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Polyploidy control in hepatic health and disease

  • Valentina C. Sladky
    Affiliations
    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Felix Eichin
    Affiliations
    Institute for Developmental Immunology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria
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  • Thomas Reiberger
    Affiliations
    Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

    Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases (LBI-RUD), 1090 Vienna, Austria

    CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria
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  • Andreas Villunger
    Correspondence
    Corresponding author. Address: Institute for Developmental Immunology, Biocenter, Medical University of Innsbruck, Innrain 80, A-6020, Innsbruck, Austria; Tel.: +43-512-9003-70380, fax: +43-512-9003-73960.
    Affiliations
    Institute for Developmental Immunology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

    Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases (LBI-RUD), 1090 Vienna, Austria

    CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria
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Open AccessPublished:July 02, 2021DOI:https://doi.org/10.1016/j.jhep.2021.06.030

      Summary

      A balanced increase in DNA content (ploidy) is observed in some human cell types, including bone-resorbing osteoclasts, platelet-producing megakaryocytes, cardiomyocytes or hepatocytes. The impact of increased hepatocyte ploidy on normal physiology and diverse liver pathologies is still poorly understood. Recent findings suggest swift genetic adaptation to hepatotoxic stress and the protection from malignant transformation as beneficial effects. Herein, we discuss the molecular mechanisms regulating hepatocyte polyploidisation and its implication for different liver diseases and hepatocellular carcinoma. We report on centrosomes’ role in limiting polyploidy by activating the p53 signalling network (via the PIDDosome multiprotein complex) and we discuss the role of this pathway in liver disease. Increased hepatocyte ploidy is a hallmark of hepatic inflammation and may play a protective role against liver cancer. Our evolving understanding of hepatocyte ploidy is discussed from the perspective of its potential clinical application for risk stratification, prognosis, and novel therapeutic strategies in liver disease and hepatocellular carcinoma.

