Karyotypic Aberrations in Oncogenesis and Cancer Therapy

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Review

Karyotypic Aberrations in Oncogenesis and Cancer Therapy Ilio Vitale,1,2,z Gwenola Manic,2 Laura Senovilla,3,4,5,6,7 Guido Kroemer,3,4,5,6,8,9,10 and Lorenzo Galluzzi3,4,5,6,7,z,* The propagation of whole-chromosome (aneuploid) or whole-genome (polyploid) defects is normally prevented by robust cell-intrinsic mechanisms. Moreover, non-diploid cells are under strict immunological surveillance. Nonetheless, tumors contain a high percentage of non-diploid genomes, indicating that malignant cells acquire the ability to bypass these control mechanisms and obtain a survival/proliferation benefit from bulky karyotypic defects. The non-diploid state imposes a significant metabolic burden on cancer cells and hence can be selectively targeted for therapeutic purposes. Here we discuss the impact of abnormal karyotypes on oncogenesis, tumor progression, and response to treatment, focusing on the biochemical and metabolic liabilities of non-diploid cells that can be harnessed for the development of novel chemo (immuno)therapeutic regimens against cancer.

Trends

Aneuploidy and Polyploidy

Non-diploidy confers on developing tumors increased plasticity and an improved capacity for adaptation to adverse microenvironmental conditions.

Programmed whole-chromosome gain/losses and whole-genome duplications, which are referred to as aneuploidy and polyploidy (see Glossary), respectively, are relatively frequent and tolerated in nature, especially among terminally differentiated cells [1,2]. Somatic polyploidy and aneuploidy have been detected even in the human brain and liver, but their physiological significance remains a matter of debate (Box 1) [1,2]. Conversely, the unscheduled generation of aneuploid and polyploid cells is strictly controlled by an armamentarium of cell-intrinsic and cellextrinsic (immunological) mechanisms (Figure 1) [3,4]. This is unsurprising because non-diploidy imposes an increased metabolic and oxidative burden on the cell, which is often accompanied by high levels of genomic instability and hence by an increased risk for malignant transformation [2]. This is believed to originate from the simultaneous alteration in the relative dosage of many genes, as it occurs in aneuploid cells, and/or from defects in the functionality of the machineries involved in DNA repair and chromosome segregation, as it occurs in polyploid cells [5]. Despite the abovementioned control mechanisms, non-diploid cells form and tend to accumulate in the course of oncogenesis and tumor progression (Box 2) [1,6]. This implies that cancer cells not only bypass the cell-intrinsic and -extrinsic mechanisms that generally prevent the propagation of non-diploid genomes, but also obtain a survival/proliferative benefits from aneuploidy or polyploidy [7]. Importantly, non-diploid malignant cells exhibit a high degree of non-oncogene addiction [8]. Thus, non-diploidy may constitute a target for the development of highly selective anticancer agents. Here we discuss the relevance of non-diploidy for human neoplasms, the cell-intrinsic and -extrinsic systems that control the generation of aneuploid and polyploid cells, the mechanisms

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Non-diploidy is the state of cells that have gained or lost entire chromosomes (aneuploidy) or experienced whole-genome duplication events (polyploidy). Several cell-intrinsic and cell-extrinsic (immunological) mechanisms are in place to prevent the insurgence and arrest the propagation of non-diploidy. In some circumstances, malignant cells can bypass these barriers and proliferate in the presence of increasingly more complex karyotypic alterations.

Non-diploidy may constitute a liability of cancer cells, representing a promising target for the development of novel antineoplastic agents.

1 Department of Biology, University of Rome ‘TorVergata’, Rome, Italy 2 Regina Elena National Cancer Institute, Rome, Italy 3 Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 4 INSERM, U1138, Paris, France 5 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6 Université Pierre et Marie Curie, Paris, France 7 Gustave Roussy Comprehensive Cancer Institute, Villejuif, France 8 Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France

http://dx.doi.org/10.1016/j.trecan.2015.08.001 © 2015 Elsevier Ltd. All rights reserved.

