Zuzana Storchova

From polyploidy to aneuploidy, genome instability and cancer

Polyploidy is a frequent phenomenon in the eukaryotic world, but the biological properties of polyploid cells are not well understood. During evolution, polyploidy is thought to be an important mechanism that contributes to speciation. Polyploid, usually non-dividing, cells are formed during development in otherwise diploid organisms. A growing amount of evidence indicates that polyploid cells also arise during a variety of pathological conditions. Genetic instability in these cells might provide a route to aneuploidy and thereby contribute to the development of cancer.

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described ‘ploidy-specific lethality’, where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.

The consequences of tetraploidy and aneuploidy.

Polyploidy, an increased number of chromosome sets, is a surprisingly common phenomenon in nature, particularly in plants and fungi. In humans, polyploidy often occurs in specific tissues as part of terminal differentiation. Changes in ploidy can also result from pathophysiological events that are caused by viral-induced cell fusion or erroneous cell division. Tetraploidization can initiate chromosomal instability (CIN), probably owing to supernumerary centrosomes and the doubled chromosome mass. CIN, in turn, might persist or soon give way to a stably propagating but aneuploid karyotype. Both CIN and stable aneuploidy are commonly observed in cancers. Recently, it has been proposed that an increased number of chromosome sets can promote cell transformation and give rise to an aneuploid tumor. Here, we review how tetraploidy can occur and describe the cellular responses to increased ploidy. Furthermore, we discuss how the specific physiological changes that are triggered by polyploidization might be used as novel targets for cancer therapy.

Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells.

Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62‐dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome.

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

Uncrossing covalently linked DNA strands DNA interstrand cross-links (ICLs) covalently link the two strands of the double helix. ICL mutations are difficult to repair, because the two DNA strands cannot be separated and so one strand cannot be used as a template to repair the other. Räschle et al. developed a mass spectrometry–based method to systematically analyze a time series of all the proteins recruited to repair ICLs in Xenopus egg extracts. They found many of the known factors required for ICL repair. They also found a number of new factors, two of which define a new repair pathway for ICL mutations. Science, this issue 10.1126/science.1253671 Surveying the battery of proteins required to repair covalently linked DNA strands reveals a new repair pathway. INTRODUCTION DNA damage encountered during DNA replication represents a major challenge to the integrity of the genome. Because replicative polymerases are unable to synthesize across DNA lesions, prolonged stalling of replisomes can lead to replication fork collapse, giving rise to gross genomic alterations. Cells have evolved intricate responses that orchestrate the reorganization of the replication fork necessary for overcoming such roadblocks, but the full set of factors involved in these processes has not been defined. Here, we performed unbiased proteomic analyses of the dynamically changing protein landscape at damaged chromatin undergoing DNA replication. This yielded mechanistic insights into the pathways that ensure genomic stability during perturbed DNA replication. RATIONALE We combined the powerful and well-established Xenopus egg extract system for cell-free DNA replication with quantitative mass spectrometry to develop CHROMASS (chromatin mass spectrometry), a simple yet robust method for the unbiased analysis of chromatin composition. Using bifunctional cross-linkers, compounds commonly applied in chemotherapy, we systematically monitored the assembly and disassembly of protein complexes on replicating chromatin containing DNA interstrand cross-links (ICLs). RESULTS We show that replication of ICL-containing chromatin templates triggers recruitment of more than 90 DNA repair and genome maintenance factors. Addition of replication inhibitors revealed the subset of proteins that accumulate in a strictly replication-dependent fashion. The quantitative readout by CHROMASS is highly lesion-specific, as the known repair factors enriched on psoralen–cross-linked templates had previously been linked to ICL repair or specific branches of DNA damage signaling. In contrast, virtually none of the proteins involved in unrelated DNA repair pathways (e.g., base excision repair or nonhomologous end joining) showed damage-specific enrichment. The temporal profiles of hundreds of proteins across an extensive time course and a variety of perturbations provided a data-rich resource that could be mined to identify previously unknown genome maintenance factors. Among such hits, we identified SLF1 and SLF2 and showed that they physically link RAD18 with the SMC5/6 complex. This defines a linear RAD18-SLF1-SLF2 recruitment pathway for the SMC5/6 complex to RNF8/RNF168-generated ubiquitylations at damaged DNA in vertebrate cells. We found that SLF2 is a distant ortholog of yeast NSE6, an SMC5/6-associated factor that is essential for targeting this complex to damaged DNA to promote faithful repair of the lesions. Consistent with pivotal functions of SMC5/6 in the suppression of replication stress-induced, illegitimate recombination intermediates, depletion of SLF1 or SLF2 led to mitotic errors and compromised cell survival in response to genotoxic agents. CONCLUSIONS CHROMASS enables rapid and unbiased time-resolved insights into the chromatin interaction dynamics of entire DNA repair pathways. Combined with specific perturbations, CHROMASS allows systems-level interrogation of the consequences of inactivating particular aspects of the repair process. We compiled comprehensive proteome-wide profiles of dynamic protein interactions with damaged chromatin. These can be mined to pinpoint genome stability maintenance factors, exemplified here by the identification of SLF1 and SLF2, which define a recruitment pathway for the SMC5/6 complex. CHROMASS can be applied to other chromatin-associated pathways and may also shed light on the dynamics of posttranslational modifications governing the regulation of these processes. CHROMASS analysis of proteins recruited to stalled replication forks reveals a specific set of DNA repair factors involved in the replication stress response. Among these, SLF1 and SLF2 are found to bridge the SMC5/6 complex to RAD18, thereby linking SMC5/6 recruitment to ubiquitylation products formed at various DNA lesions. DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