      Keywords

      Different routes to polyploidy

      Polyploidisation describes the process of whole genome duplication. Polyploid cells harbour multiple copies of the entire genome, which, in contrast to aneuploidy, allows them to maintain balanced gene expression.
      • Fox D.T.
      • Soltis D.E.
      • Soltis P.S.
      • Ashman T.L.
      • Van de Peer Y.
      Polyploidy: a biological force from cells to ecosystems.
      ,
      • Van de Peer Y.
      • Mizrachi E.
      • Marchal K.
      The evolutionary significance of polyploidy.
      Whole-organism polyploidy is commonly found in plants, insects, and fungi where it is linked to diversity and evolution.
      • Van de Peer Y.
      • Mizrachi E.
      • Marchal K.
      The evolutionary significance of polyploidy.
      • Otto S.P.
      • Whitton J.
      Polyploid incidence and evolution.
      • Albertin W.
      • Marullo P.
      Polyploidy in fungi: evolution after whole-genome duplication.
      In vertebrates, it occurs in teleost fish, reptiles and amphibia,
      • Schmid M.
      • Evans B.J.
      • Bogart J.P.
      Polyploidy in Amphibia.
      while whole-organism polyploidy in mammals has been reported only once but was subsequently heavily disputed.
      • Gallardo M.H.
      • Kausel G.
      • Jiménez A.
      • Bacquet C.
      • González C.
      • Figueroa J.
      • et al.
      Whole-genome duplications in South American desert rodents (Octodontidae).
      ,
      • Svartman M.
      • Stone G.
      • Stanyon R.
      Molecular cytogenetics discards polyploidy in mammals.
      On a cellular level, however, polyploidy can be found as a distinct feature of specific and highly differentiated mammalian cell types, such as hepatocytes or cardiomyocytes.
      • Pandit S.K.
      • Westendorp B.
      • de Bruin A.
      Physiological significance of polyploidization in mammalian cells.
      • Duncan A.W.
      Aneuploidy, polyploidy and ploidy reversal in the liver.
      • Gan P.
      • Patterson M.
      • Sucov H.M.
      Cardiomyocyte polyploidy and implications for heart regeneration.
      For many of these cell types the physiological relevance of polyploidisation is still unclear (Fig. 1A).
      Figure thumbnail gr1
      Fig. 1Physiological polyploidisation is essential for the function of various cell types.
      (A) In megakaryocytes the degree of polyploidy is associated with their capability to produce platelets. Osteoclasts, which increase their size and ploidy through fusion and cytokinesis failure, depend on polyploidy for their effector function in bone resorption. Trophoblast giant cells in the placenta, which create the barrier between the foetus and the maternal blood, endocycle and reach a DNA content of up to 512N. Whether polyploidy in these cells is beneficial for the barrier function, or a stress response to save energy when rapid growth is needed, is still unclear. In the lactating mammary gland, binucleated epithelial cells, formed by cytokinesis failure, are required for efficient milk production. The skeletal muscle and the heart are also formed by polyploid cell types. While skeletal muscle generates large multinucleated myofibers through cell-cell fusion, early postnatal cardiomyocytes undergo rounds of cytokinesis failure and endoreduplication to achieve a polyploid state. The role of hepatocyte polyploidy is not fully resolved yet but has been shown to buffer tumorigenic events that lead to HCC. (B) Polyploidisation can be achieved through different routes. Cells undergoing endocycles alternate between G1 and S-phase, as seen in trophoblast giant cells. The endomitotic cell cycle is aborted before completion of mitosis and is found in megakaryocytes. Both of these alternative cell cycle modes generate mononucleated, polyploid daughter cells. Impaired cytokinesis generates binucleated cells, as chromosome segregation and formation of the nuclear envelope are completed before the cell cycle is aborted. This occurs in hepatocytes at the time of weaning or in postnatal cardiomyocytes. All of these alternative cell cycle modes generate polyploid progeny that also accumulate the corresponding number of centrosomes. HCC, hepatocellular carcinoma.
      Polyploidisation can be achieved through different mechanisms (Fig. 1B). Endocycles and endomitosis are abortive cell cycle modes in which cells, like megakaryocytes, skip nuclear (karyokinesis) and cellular division (cytokinesis).
      • Ravid K.
      • Lu J.
      • Zimmet J.M.
      • Jones M.R.
      Roads to polyploidy: the megakaryocyte example.
      ,
      • Edgar B.A.
      • Zielke N.
      • Gutierrez C.
      Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.
      Polyploidisation of hepatocytes and cardiomyocytes is mostly the result of incomplete cytokinesis, generating binucleated cells. Both of these cell types can also undergo endocycles, which leads to an increase in nuclear ploidy.
      • Pandit S.K.
      • Westendorp B.
      • de Bruin A.
      Physiological significance of polyploidization in mammalian cells.
      ,
      • Donne R.
      • Saroul-Aïnama M.
      • Cordier P.
      • Celton-Morizur S.
      • Desdouets C.
      Polyploidy in liver development, homeostasis and disease.
      Similar to polyploid cardiomyocytes or epithelial cells found in the lactating mammary gland, the increase in cell mass and ploidy is thought to be necessary for hepatocytes to carry out their excessive, physical, secretory and metabolic workload (Fig. 1A).
      • Donne R.
      • Saroul-Aïnama M.
      • Cordier P.
      • Celton-Morizur S.
      • Desdouets C.
      Polyploidy in liver development, homeostasis and disease.