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Box 1. Physiological Instances of Non-Diploidy Constitutional polyploidy (i.e., the acquisition of a polyploid genome by gametes or embryonic cells) appears to be recurrent over evolutionary time [6]. Polyploid organisms are relatively common among plants, insects, fish, amphibians, and fungi but virtually absent in mammals, with the notable exception of the allotetraploid red viscacha rat [6]. On the contrary, constitutional aneuploidy is very often incompatible with life early during embryogenesis or causes severe developmental defects and growth retardation [11]. Somatic alterations of chromosomal content are widespread in multicellular organisms, including humans [1]. Wholegenome duplication events occur in various cell lineages within the body, mostly in response to perturbations of homeostasis (not necessarily of pathological nature) [1]. For instance, polyploidy can be detected in cardiomyocytes, hepatocytes, neurons, myoblasts, multinucleated osteoclasts, multinucleated giant cells, megakaryocytes, trophoblast giant cells, and the syncytiotrophoblast, which mainly (but not always) are terminally differentiated, nonproliferating cells [1]. Polyploidy is believed to contribute to the regulation of tissue/organ size, homeostasis, and function by: (i) constituting a relatively rapid form of growth; (ii) increasing biosynthetic and metabolic tissue activities; (iii) triggering cell differentiation or morphogenesis; (iv) constituting a storage of proteins, nutrients, and metabolites; (v) favoring the adaptation of tissues to environmental perturbations; (vi) diminishing cellular sensitivity to potentially lethal stimuli; and (vii) acting as a buffer against deleterious genetic mutations [1]. Interestingly, polyploidy is strictly required for the differentiation and function of some cell lineages, including megakaryocytes, myoblasts, and osteoclasts [1]. However, the physiological functions of programmed polyploidy in somatic cells remain to be precisely elucidated. Although an elevated fraction of asymptomatic aneuploidy has been detected in various tissues including the brain and liver (where it seems to originate from polyploidy) and is believed to contribute to phenotypic and biochemical plasticity, a recent study suggests that cells bearing aneuploid karyotypes are present at low levels (<5%) across mammalian tissues [94].

whereby malignant cells circumvent them, and the rationale behind targeting non-diploidy for cancer therapy.

Relevance of Non-Diploidy for Human Cancer According to current estimates, proliferating cancer cells fail to correctly segregate chromosomes once every five divisions, a state that is commonly referred to as chromosome instability (CIN) [9]. Thus, malignant cells are unable to maintain a stable karyotype across consecutive generations [9]. In line with this notion, aneuploid genomes can be detected in the vast majority of hematological and solid tumors [10]. Nonetheless, the true etiological role of aneuploidy in oncogenesis remains a matter of debate. Individuals with disorders linked to aneuploidy, including Down syndrome, Turner syndrome, and mosaic variegated aneuploidy (MVA), have an increased risk of developing various malignancies, but this is not a constant finding [11]. Moreover, patients with Down syndrome appear to have a decreased incidence of solid tumors [11]. Thus, although aneuploidy and CIN are currently viewed as a source of genetic/genomic defects that may promote tumor progression (i.e., the acquisition of ever-more-aggressive features by proliferating cancer cells), their involvement in malignant transformation (i.e., the conversion of a normal cell into a malignant one) remains to be formally demonstrated. About 20% of solid tumors display higher-order aneuploidy (i.e., a near-to-tetraploid chromosomal content) [7]. Moreover, advanced-stage (but not early-stage) neoplasms are often characterized by elevated intratumoral heterogeneity in chromosomal content and a very complex karyotype [12,13]. These observations suggest that aneuploidy and CIN may originate from a whole-genome doubling event followed by the missegregation of one or more chromosomes [5]. This hypothesis has been corroborated by several other lines of evidence. First, it has been estimated that approximately 37% of all neoplasms, including those displaying a near-todiploid DNA content, have experienced at least one whole-genome doubling event during their evolution [14]. Second, the progressive loss of whole chromosomes starting from a polyploid state has been proposed as the most likely mechanism for the development of aneuploid neuroblastoma (based on karyotypic data from 120 neoplastic lesions analyzed with a refined mathematical model and computer simulation) [15]. Third, accumulating preclinical and clinical

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9 Pôle de Biologie, Hopitâl Européen George Pompidou, Paris, AP-HP, France 10 Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden

*Correspondence: [email protected] (L. Galluzzi). z These authors share senior coauthorship.

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Box 2. Mechanisms Leading to Nonphysiological Non-Diploidy Somatic diploid cells can acquire a polyploid genome owing to distinct alterations of normal G1–S–G2–M cell cycle progression [5]. Such defects include: (i) endoreplication (also known as endocycling), the process whereby cells completely skip the M phase and hence glide from a diploid G2 state to a tetraploid G1 state; (ii) endomitosis, the phenomenon whereby cells initiate mitosis but cannot complete it, and slip from a prolonged block in metaphase to the subsequent interphase without segregating chromosomes (karyokinesis) and dividing the cytoplasm (cytokinesis); and (iii) cytokinesis failure, referring to the inability of some cells that complete karyokinesis and properly split into two daughter cells. In all of these cases, both the genetic and centrosome contents are doubled. However, the genome of freshly formed tetraploid cells can be contained in a single nucleus (as in the case of endoreplication and endomitosis) or in two (as in the case of cytokinesis failure). Binucleated tetraploid cells may also arise from the fusion of two somatic diploid cells [5]. One mechanism for somatic diploid cells to become aneuploid involves a polyploidization step followed by an asymmetric division coupled to chromosome missegregation, resulting in the gain or loss of one or several chromosomes [7]. Asymmetric cell divisions may also affect diploid cells, but with a very low incidence [5]. Mitotic defects leading to chromosome missegregation include (but are not limited to): (i) perturbations of the geometry, organization, or dynamics of the mitotic spindle (e.g., incomplete centrosome separation at prophase, incorrect attachments between kinetochores and microtubules); (ii) alterations in the structure of chromosomes and/or the cohesion between sister chromatids; (iii) defects in the SAC and/or other mechanisms that regulate the metaphase-to-anaphase transition; and (iv) problems at cytokinesis [5]. In addition, somatic diploid cells can become aneuploid as a consequence of the massive fragmentation and subsequent reassembly of a single chromatid, a process that can occur in mitosis as well as in interphase and is commonly known as chromothripsis [95]. Of note, mitotic issues (and hence an increased likelihood of aneuploidization) may also originate from premitotic defects, including (but not limited to): (i) centrosome amplification, which increases the frequency of multipolar mitoses, merotelic attachments, and chromosome lagging; (ii) incomplete DNA replication coupled to premature mitotic entry; (iii) activation of the DNA damage response persisting throughout mitosis; (iv) abrogation of the mechanism that prevents DNA rereplication, which may be linked to DNA synthesis in mitosis; (v) deregulation of proteins involved in the transcriptional or post-transcriptional regulation of components of the mitotic machinery; and (vi) oncogene activation, often resulting in DNA replication stress [61].

