Tolerance of Chromosomal Instability in Cancer: Mechanisms and Therapeutic Opportunities Eva Gronroos1 and Carlos Lopez-García 1,2

Published OnlineFirst November 12, 2018; DOI: 10.1158/0008-5472.CAN-18-1958 Cancer Review Research

Abstract

Chromosomal instability (CIN) is the result of ongoing additional mechanisms have been identiﬁed in CIN-surveil- changes in the number (aneuploidy) and structure of chromo- lance, resulting in a more complex network of pathways acting somes. CIN is induced by chromosome missegregation in independently or in cooperation with p53. Tolerance might also mitosis and leads to karyotypic diversity within the cancer cell be achieved by modifying aspects of the cancer cell physiology population, thereby adding to intratumor heterogeneity. in order to attenuate CIN or by adaptation to the consequences Regardless of the overall pro-oncogenic function of CIN, its of aneuploid karyotypes. In this review, we summarize the onset is typically detrimental for cell ﬁtness and thus tumors current knowledge about p53-dependent and -independent must develop CIN-tolerance mechanisms in order to propagate. mechanisms of CIN-tolerance in cancer, the adaptations There is overwhelming genetic and functional evidence linking observed in CIN cells buffering CIN levels, its consequences mutations in the tumor suppressor TP53 with CIN-tolerance. for cellular homeostasis, and the potential of exploiting However, the pathways leading to p53 activation following these adaptations in order to design new cancer therapies. chromosome missegregation remain controversial. Recently, Cancer Res; 78(23); 1–7. 2018 AACR.

Background missegregation events and generate sufficient karyotypic diversity within the population to confront the challenges that arise Most cancer types show some extent of chromosomal insta- during tumor evolution. In this review, we will focus on the bility (CIN). This hallmark of cancer, which can be defined as the current knowledge of the effects of CIN on cancer cell fitness and continual spatiotemporal alterations of the number and structure the mechanisms whereby cells override the deleterious effects of of chromosomes, fuels intercellular heterogeneity and is a major chromosome segregation errors by inactivating CIN surveillance driving force of tumor evolution and adaptation to challenges pathways or adapting their physiology to the newly acquired arising from the tumor microenvironment. Numerical CIN chromosome imbalances. (nCIN) originates from missegregation of whole chromosomes in anaphase, resulting in two daughter cells with a karyotype differing from the natural karyotype of the species, also known as How Much CIN Can a Cancer Cell Tolerate? aneuploidy. However, segregation errors can also result in struc- During the course of CIN research, several controversies tural CIN (sCIN), giving rise to structural variants of chromo- about the role of CIN in cancer have been raised. CIN is very somes. Although both types of CIN originate from different frequent in cancer and it would be easy to assume that CIN mitotic (1–7) and premitotic alterations (8), they often co-occur operates as an oncogenic event with tumor promoting capa- and are interrelated (8–13). bilities. However, this conclusion is premature. Although extensive and detailed research has been carried out Induction of segregation errors in vivo by genetic inactivation of on the origin of segregation errors in cancer, the processes that components of the spindle assembly checkpoint (SAC) or spindle allow the propagation of CIN in tumors has not been subject to attachment proteins (21) has shown variable tumorigenesis the same level of research. Perhaps the most notable observa- when these mice are crossed onto cancer predisposed models. tion about CIN and its role in cancer is its apparently paradox- Induction of CIN shows oncogenic and tumor suppressive ical negative impact on cell fitness in vitro and in vivo (14–20). capacity depending on the models of carcinogenesis and the Thus, mechanisms of tolerance (denominated CIN-tolerance) CIN drivers considered. Explanations to address these differ- remain necessary in order to allow cancer cells to survive ences converge into a "just-right" model whereby only optimal levels of CIN can promote tumorigenesis. Very similar conclusions canbedrawnwhenhumancancers 1Translational Cancer Therapeutics Laboratory, Francis Crick Institute, London, are analyzed. Stratification of patients with breast cancer United Kingdom. 2National Heart and Lung Institute, Airway Disease Group, according to levels of CIN measured by centromeric FISH Faculty of Medicine, Imperial College London, London, United Kingdom. analysis (cell-to-cell variation) or computational surrogates of Corresponding Author: Carlos Lopez-García, Imperial College London, Guy CIN (weighted genome instability index) showed that tumors Scadding Building, Dovehouse Street, London SW3 6LY, UK. Phone: 44 (0) 20 with intermediate levels CIN (mid-CIN) had worse prognostic 7594 7846; Orchid: 0000-0001-9848-8216; E-mail: features than samples with high and low-CIN, confirming the [email protected] requirement of an intermediate level of CIN for cancer cell doi: 10.1158/0008-5472.CAN-18-1958 fitness (22, 23). A similar trend has also been shown in a pan- 2018 American Association for Cancer Research. cancer analysis (24). Although empirical evidence clearly point