      Hepatocyte polyploidy – origin and function

      Up to 40% of hepatocytes in the adult human liver are polyploid, while this percentage is twice as high in rodents.
      • Wang M.-J.
      • Chen F.
      • Lau J.T.Y.
      • Hu Y.-P.
      Hepatocyte polyploidization and its association with pathophysiological processes.
      ,
      • Kudryavtsev B.N.
      • Kudryavtseva M.V.
      • Sakuta G.A.
      • Stein G.I.
      Human hepatocyte polyploidization kinetics in the course of life cycle.
      The spatial distribution of polyploidy in the liver is mostly independent of metabolic liver zonation but a tendency toward lower ploidy in the periportal region (zone 1) has been reported. Considering temporal frequency, polyploid hepatocytes usually first appear in midlobular zone 2.
      • Tanami S.
      • Ben-Moshe S.
      • Elkayam A.
      • Mayo A.
      • Bahar Halpern K.
      • Itzkovitz S.
      Dynamic zonation of liver polyploidy.
      However, this effect evens out with age and polyploidy is fairly evenly distributed across the lobule in the adult human liver.
      • Tanami S.
      • Ben-Moshe S.
      • Elkayam A.
      • Mayo A.
      • Bahar Halpern K.
      • Itzkovitz S.
      Dynamic zonation of liver polyploidy.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      • Chen F.
      • Jimenez R.J.
      • Sharma K.
      • Luu H.Y.
      • Hsu B.Y.
      • Ravindranathan A.
      • et al.
      Broad distribution of hepatocyte proliferation in liver homeostasis and regeneration.
      In mice, homeostatic proliferation is most frequently observed in zone 2, as studied in inducible Ki67-Cre or Hamp2-Cre reporter mice.
      • Wei Y.
      • Wang Y.G.
      • Jia Y.
      • Li L.
      • Yoon J.
      • Zhang S.
      • et al.
      Liver homeostasis is maintained by midlobular zone 2 hepatocytes.
      ,
      • He L.
      • Pu W.
      • Liu X.
      • Zhang Z.
      • Han M.
      • Li Y.
      • et al.
      Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.
      In addition to zone 2, diploid pericentral (zone 3) hepatocytes also contribute to liver homeostasis
      • Wang B.
      • Zhao L.
      • Fish M.
      • Logan C.Y.
      • Nusse R.
      Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver.
      although the importance of this population is unclear.
      • Sun T.
      • Pikiolek M.
      • Orsini V.
      • Bergling S.
      • Holwerda S.
      • Morelli L.
      • et al.
      AXIN2+ pericentral hepatocytes have limited contributions to liver homeostasis and regeneration.
      Hepatocyte polyploidisation is a developmental process that is regulated by transcriptional and post-transcriptional mechanisms.
      Drug- and diet-induced liver injury primarily affect the periportal (zone 1) and pericentral (zone 3) hepatocyte populations (Fig. 2). Two recent studies show that zone 2 hepatocytes can regenerate the liver upon such types of zonal injury as this hepatocyte population is shielded from the insult.
      • Wei Y.
      • Wang Y.G.
      • Jia Y.
      • Li L.
      • Yoon J.
      • Zhang S.
      • et al.
      Liver homeostasis is maintained by midlobular zone 2 hepatocytes.
      ,
      • He L.
      • Pu W.
      • Liu X.
      • Zhang Z.
      • Han M.
      • Li Y.
      • et al.
      Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.
      Upon partial hepatectomy, a type of injury that induces regeneration without a zonal bias, a wave-like propagation of proliferation from the periportal vein (zone 1) to the central vein (zone 3) can be detected.
      • He L.
      • Pu W.
      • Liu X.
      • Zhang Z.
      • Han M.
      • Li Y.
      • et al.
      Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.
      ,
      • Rabes H.M.
      Kinetics of hepatocellular proliferation as a function of the microvascular structure and functional state of the liver.
      Figure thumbnail gr2
      Fig. 2Liver zonation and the proliferative response to damage.
      Hepatocytes fulfil different metabolic tasks depending on their zonal localisation. Periportal zone 1 hepatocytes are surrounded by oxygen-rich blood and handle oxidative processes (i.e. fatty acid oxidation), whereas pericentral zone 3 hepatocytes are important for drug detoxification and metabolic functions, including glycolysis. Zone 2 hepatocytes have high proliferative activity upon zonal liver injury and are important for liver homeostasis and regeneration. (A: hepatic artery, B: bile duct)
      The polyploidisation process has mostly been studied in mice and rats. It was shown to be triggered by weaning, giving rise to binucleated tetraploid daughter cells, but it is limited to a short window of time during this period of development.