evidence indicates that whole-genome duplication events occur at least once during oncogenesis and tumor progression [13,16,17]. Of note, these events often (but not always) occur early in the evolution of neoplastic lesions and rapidly allow the outgrowth of an aggressive population of malignant cells that de facto drive disease progression (while acquiring further genomic defects) [18,19]. Fourth, bona fide tetraploid cells (i.e., cells that have undergone only one event of genome duplication) are found at increased frequency in early-stage colorectal, breast, and cervical carcinomas, as well as in premalignant lesions like Barrett esophagus [7]. Finally, it has been shown that tetraploid, but not diploid, mammary epithelial cells lacking the mouse homolog of tumor protein 53 (TP53) (see below) survive and proliferate on inoculation into immunodeficient mice, forming tumors with high CIN levels [20]. Thus, at least in some settings, polyploidization may represent a gate for oncogenic aneuploidization. Aneuploidy and genome-doubling events have been correlated with poor disease outcome in cohorts of patients affected by various neoplasms, including breast, colorectal, and lung carcinomas [13,21–23]. This is in line with an abundant preclinical literature demonstrating that aneuploidy generally supports tumor progression and chemotherapy resistance, possibly because it rapidly endows evolving neoplastic lesions with an elevated genetic heterogeneity and hence an increased capacity for adaptation [24–28]. Non-diploid cancer cells are markedly less sensitive than their diploid counterparts to a wide panel of conditions frequently encountered in the course of oncogenesis and tumor progression, including (but not limited to) hypoxia, glucose deprivation, acidification of the local microenvironment, and exposure to therapeutic agents [29,30]. Apparently at odds with these observations, a large meta-analysis encompassing 13 cohorts of patients with different tumors identified a paradoxical correlation between CIN and improved disease outcome [31]. These apparently discrepant observations reflect the fact that moderate levels of CIN are generally tolerated and endow cancer cells with an elevated plasticity and adaptive capacity, while excessive levels of CIN are generally incompatible with cell survival and proliferation. Moreover, it should be noted that, besides conferring increased

Glossary Aneuploidy: the state of cells bearing a non-diploid number of chromosomes that is not a multiple of the haploid (n) number. Autophagy: an evolutionarily old mechanism for the lysosomal degradation of ectopic, supernumerary, or damaged intracellular entities. Autophagy is critically required for optimal adaptive responses to most, if not all, perturbations of intracellular and extracellular homeostasis. Centrosome clustering: a process whereby supernumerary centrosomes coalesce to form a near-to-normal bipolar mitotic spindle. Chromosome instability (CIN): a type of genomic instability characterized by the loss or gain of whole chromosomes or large parts thereof at a high rate. Chromosome lagging: the situation in which one or more chromatids persist at (or near) the spindle equator when most have already reached the poles during anaphase. Chromosome lagging is a major source of chromosome missegregation and cytokinesis failure. Chromothripsis: a process involving the extensive fragmentation of a single chromatid followed by its aberrant repair. Chromothripsis can occur when DNA is condensed (i.e., during the M phase of the cell cycle) and has been etiologically linked to congenital disorders and oncogenesis. DNA replication stress: inefficient DNA duplication resulting in replication forks progressing slowly or stalling. Several factors can cause DNA replication stress, including limited dNTP availability and altered expression levels of proteins involved in DNA replication. Down syndrome: an inherited genetic disorder characterized by the presence of an extranumerary chromosome 21 or large parts thereof (also known as trisomy 21). It is generally associated with growth retardation, peculiar facial traits, cardiac disorders, and mild-tomoderate intellectual disability. Merotelic attachment: the situation in which microtubules from the same pole of the mitotic spindle are attached to both sister chromatids while microtubules from the other pole contact one chromatid only.