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at low- and high-CIN as good prognostic markers, a more mulation when cells are treated with SAC inhibitors (40). More detailed analysis is necessary to elucidate the cancer types that recently, in a model that used cold-shock to induce missegrega- are more sensitive to CIN and to design new therapeutic tion, it was observed that p53 accumulation was dependent on strategies to target cancer types that do not tolerate extreme histone H3.3 Ser31 phosphorylation on lagging chromosomes levels of CIN. (41). Finally, evidence drawn from SAC inactivation with Mps1 inhibitors showed that p53 accumulation was dependent on Tolerance of Chromosome Missegregation activation of caspase-2 (Fig. 1; ref. 42). Thus, it seems that multiple mechanisms to stabilize p53 after CIN exist and, in Cancer perhaps, different types of segregation errors, reflecting the The p53/MDM2 pathway various processes that can induce them, trigger p53 accumu- The most frequently mutated gene in cancer is TP53, encoding lation through different pathways. Taken into account that p53, a protein that prevents the propagation of genomically some of these mechanisms could occur concomitantly, the link unstable cells. The relationship between TP53 loss-of-function between p53 activation and CIN remains an open field that and CIN-tolerance in cancer derives primarily from the strong requires further insight. correlations found between TP53 mutations and aneuploidy in p53 also restricts the proliferation of tetraploid cells. Genome- multiple tumor types. In endometrial, gastric, and colorectal doubling (also known as tetraploidization) is common in CIN cancer, TP53-mutant tumors present more unstable and complex tumors and this process has been proposed as an intermediate karyotypes than TP53-proficient tumors (25–29). Notwithstand- stage in the development of CIN with high rates of missegregation ing this abundant genetic evidence, it is not possible to assign a and enhanced CIN tolerance (14, 43). Evidence drawn from CIN-tolerance function to TP53 mutations in all tumor types: for genome wide loss-of-function screens has documented a clear instance, microsatellite-unstable colorectal cancer has a high connection between tolerance of tetraploidization and the fraction of TP53 mutations (10%–20%) while remaining dip- Hippo pathway (Fig. 1). Cytokinesis failure triggers activation loid, possibly reflecting the pleiotropic effects of p53. of this pathway with downstream phosphorylation of LATS2 Experimental cell biology and mouse models, additionally that specifically binds and inactivates MDM2, resulting in p53 support the role of p53 in CIN tolerance derived from cancer stabilization (44). genetics. Diploid cell lines treated with segregation error inducing drugs or bearing mutations in components of the SAC become Beyond p53: Other Mechanisms Driving aneuploid only in a TP53-mutant background (20, 30, 31). Also, ablation of TP53 in certain cancer predisposed mouse models CIN Tolerance promotes CIN and overall aneuploidy (32, 33). More recently, Although inactivation of TP53 seems to dominate the muta- genome-editing of human derived colon organoids revealed that tional landscape associated with CIN tolerance, TP53 wild-type when TP53 is mutated, organoids spontaneously develop CIN tumors are frequently aneuploid and therefore other mechanisms and a more aggressive phenotype (34). of tolerance are likely to take place. p53 is primarily regulated by a posttranslational mechanism BCL9L is one example of a CIN-tolerance gene that functions that involves MDM2, an ubiquitin-E3-ligase targeting p53 for either independently of, or in cooperation with p53. BCL9L is a proteasomal degradation. Although MDM2 is rarely mutated in Wnt signaling component that facilitates b-catenin transcriptional cancer, transgenic MDM2 expression in mouse models induces activity. Loss of BCL9L function in colorectal cancer reduces CIN (35) and MDM2 gene amplification and overexpression are the expression of caspase-2, an "orphan" caspase that can cleave common in some solid tumors and hematological malignancies substrates controlling cell viability such as the p53 regulator (36). However, the general role of MDM2 in tumorigenesis MDM2 and the pro-apoptotic protein BID (Fig. 1; ref. 42). Impor- and CIN is more complicated as this protein can also mediate tantly, this role of BCL9L as a CIN tolerance gene might shed light the degradation of other targets such as the tumor suppressor on the "just-right" theory of Wnt mediated tumorigenesis (45). Rb (37–39). An attenuation of Wnt signaling levels in cancer cells (e.g., by BCL9L dysfunction or selection of certain APC mutant alleles) Pathways leading to p53 activation could modulate the expression of caspase-2 to levels compatible Although the role of p53 in CIN-surveillance has been solidly with CIN. demonstrated, the mechanisms whereby p53 senses chromo- Dysfunction of caspase-2 has previously been shown to some missegregation require a better explanation. The p38 drive karyotypic alterations in various cancer predisposed stress kinase has been reported as necessary for p53-mediated models (46, 47) and to prevent p53 stabilization (46, 48, arrest in experimental models with cytoskeletal or centrosome 49). However, the connection between caspase-2 and BCL9L disruption (Fig. 1; ref. 20). Additionally, DNA damage follow- reinforced the relevance of caspase-2 for CIN tolerance in ing chromosome missegregation has also been proposed as the human tumors. Caspase-2 is activated by cleavage of the event triggering p53 stabilization. Janssen and colleagues (11) proenzyme generating the catalytically active p19 peptide. demonstrated that missegregated chromosomes are subject to Oliver and colleagues (48) proposed a p53-dependent mech- mechanical stress and DNA damage, leading to ATM-dependent anism whereby p53 transcriptionally activates PIDD, a protein p53 stabilization (Fig. 1). Other authors have postulated that mediating caspase-2 dimerization and self-cleavage. Thomp- micronuclei (extranuclear chromatin formed by missegregated son and colleagues (50) proposed a different mechanism chromosomes) are more prone to DNA damage than nuclear involving ATM dependent phosphorylation of PIDD that is DNA (10). But the dependency of p53-mediated arrest on DNA subsequently recruited by BubR1 to the kinetochore thereby damage does not seem to apply in every experimental context. preventing caspase-2 activation and apoptosis. Andersen and For instance, inhibition of ATM does not prevent p53 accu- colleagues (51) also showed that phosphorylation of caspase-2