      • Margall-Ducos G.
      • Celton-Morizur S.
      • Couton D.
      • Bregerie O.
      • Desdouets C.
      Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
      ,
      • Celton-Morizur S.
      • Merlen G.
      • Couton D.
      • Margall-Ducos G.
      • Desdouets C.
      The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.
      Further proliferation of such a polyploid cell either generates 2 mononucleated tetraploids or a binucleated octaploid cell, if cytokinesis fails again. After this initial wave, hepatocyte polyploidy increases only modestly over the lifetime of the organism. Consistent with this, a recent study shows that homeostatic proliferation is very low (an estimated cell fraction of only 0.042% in zone 2) but coincides with successful cytokinesis in >90% of proliferation events.
      • He L.
      • Pu W.
      • Liu X.
      • Zhang Z.
      • Han M.
      • Li Y.
      • et al.
      Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.
      Hence, the aged liver contains hepatocytes of all ploidy states reaching up to 16 copies in the wild-type mouse.
      • Pandit S.K.
      • Westendorp B.
      • de Bruin A.
      Physiological significance of polyploidization in mammalian cells.
      ,
      • Wang M.-J.
      • Chen F.
      • Lau J.T.Y.
      • Hu Y.-P.
      Hepatocyte polyploidization and its association with pathophysiological processes.
      Strikingly, in response to injury, quiescent polyploid hepatocytes re-enter the cell cycle to regenerate the liver. Although the overall liver ploidy is maintained in the regenerated liver, the ratio between mononucleated and binucleated hepatocytes changes. Dependent on the degree of injury, hepatocytes can undergo endoreduplication which increases the fraction of mononuclear hepatocytes.
      • Miyaoka Y.
      • Ebato K.
      • Kato H.
      • Arakawa S.
      • Shimizu S.
      • Miyajima A.
      Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.
      At the same time, diploid hepatocytes show increased proliferation rates, and thus contribute more to liver regeneration.
      • Wilkinson P.D.
      • Delgado E.R.
      • Alencastro F.
      • Leek M.P.
      • Roy N.
      • Weirich M.P.
      • et al.
      The polyploid state restricts hepatocyte proliferation and liver regeneration.
      Lack of core components of the p53 response machinery can increase hepatocyte ploidy up to 32 in steady state, or even 64 copies, after regeneration from a two-thirds partial hepatectomy.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Kurinna S.
      • Stratton S.A.
      • Coban Z.
      • Schumacher J.M.
      • Grompe M.
      • Duncan A.W.
      • et al.
      P53 regulates a mitotic transcription program and determines ploidy in normal mouse liver.
      Astonishingly, this all happens without an obvious impact on organ function.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      The exact timing of polyploidisation in the human liver is less clear but, as in mice, it increases with age.
      • Kudryavtsev B.N.
      • Kudryavtseva M.V.
      • Sakuta G.A.
      • Stein G.I.
      Human hepatocyte polyploidization kinetics in the course of life cycle.
      In line with reports analysing mouse livers,
      • Wang B.
      • Zhao L.
      • Fish M.
      • Logan C.Y.
      • Nusse R.
      Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver.
      ,
      • Wilkinson P.D.
      • Delgado E.R.
      • Alencastro F.
      • Leek M.P.
      • Roy N.
      • Weirich M.P.
      • et al.
      The polyploid state restricts hepatocyte proliferation and liver regeneration.
      homeostasis is mainly driven by diploid human hepatocytes.
      • Heinke P.
      • Rost F.
      • Rode J.
      • Welsch T.
      • Alkass K.
      • Feddema J.
      • et al.
      Diploid hepatocytes drive physiological liver renewal in adult humans.
      Several pathways have been implicated in hepatic ploidy control.
      • Hsu S.H.
      • Delgado E.R.
      • Otero P.A.
      • Teng K.Y.
      • Kutay H.
      • Meehan K.M.
      • et al.
      MicroRNA-122 regulates polyploidization in the murine liver.
      ,
      • Zhang S.
      • Chen Q.
      • Liu Q.
      • Li Y.
      • Sun X.
      • Hong L.
      • et al.
      Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2.
      The main mechanisms regulating polyploidisation are linked to insulin signalling in response to nutritional changes and transcriptional control of cytokinesis effector genes, but post-transcriptional effects involving microRNA (miR) action or direct interference with the process of cytokinesis have also been reported (see below).