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C1 Spindle assembly checkpoint

C2

Mitoc catastrophe

Nucleus

C3 Diploid (2n)

C1

C1 Aneuploid (2n+/–C)

Karyotypic aberraons

C2

C2

Tetraploid (4n)

C3

C3

NK cell

ATP T cell

Gene deregulaon

Metabolic burden

RCD

Immune response

Figure 1. Mechanisms that Limit Non-Diploidy. Two major mechanisms are in place to prevent the generation of nondiploid cells, the so-called ‘spindle assembly checkpoint’ and mitotic catastrophe. The former prevents the metaphase-toanaphase transition until all chromosomes are properly aligned on the metaphase plate and correctly attached to the mitotic spindle. The latter can eliminate cells that experience mitotic problems before they reach the subsequent interphase. Moreover, gross karyotypic alterations are generally incompatible with the survival or proliferation of newly formed aneuploid cells because: (i) key genes are missing, overexpressed, or underexpressed as a direct consequence of the aberrant chromosomal setup; (ii) non-diploidy is associated with an unbearable metabolic burden; (iii) non-diploidy is sensed by oncosuppressive mechanisms, resulting in the activation of regulated cell death (RCD) or cellular senescence; or (iv) nondiploidy promotes the emission of signals that trigger an innate or adaptive immune response. Abbreviations: C, chromosome; NK, natural killer.

adaptive potential on cancer cells, CIN boosts their antigenicity, perhaps rendering them susceptible to immunosurveillance [21]. Corroborating this notion, it has recently been demonstrated that only colorectal carcinoma patients bearing mismatch DNA repair-deficient lesions (which display a high amount of somatic mutations similar to lesions with elevated CIN) respond to checkpoint blockade with a monoclonal antibody targeting programmed cell death 1 (PDCD1, best known as PD-1) [32]. Blocking PD-1 (re)activates an anticancer immune response that is particularly vigorous against mismatch DNA repair-deficient cancers, presumably because the large amounts of somatic mutations accumulating in these tumors create a large panel of potentially reactive antigenic determinants [32]. Taken together, these observations indicate that non-diploidy and CIN constitute robust drivers of tumor progression. However, cancer cells may pay for the elevated adaptive potential conferred by non-diploidy and CIN with an increased susceptibility to immunological control.

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Merotelic attachments not recognized by the SAC result in chromosome missegregation. Mismatch DNA repair: a system for the recognition and repair of erroneous insertion, deletion, and misincorporation of bases that arise during DNA replication and recombination. Mitotic catastrophe: an oncosuppressive mechanism that eliminates or permanently controls mitosis-incompetent cells by activating regulated cell death or cell senescence. Mitotic slippage: a phenomenon whereby cells can adapt to persistent SAC activation without undergoing mitotic catastrophe and reach the G1 phase of the cell cycle with a tetraploid genome. Mitotic slippage ultimately leads to chromosome missegregation and aneuploidy. Mosaic variegated aneuploidy (MVA): a rare genetic disorder characterized by an increased predisposition of chromosomes to mitotic nondisjunction, resulting in a very high proportion of aneuploid cells in various tissues. MVA patients exhibit severe physical abnormalities, developmental delays, and intellectual disability. Mutator phenotype: a phenotype often displayed by malignant cells and characterized by a very high rate of acquisition of somatic mutations. Non-oncogene addiction: the addition of cancer cells to the constitutive activation of adaptive response pathways that: (i) are not activated by nonmalignant cells under physiological conditions; and (ii) per se are not oncogenic. Poly(ADP-ribose) polymerase 1 (PARP1): a NAD+-dependent enzyme that attaches poly(ADPribose) moieties to various substrates. PARP1 participates in DNA damage responses, but can also promote a regulated variant of necrosis known as parthanatos. Polyploidy: the state of cells bearing a non-diploid number of chromosomes that is a multiple of the haploid (n) number. Tetraploidy (4n) is the most common form of polyploidy. Programmed cell death 1 (PDCD1 or PD-1): an immunosuppressive receptor expressed on the surface of pro-B cells and activated T lymphocytes. FDA-approved monoclonal antibodies targeting PD-1 mediate robust antineoplastic effects in patients with various tumors by

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Mechanisms Limiting Non-Diploidy One of the major mechanisms counteracting the formation of non-diploid cells is commonly known as the spindle assembly checkpoint (SAC) or mitotic checkpoint [33]. The SAC relies on a large multiprotein complex that monitors the proper connection of kinetochores to spindle microtubules, preventing the start of anaphase until all chromosomes are properly attached and oriented on the metaphase plate [33]. In the presence of irresolvable mitotic defects, prolonged SAC signaling generally activates mitotic catastrophe, an oncosuppressive mechanism resulting in permanent proliferative arrest (cell senescence) or regulated cell death (RCD) [34]. Corroborating the importance of the SAC for the maintenance of genome integrity, altering the levels of various SAC components including MAD2 mitotic arrest deficient-like 1 (MAD2L1, best known as MAD2), BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B, best known as BUBR1), and centromere protein E (CENPE) has been linked to non-diploidy, CIN, and suppressed or accelerated oncogenesis [5]. In some instances, cells are capable of adapting to persistent SAC activation (while avoiding mitotic catastrophe) and reach the G1 phase of the cell cycle with a tetraploid genome, a process that is called mitotic slippage. Most often, such tetraploid cells form daughter cells that are not viable owing to intrinsically lethal monosomies or trisomies [34]. Alternatively, slippage ultimately results in the formation of cells bearing a chromosomal content that is compatible with life but imposes an excessive metabolic burden on them, de facto blocking (or at least significantly retarding) proliferation [2]. This also applies to polyploid cells forming on cytokinesis failure or as a result of unscheduled cell-to-cell fusion events (Box 2). Thus, the metabolic alterations imposed by aneuploidy and polyploidy may be viewed as a passive, cell-intrinsic barrier to the propagation of non-diploid cells. In line with this notion, viable aneuploid and polyploid cells (which de facto survive the metabolic burden imposed by non-diploidy) exhibit consistent metabolic alterations [2]. These encompass (but are not limited to): (i) elevated uptake of glucose and glutamine, correlating with increased metabolic flux through glycolysis and the Krebs’ cycle as well as with abundant secretion of lactate [2,35]; (ii) reduced mitochondrial transmembrane potential but increased ATP content (further corroborating the importance of glycolysis in the maintenance of bioenergetic metabolism in non-diploid cells) [36]; and (iii) altered redox homeostasis, which may constitute a cause as well as a consequence of mitochondrial defects [37].