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Segregation error

Caspase-2 DNA damage p38 MAPK activation

BID MDM2 ATM cleavage cleavage phosphorylation

Apoptosis p21 Arrest Figure 1. Pathways involved in CIN-surveillance found p53 deregulated in cancer. The majority of pathways result in stabilization of p53 (blue), whereas some pathways operate independently of, or in cooperation with, p53 (red). MDM2 inhibition

Low Wild-type LATS2 Cyclin D1/D2 PIK3CA

Genome doubling

p53-dependent pathways p53-independent pathways

on serine 340 prevents caspase-2 activation during mitosis. As agation of aneuploid cells. For this reason, other mechanisms with p53 stabilization, modes of caspase-2 activation seem to of CIN tolerance have been proposed based on adaptations of be dependent on the experimental systems used and additional the cellular physiology that partially compensate mitotic altera- research is needed to fully elucidate the mechanisms of caspase-2 tions and the impact of chromosomal imbalances on cellular activation. homeostasis (Fig. 2). p53-independent mechanisms can also induce tolerance of tetraploidization. Two independent reports found that overex- CIN stabilization pression of cyclin D1 and D2, two cyclins frequently deregulated The proposed "just-right" level of CIN described above might in cancer, counteracted p53 activity by sequestering p21 and be the explanation for the intermediate levels of CIN found in thereby preventing p21-mediated arrest (Fig. 1; refs. 52, 53). patients with reduced survival and enhanced disease progres- Berenjeno and colleagues have also shown that oncogenic sion. This observation has motivated efforts to elucidate PIK3CA mutations mediate tolerance of tetraploidization in CIN modulatory mechanisms selected for in tumors. Loss-of- TP53-wild-type cells, although it is unclear whether this effect function mutations in the anaphase promoting complex/ involves a deficient p53 stabilization or a blockade of down- cyclosome complex (APC/C) have been recently found to stream effectors (54). counteract the effects of various mitotic alterations on mitotic fidelity (40, 55). The APC/C complex remains inhibited by the SAC until chromosomes achieve the appropriate biorientation Adaptation to CIN in Cancer Cells in metaphase. Hence, inactivation of the APC/C complex Although the mechanisms described above appear indis- results in a prolonged mitosis, enabling cells to correct mitotic pensable in many cancer types, newly acquired aneuploidies alterations, enhancing mitotic fidelity, and limiting CIN might still be detrimental for cell fitness and restrict the prop- (Fig. 2). Of note, a pan-cancer analysis showed a higher