The presence of extra chromosomes leads to genomic instability

Aneuploidy is a hallmark of cancer and underlies genetic disorders characterized by severe developmental defects, yet the molecular mechanisms explaining its effects on cellular physiology remain elusive. Here we show, using a series of human cells with defined aneuploid karyotypes, that gain of a single chromosome increases genomic instability. Next-generation sequencing and SNP-array analysis reveal accumulation of chromosomal rearrangements in aneuploids, with break point junction patterns suggestive of replication defects. Trisomic and tetrasomic cells also show increased DNA damage and sensitivity to replication stress. Strikingly, we find that aneuploidy-induced genomic instability can be explained by the reduced expression of the replicative helicase MCM2-7. Accordingly, restoring near-wild-type levels of chromatin-bound MCM helicase partly rescues the genomic instability phenotypes. Thus, gain of chromosomes triggers replication stress, thereby promoting genomic instability and possibly contributing to tumorigenesis.

Unique features of the transcriptional response to model aneuploidy in human cells

BackgroundAneuploidy, a karyotype deviating from multiples of a haploid chromosome set, affects the physiology of eukaryotes. In humans, aneuploidy is linked to pathological defects such as developmental abnormalities, mental retardation or cancer, but the underlying mechanisms remain elusive. There are many different types and origins of aneuploidy, but whether there is a uniform cellular response to aneuploidy in human cells has not been addressed so far.ResultsHere we evaluate the transcription profiles of eleven trisomic and tetrasomic cell lines and two cell lines with complex aneuploid karyotypes. We identify a characteristic aneuploidy response pattern defined by upregulation of genes linked to endoplasmic reticulum, Golgi apparatus and lysosomes, and downregulation of DNA replication, transcription as well as ribosomes. Strikingly, complex aneuploidy elicits the same transcriptional changes as trisomy. To uncover the triggers of the response, we compared the profiles with transcription changes in human cells subjected to stress conditions. Interestingly, we found an overlap only with the response to treatment with the autophagy inhibitor bafilomycin A1. Finally, we identified 23 genes whose expression is significantly altered in all aneuploids and which may thus serve as aneuploidy markers.ConclusionsOur analysis shows that despite the variability in chromosome content, aneuploidy triggers uniform transcriptional response in human cells. A common response independent of the type of aneuploidy might be exploited as a novel target for cancer therapy. Moreover, the potential aneuploidy markers identified in our analysis might represent novel biomarkers to assess the malignant potential of a tumor.

HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells

Aneuploidy is a hallmark of cancer and is associated with malignancy and poor prognosis. Recent studies have revealed that aneuploidy inhibits proliferation, causes distinct alterations in the transcriptome and proteome and disturbs cellular proteostasis. However, the molecular mechanisms underlying the changes in gene expression and the impairment of proteostasis are not understood. Here, we report that human aneuploid cells are impaired in HSP90‐mediated protein folding. We show that aneuploidy impairs induction of the heat shock response suggesting that the activity of the transcription factor heat shock factor 1 (HSF1) is compromised. Indeed, increased levels of HSF1 counteract the effects of aneuploidy on HSP90 expression and protein folding, identifying HSF1 overexpression as the first aneuploidy‐tolerating mutation in human cells. Thus, impaired HSF1 activity emerges as a critical factor underlying the phenotypes linked to aneuploidy. Finally, we demonstrate that deficient protein folding capacity directly shapes gene expression in aneuploid cells. Our study provides mechanistic insight into the causes of the disturbed proteostasis in aneuploids and deepens our understanding of the role of HSF1 in cytoprotection and carcinogenesis.

Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells

Up to 80% of human cancers, in particular solid tumors, contain cells with abnormal chromosomal numbers, or aneuploidy, which is often linked with marked chromosomal instability. Whereas in some tumors the aneuploidy occurs by missegregation of one or a few chromosomes, aneuploidy can also arise during proliferation of inherently unstable tetraploid cells generated by whole genome doubling from diploid cells. Recent findings from cancer genome sequencing projects suggest that nearly 40% of tumors underwent whole genome doubling at some point of tumorigenesis, yet its contribution to cancer phenotypes and benefits for malignant growth remain unclear. Here, we investigated the consequences of a whole genome doubling in both cancerous and non-transformed p53 positive human cells. SNP array analysis and multicolor karyotyping revealed that induced whole-genome doubling led to variable aneuploidy. We found that chromosomal instability (CIN) is a frequent, but not a default outcome of whole genome doubling. The CIN phenotypes were accompanied by increased tolerance to mitotic errors that was mediated by suppression of the p53 signaling. Additionally, the expression of pro-apoptotic factors, such as iASPP and cIAP2, was downregulated. Furthermore, we found that whole genome doubling promotes resistance to a broad spectrum of chemotherapeutic drugs and stimulates anchorage-independent growth even in non-transformed p53-positive human cells. Taken together, whole genome doubling provides multifaceted benefits for malignant growth. Our findings provide new insight why genome-doubling promotes tumorigenesis and correlates with poor survival in cancer.

Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.