The genomic landscape of 2,023 colorectal cancers.

Colorectal carcinoma (CRC) is a common cause of mortality1, but a comprehensive description of its genomic landscape is lacking2-9. Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichia colipks+ colibactin in rectal cancers10 and the importance of the SBS93 signature11-13, which suggests that diet or smoking is a risk factor. Immune-escape driver mutations14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1), highlighting the role of whole-genome sequencing in optimizing patient care.

Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics.

Centrosomal Protein 55 (CEP55) exhibits various oncogenic activities; it regulates the PI3K-Akt-pathway, midbody abscission, and chromosomal instability (CIN) in cancer cells. Here, we analyzed the mechanism of how CEP55 controls CIN in ovarian and breast cancer (OvCa) cells. Down-regulation of CEP55 reduced CIN in all cell lines analyzed, and CEP55 depletion decreased spindle microtubule (MT)-stability in OvCa cells. Moreover, recombinant CEP55 accelerated MT-polymerization and attenuated cold-induced MT-depolymerization. To analyze a potential relationship between CEP55-controlled CIN and its impact on MT-stability, we identified the CEP55 MT-binding peptides inside the CEP55 protein. Thereafter, a mutant with deficient MT-binding activity was re-expressed in CEP55-depleted OvCa cells and we could show that this mutant did not restore reduced CIN in CEP55-depleted cells. This finding strongly indicates that CEP55 regulates CIN by controlling MT dynamics.

Contribution of AurkA/TPX2 Overexpression to Chromosomal Imbalances and Cancer.

The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.

Inference of chromosome selection parameters and missegregation rate in cancer from DNA-sequencing data.

Aneuploidy is frequently observed in cancers and has been linked to poor patient outcome. Analysis of aneuploidy in DNA-sequencing (DNA-seq) data necessitates untangling the effects of the Copy Number Aberration (CNA) occurrence rates and the selection coefficients that act upon the resulting karyotypes. We introduce a parameter inference algorithm that takes advantage of both bulk and single-cell DNA-seq cohorts. The method is based on Approximate Bayesian Computation (ABC) and utilizes CINner, our recently introduced simulation algorithm of chromosomal instability in cancer. We examine three groups of statistics to summarize the data in the ABC routine: (A) Copy Number-based measures, (B) phylogeny tip statistics, and (C) phylogeny balance indices. Using these statistics, our method can recover both the CNA probabilities and selection parameters from ground truth data, and performs well even for data cohorts of relatively small sizes. We find that only statistics in groups A and C are well-suited for identifying CNA probabilities, and only group A carries the signals for estimating selection parameters. Moreover, the low number of CNA events at large scale compared to cell counts in single-cell samples means that statistics in group B cannot be estimated accurately using phylogeny reconstruction algorithms at the chromosome level. As data from both bulk and single-cell DNA-sequencing techniques becomes increasingly available, our inference framework promises to facilitate the analysis of distinct cancer types, differentiation between selection and neutral drift, and prediction of cancer clonal dynamics.

YY2/BUB3 Axis promotes SAC Hyperactivation and Inhibits Colorectal Cancer Progression via Regulating Chromosomal Instability.

Spindle assembly checkpoint (SAC) is a crucial safeguard mechanism of mitosis fidelity that ensures equal division of duplicated chromosomes to the two progeny cells. Impaired SAC can lead to chromosomal instability (CIN), a well-recognized hallmark of cancer that facilitates tumor progression; paradoxically, high CIN levels are associated with better therapeutic response and prognosis. However, the mechanism by which CIN determines tumor cell survival and therapeutic response remains poorly understood. Here, using a cross-omics approach, YY2 is identified as a mitotic regulator that promotes SAC activity by activating the transcription of budding uninhibited by benzimidazole 3 (BUB3), a component of SAC. While both conditions induce CIN, a defect in YY2/SAC activity enhances mitosis and tumor growth. Meanwhile, hyperactivation of SAC mediated by YY2/BUB3 triggers a delay in mitosis and suppresses growth. Furthermore, it is revealed that YY2/BUB3-mediated excessive CIN causes higher cell death rates and drug sensitivity, whereas residual tumor cells that survived DNA damage-based therapy have moderate CIN and increased drug resistance. These results provide insights into the role of SAC activity and CIN levels in influencing tumor cell survival and drug response, as well as suggest a novel anti-tumor therapeutic strategy that combines SAC activity modulators and DNA-damage agents.

A survey of chromosomal instability measures across mechanistic models.

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway.

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.

The two sides of chromosomal instability: drivers and brakes in cancer.