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APC/C Mitotic delay dysfunction (error correction)

Figure 2. Adaptations of the cellular physiology and homeostasis to attenuate the p62 effects of CIN. Top, APC/C dysfunction (autophagy) allows segregation error correction and prevents excessive CIN. Middle, Gene Protein aneuploid cells restrict the effects of Aneuploidy chromosome imbalances on the tolerance proteome by activating autophagy- Copy related protein p62. Bottom, transition Proteotoxic Protein number to near-triploid karyotypes enhances stress homeostasis ﬁ gain cancer cell tness.

2N Near-triploid 3N transition 4N

frequency of mutations in APC/C subunits in aneuploid suggested that aneuploid cells mitigate the detrimental effects tumors, indicating that a longer mitosis is necessary to avoid of certain copy number changes by modulating the expression excessive genomic chaos (40). This "buffering" of CIN by of those genes or activating the degradation of the surplus regulating mitosis and enhancing error correction could be protein, to achieve levels compatible with cell survival. In selected in tumors as a mechanism that promotes the expan- particular, Hose and colleagues (62) documented a poor stoi- sion of cancer cells with intermediate and viable levels of CIN. chiometric correlation between mRNA and copy number in Of note, these studies predict that APC/C dysfunction could be aneuploid yeast strains. Chromosome imbalances also generate a mechanism of resistance to Mps1 inhibitors (BOS172722, changes in the stoichiometry of the cellular protein content, BAY1217389, BAY1161909, and CFI402257), a group of novel known as proteotoxic stress, that compromises the propagation anticancer drugs that induce CIN by inhibiting the SAC of CIN cells. The buffering of this process has been proposed as (56, 57), resulting in excessive CIN and cell death. a mechanism of propagation of aneuploidy. Seminal work by Inactivation of APC/C is not the only mechanism for CIN Torres and colleagues (63) reported that inactivation in the buffering. For instance, some cancer types have telomere gene encoding the deubiquitinating enzyme Ubp6 increased attenuation in early phases of the disease, with telomerase the proliferation rates of aneuploid yeast and attenuated pro- reactivation at later stages thereby preventing excessive CIN tein changes caused by aneuploidy. (58–61). The relevance of these observations for mammalian cells was later confirmed with artificial introduction polysomy in Correcting the Consequences of CIN: near-diploid cancer cells. Transcriptomic analysis of viable Dosage Compensation and Protein polysomic strains showed a good correlation between copy number changes and mRNA abundance, whereas correlation of Homeostasis copy number with protein abundance showed protein levels CIN drives phenotypic evolution by altering dosage and similar to the parental near-diploid cells (64). Pathway analysis expression of genes located on imbalanced chromosomes of uncovered activation of the p62-dependent autophagic path- aneuploid cells. However, it is likely that subsets of gene dosage way, which conceivably contributedtocounteracttheeffect imbalances are advantageous for the aneuploid cell, whereas of copy number changes in protein homeostasis (Fig. 2). others impair cell fitness or are simply neutral. It has been Similarly, recent analysis of aneuploid cancer cells has shown

