistant PIGN-GFP and ZNF516-GFP reduced segregation errors induced by depletion of endogenous PIGN or ZNF516 (Supplementary Fig. 8f). Silencing each CIN-suppressor also induced segregation errors in two additional CIN− CRC cell lines, DLD1 and RKO, and in 18q-normal CIN+ NCIH508 cells (Supplementary Fig. 9a-c). Induction of segregation errors was independent of oncogenic KRAS signalling in HCT-116 cells, as segregation errors were also induced following CIN-suppressor gene silencing in a KRAS wild-type isogenic cell line (Supplementary Fig. 9d).

CIN-suppressor gene silencing in HCT-116 cells primarily induced acentric chromosomes and anaphase bridges (Figure 3a) in the absence of gross mitotic defects (data not shown), similar to observations in CIN+ CRC cell lines with 18q loss (Figure 1b). This suggested a pre-mitotic origin for these chromosome segregation errors and accordingly, we observed an increased frequency of structurally abnormal chromosomes (Figure 3a,b, Supplementary Fig. 10a). To assess chromosome non-disjunction, HCT-116 cell lines stably expressing small-hairpin RNAs (shRNAs) were constructed. shRNA-mediated silencing of each CIN-suppressor gene increased segregation error frequency (Supplementary Fig. 10b), and single cell clones grown from each cell line displayed significantly increased intra-colony deviation for chromosomes 2 and 15 (Figure 3c,d and Supplementary Fig. 10c), indicating that CIN-suppressor gene silencing induces both structural and numerical instability.

We next examined replication stress following CIN-suppressor gene silencing. Elevated prometaphase DNA damage was observed (Figure 3e), and this increased concomitantly with, rather than after, the rise in segregation error frequency (Supplementary Fig. 10d), supporting the hypothesis that the observed DNA damage reflects a cause, rather than a consequence, of segregation errors. Silencing each CIN-suppressor gene also resulted in elevated 53BP1 bodies in G1 cells (Figure 3f), which were unaffected by cytokinesis-inhibition, and therefore unlikely to reflect cytokinesis-induced chromosome damage^7,15 (Supplementary Fig. 10e-g). Silencing PIGN and ZNF516 also significantly increased the frequency of UFBs (Figure 3g). Consistent with loss of CIN-suppressor genes contributing to replication stress in CIN+ cells, transient co-expression of PIGN, RKHD2 and ZNF516 resulted in a partial reduction in G1 53BP1 bodies in 3 CIN+ cell lines with 18q loss (Supplementary Fig. 11a-d). DNA fibre assays revealed a shift in distribution towards reduced replication fork speeds upon silencing each of the CIN-suppressor genes in HCT-116 cells, with reduced average fork rates after silencing PIGN and ZNF516 (Figure 3h,i and Supplementary Fig. 12a,b). Furthermore, we observed increased sister fork asymmetry after silencing each of the three genes (Supplementary Fig. 12c) consistent with impaired replication fork progression.

These data suggest that segregation errors resulting from CIN-suppressor gene silencing are driven by replication stress. To further test this hypothesis, HCT-116 cells transfected with siRNAs against CIN-suppressor genes were supplemented with nucleosides, previously shown to reduce replication-induced DNA damage⁵. Nucleosides significantly reduced the induction of segregation errors following silencing of PIGN (62% to 32%), RKHD2 (57% to 36%) and ZNF516 (43% to 34%) whilst control segregation errors were unaffected (Figure 4a). We then tested whether nucleoside supplementation could attenuate chromosome missegregation in CIN+ cell lines with 18q loss. Nucleoside supplementation significantly reduced segregation error frequency by 45-55% and attenuated prometaphase DNA damage by 28-43% in four CIN+ cell lines (Figure 4b,c, Supplementary Fig. 13a), indicating suppression of replication-induced DNA damage and subsequent chromosome missegregation. The extent of the nucleoside-mediated reduction in segregation errors implies that de novo generation of structural chromosome aberrations is responsible for a large proportion of chromosome missegregation events in CIN+ cells. In contrast, nucleoside supplementation did not affect segregation error frequency in four CIN− cell lines or 18q-normal CIN+ NCIH508 cells (Supplementary Fig. 13b,c). Nucleoside supplementation did not affect proliferation, cell cycle distribution, or cellular ATP levels (Supplementary Fig. 14a-f)⁵.

In conclusion, our findings implicate replication stress as a major driver of chromosomal instability in CRC. In addition to impaired replication fork progression, CIN+ cells exhibit replication stress-associated DNA damage and structurally abnormal chromosomes that missegregate during mitosis, linking structural and numerical instability. Supplementing CIN+ cells with nucleosides reduced both DNA damage and segregation errors, supporting a direct role for replication stress in driving CIN. Given the complex nature of replication stress and CIN, it is likely that there are multiple genetic aberrations contributing to these phenotypes within an individual tumour. Here, we suggest a putative genetic mechanism that may contribute to CIN in CRC, through the recurrent loss of a region on chromosome 18q, encoding three newly identified suppressors of replication stress and chromosome missegregation. The loss of chromosome 18q in many solid tumour types suggests the potential contribution of this locus to CIN in tumours beyond CRC. Furthermore, reports of DNA replication stress across multiple tumour types suggest replication stress may be a common route to CIN and intratumour heterogeneity^13,26. Efforts to target or restrain replication stress may therefore provide a rational approach to limit tumour diversity, genome evolution and adaptation.