reinstating anticancer immunosurveillance. Spindle assembly checkpoint (SAC): a complex molecular mechanism that prevents the metaphase-to-anaphase transition until all chromosomes are properly aligned on the metaphase plate and correctly attached to the mitotic spindle. Tetraploidy checkpoint: a TP53dependent molecular mechanism promoting the permanent proliferative arrest or death of cells that, despite other control systems, reach the G1 phase of the cell cycle with a tetraploid genome. The existence of the tetraploidy checkpoint remains controversial. Tumor mutanome: the ensemble of somatic mutations accumulated by malignant cells in the course of oncogenesis and tumor progression. Turner syndrome: an inherited genetic disorder characterized by the lack of the X chromosome or large parts thereof (also known as 45,X syndrome). It is compatible with life only in females and associated with sterility, heart defects, and diabetes, but not intellectual disability.

Aneuploidy and polyploidy can also be detected by sensors of genetic/genomic defects, including ATM serine/threonine kinase (ATM) and ATR serine/threonine kinase (ATR), generally resulting in the delivery of a TP53-dependent cell cycle-arresting or RCD-inducing signal [38,39]. The precise molecular mechanisms underlying such a ‘tetraploidy checkpoint’ (which reportedly operates in the G1 phase of the cell cycle) remain a matter of debate. Recent data indicate that components of the Hippo signaling pathway, notably large tumor suppressor kinase 2 (LATS2), are required for the activation and stabilization of TP53 by supernumerary centrosomes in polyploid cells [40]. It remains to be demonstrated whether this pathway also operates in aneuploid cells. Finally, non-diploidy can be recognized by components of the innate immune system. Compared with their diploid counterparts, polyploid cells expose an increased amount of the endoplasmic reticulum chaperone calreticulin (CALR) on their surface, rendering them susceptible to recognition by antigen-presenting cells and elimination by cytotoxic T lymphocytes as well as by CD226+ natural killer (NK) cells [4,41]. Moreover, polyploid cells undergoing TP53dependent senescence express: (i) elevated amounts of ligands for activating NK cell receptors, including CD226 and killer cell lectin-like receptor subfamily K, member 1 (KLRK1, best known as NKG2D) [42]; (ii) many immunostimulatory and chemotactic cytokines, including chemokine (C-C motif) ligand 2 (CCL2) [43]; and (iii) multiple cell adhesion factors, like intercellular adhesion molecule 1 (ICAM1) [44].

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Taken together, these observations indicate that several cell-intrinsic and -extrinsic (immunological) systems are in place not only to prevent the insurgence of aneuploid and polyploid cells, but also to avoid their survival and proliferation (Figure 1).

Non-Diploidy and Tumor Evolution The elevated proportion of non-diploid genomes found in human tumors suggests not only that malignant cells can overcome the cell-intrinsic and -extrinsic controls that normally preserve diploidy, but also that they obtain an advantage from (at least some degree of) karyotypic aberration (Figure 2). Several mechanisms have been shown to increase the cellular and organismal tolerance to nondiploidy in the course of oncogenesis, including the loss of tumor suppressor genes and the activation of oncogenes. The absence of TP53 favors the generation, survival, and/or proliferation of aneuploid as well as polyploid cells [26,30,38–40]. Mutations in retinoblastoma 1 (RB1) or adenomatous polyposis coli (APC) are generally accompanied by an increase in the proportion of polyploid cells and CIN, as they compromise mitotic fidelity by upregulating MAD2 or impair cytokinesis, respectively [45,46]. Of note, the combined loss of TP53 and APC is sufficient for human intestinal stem cells to accumulate major degrees of aneuploidy [47]. The capacity to promote non-diploidy and CIN has been ascribed to various oncogenes, including aurora kinase A (AURKA), B-Raf proto-oncogene, serine/threonine kinase (BRAF), cyclin-dependent kinase 4 (CDK4), v-mos Moloney murine sarcoma viral oncogene homolog (MOS), v-myc avian myelocytomatosis viral oncogene homolog (MYC), and Harvey rat sarcoma viral oncogene homolog (HRAS) [5,21]. Thus, there appears to be an intimate link between oncogenesis and nondiploidy. Compelling evidence demonstrates that aneuploidy and polyploidy may confer a significant survival or proliferative advantage on cells facing adverse microenvironmental conditions (such as malignant cells in developing tumors) [2,48–50]. In this setting, whole-chromosome and/or whole-genome duplication appears as a quick (but transient) evolutionary solution that confers genetic and phenotypic plasticity for adaptation on a wide range of selective pressures [51,52]. In particular, extra chromosomes may constitute a buffer against deleterious mutations while increasing the likelihood of insurgence of beneficial mutations that improve fitness [50,53]. Aneuploidy is a major driver of genomic instability, favoring the acquisition of both large-scale (CIN) and small-scale mutations (mutator phenotype) [53–56]. Such an effect has been