Chromosomal instability (CIN) is a hallmark of cancer and is associated with tumor cell malignancy. CIN triggers a chain reaction in cells leading to chromosomal abnormalities, including deviations from the normal chromosome number or structural changes in chromosomes. CIN arises from errors in DNA replication and chromosome segregation during cell division, leading to the formation of cells with abnormal number and/or structure of chromosomes. Errors in DNA replication result from abnormal replication licensing as well as replication stress, such as double-strand breaks and stalled replication forks; meanwhile, errors in chromosome segregation stem from defects in chromosome segregation machinery, including centrosome amplification, erroneous microtubule-kinetochore attachments, spindle assembly checkpoint, or defective sister chromatids cohesion. In normal cells, CIN is deleterious and is associated with DNA damage, proteotoxic stress, metabolic alteration, cell cycle arrest, and senescence. Paradoxically, despite these negative consequences, CIN is one of the hallmarks of cancer found in over 90% of solid tumors and in blood cancers. Furthermore, CIN could endow tumors with enhanced adaptation capabilities due to increased intratumor heterogeneity, thereby facilitating adaptive resistance to therapies; however, excessive CIN could induce tumor cells death, leading to the "just-right" model for CIN in tumors. Elucidating the complex nature of CIN is crucial for understanding the dynamics of tumorigenesis and for developing effective anti-tumor treatments. This review provides an overview of causes and consequences of CIN, as well as the paradox of CIN, a phenomenon that continues to perplex researchers. Finally, this review explores the potential of CIN-based anti-tumor therapy.

Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response.

Inactivating TP53 mutations leads to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promotes tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome and find that by targeting minichromosome maintenance complex components (MCMs), GOF mutp53 predisposes cells to replication stress and chromosomal instability (CIN), leading to a tumor cell-autonomous and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Consequently, GOF mutp53-MCMs-CIN-cytosolic DNA-cGAS-STING-NC-NF-κB signaling promotes tumor cell metastasis and an immunosuppressive tumor microenvironment through antagonizing interferon signaling and regulating genes associated with pro-tumorigenic inflammation. Our findings have important implications for understanding not only the GOF activities of TP53 mutations but also the genome-guardian role of p53 and its inactivation during tumor development and progression.

Characterizing chromosomal instability-driven cancer evolution and cell fitness at a glance.

Chromosomal instability (CIN), an increased rate of chromosome segregation errors during mitosis, is a hallmark of cancer cells. CIN leads to karyotype differences between cells and thus large-scale heterogeneity among individual cancer cells; therefore, it plays an important role in cancer evolution. Studying CIN and its consequences is technically challenging, but various technologies have been developed to track karyotype dynamics during tumorigenesis, trace clonal lineages and link genomic changes to cancer phenotypes at single-cell resolution. These methods provide valuable insight not only into the role of CIN in cancer progression, but also into cancer cell fitness. In this Cell Science at a Glance article and the accompanying poster, we discuss the relationship between CIN, cancer cell fitness and evolution, and highlight techniques that can be used to study the relationship between these factors. To that end, we explore methods of assessing cancer cell fitness, particularly for chromosomally unstable cancer.

Chromosomal Instability-Driven Cancer Progression: Interplay with the Tumour Microenvironment and Therapeutic Strategies.

Chromosomal instability (CIN) is a prevalent characteristic of solid tumours and haematological malignancies. CIN results in an increased frequency of chromosome mis-segregation events, thus yielding numerical and structural copy number alterations, a state also known as aneuploidy. CIN is associated with increased chances of tumour recurrence, metastasis, and acquisition of resistance to therapeutic interventions, and this is a dismal prognosis. In this review, we delve into the interplay between CIN and cancer, with a focus on its impact on the tumour microenvironment-a driving force behind metastasis. We discuss the potential therapeutic avenues that have resulted from these insights and underscore their crucial role in shaping innovative strategies for cancer treatment.

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Insights into common fragile site instability: DNA replication challenges at DNA repeat sequences.

Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.

Genomic signatures of past and present chromosomal instability in Barrett's esophagus and early esophageal adenocarcinoma.

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

Chromosome instability region analysis and identification of the driver genes of the epithelial ovarian cancer cell lines A2780 and SKOV3.

Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers worldwide. The molecular mechanisms of serous ovarian cancer (SOC) remain unclear and not well understood. SOC cases are primarily diagnosed at the late stage, resulting in a poor prognosis. Advances in molecular biology techniques allow us to obtain a better understanding of precise molecular mechanisms and to identify the chromosome instability region and key driver genes in the carcinogenesis and progression of SOC. Whole-exome sequencing was performed on the normal ovarian cell line IOSE80 and the EOC cell lines SKOV3 and A2780. The single-nucleotide variation burden, distribution, frequency and signature followed the known ovarian mutation profiles, without chromosomal bias. Recurrently mutated ovarian cancer driver genes, including LRP1B, KMT2A, ARID1A, KMT2C and ATRX were also found in two cell lines. The genome distribution of copy number alterations was found by copy number variation (CNV) analysis, including amplification of 17q12 and 4p16.1 and deletion of 10q23.33. The CNVs of MED1, GRB7 and MIEN1 located at 17q12 were found to be correlated with the overall survival of SOC patients (MED1: p = 0.028, GRB7: p = 0.0048, MIEN1: p = 0.0051), and the expression of the three driver genes in the ovarian cell line IOSE80 and EOC cell lines SKOV3 and A2780 was confirmed by western blot and cell immunohistochemistry.

CD30 induces Reed-Sternberg cell-like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines.

Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.