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that these cells modulate their phospho-proteome and the and NCT02792465, clinicaltrials.gov). However, the success of this microRNAome to adapt their physiology to the adverse effects strategy depends heavily on stratifying patients according to levels of aneuploidy (65, 66). Although evidence of proteome of CIN as treatment of tumors with low CIN might induce homeostasis as an aneuploidy maintenance mechanism has intermediate levels of CIN and a negative impact on patient been gained from in vitro systems, studies in breast cancer survival. Similarly, treatment of tumors bearing mutations in supports the notion that attenuation of the effect of chromo- CIN-tolerance genes such as TP53 and BCL9L should be carefully some imbalances on protein levels also occurs in human evaluated as they might reduce the therapeutic benefit. Thus, the tumors (67). success of Mps1 inhibitors will greatly depend on the development of methods to classify patients according to levels of CIN and existence of CIN-tolerance mutations in order to avoid Exploring Advantageous Karyotypes treatment of patients that will not benefit from treatment with Studies of the intrinsic tolerance of specifickaryotypesin these compounds. yeast and mammalian cells have also been performed; Pavelka Combination of Mps1 inhibitors with chemotherapy agents and colleagues (18) derived a wide range of aneuploid yeast must also be carefully evaluated. Conceivably, increasing strains from euploid laboratory strains. Most aneuploid strains genetic heterogeneity in tumors using Mps1 inhibitors might showed impaired fitness, but some aneuploid strains did not enhance drug resistance by selection of advantageous karyo- show any proliferation defects when compared with their types. However, combination of Mps1 inhibitors with low- euploid parental strains, supporting that certain karyotypes do dose taxanes resulted in a synergistic effect due to an increase not compromise cell fitness and can be tolerated. In contrast, in aberrant mitoses (57, 70). This synergy is currently being aneuploid strains showed a general growth advantage under tested in clinical trials and could lead to a reduction in taxane restrictive growth conditions or during treatment with drugs. doses, thereby preventing toxicity and drug-resistance. This report highlighted that the effect of aneuploidy on cell Although a good overall tolerability has already been con- fitness is not general, but context specific, and depends on firmed in vivo, with gastrointestinal and hematologic toxicity defined aneuploidies and selective pressures. as the most important toxicity effects (56, 57), the safety Similar approaches have been carried out in mammalian cells. profile of Mps1 inhibitors will be confirmed when the current Sheltzer and colleagues (68) generated a number of aneuploid trials are finally completed. Specifically, the long-term expan- mouse embryonic fibroblasts and aneuploid lines from diploid sion of de novo aneuploid cells in healthy tissues should be parental cell lines and showed that single chromosome trisomies carefully assessed. limit proliferation and tumorigenic capacity in the short term. The mechanisms whereby cells sense missegragation remain However, prolonged growth induced accumulation of additional an open question in the field of CIN-tolerance. A clear example chromosomal alterations and increased fitness and tumorigene- of this controversy is p53 accumulation. So far, the pathways sis. Regardless of the technical limitations of this elegant study, leading to p53 stabilization depend on the experimental model this report clearly shows the prominent short-term tumor sup- used to induce segregation errors. Perhaps, additional efforts to pressor role of aneuploidy in mammalian cells. Only when classify the types of missegregation that occur in tumors, additional karyotypes arise are cells able to explore optimal instead of cell lines, could clarify the elusive processes ulti- karyotypes, resulting in adaptation and tumorigenesis. mately driving p53 accumulation. A similar problem applies to The selection of karyotypes that are nondetrimental for caspase-2, where the mechanism of segregation error detection cell fitness has also been studied using a computational remains unclear. Recent evidence reporting that cytosolic approach that integrates the distribution of tumor suppressor dsDNA leaked from micronuclei is sensed by the NF-kBpath- genes and oncogenes on human chromosomes (69). This way (71, 72), suggest that this system should be further approach showed that a near-triploid karyotype maximizes explored as a potential mechanism to detect missegregation cell fitness, which is supported by the high frequency of this and limit the propagation of CIN. An additional aspect of karyotypes in many cancer types (Fig. 2). The same study caspase-2 in the context of CIN tolerance, is its regulation by also predicted that this near-triploid karyotype is more easily Wnt-signaling. Therapies targeting this oncogenic pathway achieved from an initial tetraploid karyotype (Fig. 2), an ob- might lead to caspase-2 depletion rendering cells unprotected servation that is compatible with the frequency of genome- against CIN. A better understanding of the link between Wnt- doubling in cancer. signaling and caspase-2 activity will shed light on the potential shortcomings of targeting the Wnt-signaling pathway to treat cancer. Future Directions Understanding the levels of CIN tolerated by cancer cells, along with the mechanisms allowing cells to survive missegregation Disclosure of Potential Conflicts of Interest has an obvious interest in cancer therapeutics. Treatment with No potential conflicts of interest were disclosed. Mps1 inhibitors to induce toxic levels of CIN is a promising therapeutic approach currently being evaluated in four phase I Received June 25, 2018; revised August 24, 2018; accepted September 12, clinical trials (NCT03328494, NCT02366949, NCT02138812, 2018; published first November 12, 2018.

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www.aacrjournals.org Cancer Res; 78(23) December 1, 2018 OF7

Tolerance of Chromosomal Instability in Cancer: Mechanisms and Therapeutic Opportunities

Eva Gronroos and Carlos López-García

Cancer Res Published OnlineFirst November 12, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1958

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