Diploid cells

Control of non-diploid cells

Increased plascity

Adaptability to microenvironment

Tumor progression and resistance to therapy

Figure 2. Non-Diploidy in Oncogenesis and Tumor Progression. Most often, non-diploid cells that arise illicitly within a diploid cell population are not viable, cannot proliferate, or are actively eliminated by cell-intrinsic or -extrinsic control mechanisms. In some circumstances, however, non-diploidy is tolerated and confers on proliferating cells a considerable degree of plasticity. Thus, non-diploidy can endow malignant cells with an accrued capacity for adaptation to adverse microenvironmental conditions, promoting tumor progression and resistance to therapy.

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ascribed multiple mechanisms, including altered expression of components of the machineries for cell cycle progression, DNA repair, and chromosome segregation and an imbalance between prosurvival and prodeath signals [2,54,55,57]. High levels of DNA replication stress have also been documented in aneuploid cancer cells [58]. However, this is not always the case, and aneuploid cells can display a relatively stable genome [7]. This latter finding indicates the existence of back-up mechanisms that, at least in some circumstances, stabilize the genome of aneuploid cells. Although the precise nature of these mechanisms remains obscure, they may be related to post-translational compensation in the levels of key gene products [2]. In polyploid cells (which bear a balanced, although duplicated, genome), genomic instability mainly stems from the duplication of DNA and centrosomes in the absence of a proportional increase of cell volume, surface, and nuclear size [5,55]. This is believed to affect not only intracellular trafficking and the interaction between subcellular structures, but also the regulation and function of macrocomplexes involved in the preservation of a stable genome (e.g., DNA repair machinery, mitotic spindle) [5,6]. One of the main mechanisms to preserve genomic stability in polyploid cells is known as centrosome clustering, which allows spindle bipolarity in the presence of supernumerary centrosomes [27]. Nonetheless, this process is error prone and often results in merotelic attachments or chromosome lagging [27]. In line with this notion, extra centrosomes (which can also arise from mechanisms other than polyploidization) are a frequent finding in human tumors and have been linked to advanced disease grade [59]. Interestingly, recent evidence suggests that polyploidy increases the ability of cells to tolerate CIN, rather than promoting it [13]. According to this study, whole-genome doubling events would arise to mitigate the deleterious effect of aneuploidy. In summary, the adaptive potential and genomic instability conferred by optimal, tolerated degrees of aneuploidy and polyploidy explain (at least in part) the link between non-diploidy and cancer (Figure 2).

Targeting Non-Diploidy for Cancer Therapy With a few exceptions [56], abnormal karyotypes and high degrees of CIN protect cancer cells from a wide panel of therapeutic interventions, including genotoxic agents [30,60]. Harnessing non-diploidy for the development of novel antineoplastic agents may therefore be challenging. At least on theoretical grounds, it may be possible to selectively target specific karyotypes (e.g., the gain of chromosome 8 that characterizes many cases of acute myeloid leukemia) based on the consequent alteration in the expression levels of one or more proteins [7]. Although compelling evidence is still missing [61], it seems that most aneuploid cancer cells gain or lose chromosomes in a nonrandom pattern [21,62], which may be compatible with the development of selective therapeutic strategies. However, the approaches proposed so far for targeting nondiploid malignant cells primarily exploit features linked to aneuploidy or polyploidy in general rather than specific karyotypic alterations (Table 1). Reflecting their increased metabolic burden, non-diploid cells generally manifest consistent metabolic rewiring, encompassing increased glucose and glutamine uptake and abundant lactate secretion [63]. In line with this notion, non-diploid cells are more sensitive than their diploid counterparts to agents that interfere with energetic metabolism, such as the AMPactivated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), resveratrol, and aspirin [64–66], the glycolysis inhibitor 2-deoxyglucose (2-DG) [67], and the mechanistic target of rapamycin (MTOR) inhibitor AZD8055, when combined with aurora kinase B (AURKB) inhibitors [68]. Moreover, non-diploid cells exhibit consistent alterations in redox homeostasis, rendering them more dependent on reactive oxygen species (ROS) for survival than their diploid counterparts [37].

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Table 1. Possible Strategies for the Selective Targeting of Non-Diploid Cancer Cells. Strategy

Molecule

Target

Note

Refs

Targeting bioenergetic liabilities

AICAR Aspirin Resveratrol

AMPK

AICAR is particularly active against nondiploid cells when combined with the HSP90AA1 inhibitor 17-AAG

[64–66]

2-DG

Hexokinase

Non-diploid cells appear to be exquisitely dependent on glycolysis for survival

[67]

AZD8055

MTOR

In combination with AURKB inhibitors

[68]

Targeting redox homeostasis

NAC

ROS

NAC specifically targeted non-diploid cells via an AKT1-dependent mechanism

[37]

Targeting protein homeostasis

Cycloheximide Hygromycin Thiolutin

Protein synthesis

Aneuploidy as such (rather than specific chromosomal arrangements) was linked to high sensitivity to protein synthesis inhibitors

[49]

17-AAG Radicicol

HSP90AA1

17-AAG is particularly active against nondiploid cells when combined with the AMPK activator AICAR

[49,65,69,70]

Chloroquine

Lysosomal acidification

Chloroquine inhibits lysosomal degradation, hence blocking autophagy

[65]

MG132

Proteasome

Another proteasome inhibitor (i.e., bortezomib) is currently approved for therapy of multiple myeloma

[49]

Targeting non-oncogene addiction

ABT-263 siRNA

BCL-XL

BCL-XL is required for cells to cope with polyploidization-associated stress

[73,74]

Exacerbating genomic instability

MLN8237 ZM447439 siRNA

AURKB

Tetraploid cells were exquisitely sensitive to AURKB, but not AURKA, inhibition

[81]

shRNA

BUBR1

Inhibition of BUBR1 promoted TP53dependent cell death in polyploid cells

[76,82]

SD1825 UCN-01 siRNA dnCHK1

CHK1

CHK1 inhibition abolished SAC and caused premature and abnormal mitoses that led to TP53 activation and cell death

[82]

Dimethylenastron siRNA

EG5

Inhibition of EG5 in tetraploid cells promoted mitotic catastrophe and cell death

[80]

siRNA

HSET

HSET was essential for the viability of aneuploidy cancer cells

[79]

siRNA

MAD2

Downregulation of MAD2 promoted TP53-dependent death in tetraploid cells

[82]

SP600125 shRNA

MPS1

Inhibition of MPS1 promoted polyploidization followed by mitotic catastrophe

[76,83]

Inducing terminal differentiation/ senescence

H-1152P Dimethylfasudil MLN8237

AURKA AURKB

Possible approach for the treatment of acute megakaryocytic leukemia

[84]

Empirical

siRNA

ENDOG

It remains unclear why polyploid cells critically rely on ENDOG

[85]

Checkpoint blockage

Nivolumab Pembrolizumab

PD-1

Non-diploid cells may be very sensitive to checkpoint blockers owing to their mutanome

[32]

AKT1, v-akt murine thymoma viral oncogene homolog 1; dn, dominant negative; NAC, N-acetyl-L-cysteine; shRNA, short hairpin RNA; siRNA, small interfering RNA.

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An alternative approach for targeting non-diploidy harnesses the elevated reliance of aneuploid and polyploid cells on protein chaperones, proteasomal degradation, and autophagy [65,69,70]. An unbalanced chromosomal content has been shown to overload the protein quality control machinery, resulting in accrued levels of proteotoxic stress [69,70]. In line with this notion, non-diploid cells are exquisitely sensitive to agents that perturb protein synthesis, folding, or degradation, including: (i) the RNA polymerase inhibitor thiolutin [49]; (ii) the protein synthesis inhibitors cycloheximide and hygromycin [49]; (iii) the heat shock protein 90 kDa alpha (cytosolic), class A member 1 (HSP90AA1) inhibitors 17-demethoxygeldanamycin (17-AAG) and radicicol [49,65,69,70]; (iv) hyperthermia [49]; (v) the proteasomal inhibitor MG132 [49]; and (vi) the lysosomal inhibitor chloroquine [65]. Accordingly, non-diploid cells have been shown to exhibit constitutively high levels of autophagy [65,71] and to obtain a proliferative advantage from molecules that improve protein homeostasis, such as the autophagy inducer rapamycin [48]. Reflecting their high degree of non-oncogene addiction, aneuploid and polyploid cells also exhibit increased dependency on antiapoptotic molecules like the Bcl-2 family member BCL2like 1 (BCL2L1, best known as BCL-XL) [72]. In line with this notion, non-diploid cells are generally less resistant to BCL-XL inhibition (by pharmacological and genetic measures) than their diploid counterparts [73,74]. Along similar lines, the survival and proliferation of aneuploid and polyploid cells are intimately linked to the processes involved in the maintenance of genome stability, including DNA repair and mitosis [75,76]. In non-diploid cells, these processes are not only deregulated or weakened but also overstimulated, owing to the high levels of DNA replication stress and mitotic constraints (see above). Moreover, many tumors (irrespective of karyotype) strictly rely on the function of mitotic regulators [77] and exhibit considerable rewiring of the DNA repair machinery [78]. In line with this notion, various means to exacerbate genomic instability have been shown to efficiently deplete non-diploid cancer cells but less so their diploid counterparts. These means include the genetic or pharmacological inhibition of: (i) components of the centrosome clustering machinery, like kinesin family member C1 (KIFC1, best known as HSET) [79]; (ii) components of the mitotic apparatus, including BUBR1, aurora kinase A (AURKA), AURKB, TTK protein kinase [TTK, best known as monopolar spindle 1 (MPS1)], and kinesin family member 11 (KIF11, best known as EG5) [76,80,81]; and (iii) proteins involved in DNA damage signaling, such as checkpoint kinase 1 (CHEK1, best known as CHK1) [82]. Finally, strategies that promote polyploidization such as the inhibition of MPS1 may be lethal for various types of cancer, either because they drive an unscheduled and uncontrollable hyperploidization program (especially when cell cycle checkpoints are defective, such as in TP53-deficient cells) [83] or because they promote terminal differentiation and/or senescence [84]. Of note, some of these approaches are efficient only against malignant (as opposed to normal or immortalized) non-diploid cells [76]. Thus, the transformed and non-diploid state may synergize to render cells particularly susceptible to genome-destabilizing agents. Interestingly, polyploid cells are also particularly sensitive to the depletion of a mitochondrial endonuclease; namely, endonuclease G (ENDOG) [85]. Although ENDOG is involved in the execution of some instances of mitochondrial cell death [86], the reasons why polyploid cells exhibit accrued reliance on ENDOG remains to be identified. Despite this unknown, accumulating evidence suggests that targeting non-diploidy may lead to the development of novel and selective anticancer agents. Importantly, recent clinical findings suggest that the therapeutic efficacy of checkpoint blockers depends to a large extent on the tumor mutanome; that is, the ensemble of mutations accumulated in the course of oncogenesis and disease progression [32,87]. Thus, checkpoint blockers (and perhaps other types of immunotherapy) may also constitute an efficient means of specifically targeting non-diploid cancer cells for therapeutic purposes.

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Concluding Remarks

Outstanding Questions

When compatible with cellular life, karyotypic alterations may favor tumor progression and resistance to therapy by conferring on malignant cells the phenotypic and biochemical heterogeneity that is required for rapid adaptation to adverse microenvironmental conditions. However, the major consequences of non-diploidy, including extensive metabolic rewiring, perturbed proteome and redox homeostasis, and accrued genomic instability, make aneuploid and polyploid cells a potential target for treatment. In addition to the therapeutic strategies outlined above, we surmise that targeting pH regulation, which is intimately connected to increased lactate secretion [88], poly(ADP-ribose) polymerase 1 (PARP1), which is important for the preservation of genome stability [89], and ras-related C3 botulinum toxin substrate 1 (RAC1), which is activated by centrosome amplification and promotes invasion [59], constitute promising approaches for the therapeutic targeting of non-diploidy. To widen even further this therapeutic armamentarium, it will be interesting to characterize the interplay between metabolic alterations and major oncosuppressive pathways like autophagy and RCD in non-diploid cells [90,91].

What are the oncogenic signaling pathways that are linked to the acquisition of non-diploidy? Various oncogenic events (e.g., the loss of TP53) have been shown to favor the insurgence of non-diploidy, but the precise molecular mechanisms remain unclear.

Two additional considerations should be made in this context. First, the effect of major karyotypic alterations on cell biology displays a high degree of context dependency [2]. Second, neoplastic lesions are complex and dynamic entities that are characterized by: (i) considerable spatial and temporal genomic heterogeneity [92]; and (ii) continuous crosstalk between malignant cells and non-transformed tumor components [93]. Therapeutic approaches efficiently targeting non-diploidy may therefore need to be tailored based on tumor type, stage, or therapeutic history. Moreover, several questions remain to be answered for the development of anticancer therapies targeting non-diploidy (see Outstanding Questions). Clarifying these points may pave the way to a novel generation of antineoplastic agents that specifically harness the liabilities of non-diploid cancer cells. Acknowledgments The authors are indebted to José Manuel Bravo-San Pedro (Centre de Recherche des Cordeliers, Paris, France) for help with figure preparation. I.V. is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC: MFAG 2013 #14641), the Ministero Italiano della Salute (RF_GR-2011-02351355), and the Programma per i Giovani Ricercatori ‘Rita Levi Montalcini’ 2011. G.M. is supported by AIRC (Triennial Fellowship ‘Antonietta Latronico’, 2014). G.K. is supported by: the Ligue contre le Cancer (équipe labellisée); the Agence National de la Recherche (ANR) – Projets Blancs; the ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; the Association pour la Recherche sur le Cancer (ARC); the Cancéropôle Ile-de-France; the Institut National du Cancer (INCa); the Fondation Bettencourt–Schueller; the Fondation de France; the Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI).

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