      Transcriptional control of liver ploidy

      E2F transcription factors play a crucial role in the regulation of hepatocyte polyploidy.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      • Conner E.A.
      • Lemmer E.R.
      • Sánchez A.
      • Factor V.M.
      • Thorgeirsson S.S.
      E2F1 blocks and c-Myc accelerates hepatic ploidy in transgenic mouse models.
      The E2F family comprises the canonical transcriptional activators E2F1-3 and repressors E2F4-6, all of which are regulated by the retinoblastoma pocket proteins, Rb, p107 or p130.
      • Bertoli C.
      • Skotheim J.M.
      • de Bruin R.A.M.
      Control of cell cycle transcription during G1 and S phases.
      Canonical E2Fs contain only one DNA binding domain, hence they heterodimerise with their binding partners DP1, DP2 or DP3 to initiate transcription.
      • Trimarchi J.M.
      • Lees J.A.
      Sibling rivalry in the E2F family.
      The non-canonical repressors E2F7 and E2F8 comprise 2 DNA binding domains and thus function independently of DPs and pocket proteins.
      • Lammens T.
      • Li J.
      • Leone G.
      • De Veylder L.
      Atypical E2Fs: new players in the E2F transcription factor family.
      • Di Stefano L.
      • Jensen M.R.
      • Helin K.
      E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes.
      • Christensen J.
      • Cloos P.
      • Toftegaard U.
      • Klinkenberg D.
      • Bracken A.P.
      • Trinh E.
      • et al.
      Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription.
      Upon cell cycle entry, activators E2F1-3 induce transcription of their target genes, including the S-phase cyclins E and A, and several cytokinesis effectors. Additionally, E2F1-3 activate the expression of their own antagonists, E2F7/8, which immediately repress the same set of target genes.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      ,
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      ,
      • Bertoli C.
      • Skotheim J.M.
      • de Bruin R.A.M.
      Control of cell cycle transcription during G1 and S phases.
      ,
      • Trimarchi J.M.
      • Lees J.A.
      Sibling rivalry in the E2F family.
      This feedback loop between E2F7/8 and E2F1 results in oscillating protein levels over the cell cycle.
      • Bertoli C.
      • Skotheim J.M.
      • de Bruin R.A.M.
      Control of cell cycle transcription during G1 and S phases.
      In the mouse, deficiency in the activating E2Fs 1-3, or even just E2F1 alone, leads to increased hepatocyte ploidy, while its overexpression results in hypoploidy.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      • Conner E.A.
      • Lemmer E.R.
      • Sánchez A.
      • Factor V.M.
      • Thorgeirsson S.S.
      E2F1 blocks and c-Myc accelerates hepatic ploidy in transgenic mouse models.
      Conversely, livers deficient in the inhibitory E2Fs 7 and 8 are mostly diploid, as the absence of these transcriptional repressors favours normal cytokinesis.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      ,
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      This could be ascribed to the fact that some of the top transcriptional targets of E2F7/8 are essential cytokinesis factors: the guanine nucleotide exchange factor ECT2 (epithelial cell transforming 2), Aurora B kinase, RACGAP1 (Rac GTPase activating protein 1), MKLP1 (or KIF23), and other genes involved in DNA replication, mitosis, and p53-dependent cell cycle arrest.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      The postnatal peak in E2F7/8 expression occurs around the time of weaning, when the first wave of hepatocyte polyploidisation occurs.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      ,
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      Upregulation of these E2Fs was also observed during liver regeneration, consistent with their role in cell cycle regulation (Fig. 3A,B).
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      Figure thumbnail gr3
      Fig. 3Pathways regulating hepatocyte polyploidy.
      (A) Several pathways target the formation of the contractile ring to prevent successful cytokinesis. Increased insulin levels at weaning activate AKT signalling. This, in turn, impairs localisation of the small GTPase RhoA to the equatorial cortex. The transcriptional repressors E2F7/8 downregulate essential cytokinesis effectors and counteract the activating function of E2F1-3. Their transcriptional targets include the RhoA GEF ECT2, Aurora B kinase, and members of the centralspindlin complex. miR-122 targets the transcripts of cytokinesis effectors, such as RhoA and Cux1. Like E2F1, CUX1 is a transcriptional activator of ECT2 and centralspindlin complex components. The core components of the circadian clock Per1-3 promote faithful cell division. Per1 transcriptionally represses Mkp1, which increases the levels of phospho-ERK1/2, which is needed to complete abscission. (B) In addition to pathways that act on cytokinesis, others control hepatocyte ploidy by regulating CDK inhibitors. The Hippo pathway component YAP sequesters cytoplasmic Skp2 and, thus, its target p27 accumulates and causes polyploidisation. Centriole distal appendages trigger assembly of the PIDDosome multiprotein complex in response to centrosome accumulation in polyploid hepatocytes. This activates caspase-2, which proteolytically inactivates MDM2, the inhibitor of p53. Stabilisation of p53 restricts further polyploidisation of hepatocytes via p21. As the expression of PIDD1 and caspase-2 is controlled by E2F transcription factors, the PIDDosome ploidy-regulating mechanism is part of the E2F regulatory circuit that ensures balanced hepatocyte polyploidisation. Square boxes identify transcription factors. Cux1, cut like homeobox 1; Ect2, epithelial cell transforming 2; miR, microRNA; Mkp1, mitogen-activated protein kinase phosphatase 1; PIDD1, p53-induced death domain protein 1; RhoA, Ras homolog family member A; SKP2, S-phase kinase associated protein 2; YAP, yes-associated protein.
      The tumour suppressor p53 and its most prominent transcriptional target, the cyclin-dependent kinase (CDK) inhibitor p21, have also been shown to regulate hepatic ploidy. Consistently, loss of either p53 or p21 promote hepatocyte polyploidisation during both normal development and regeneration.
      • Kurinna S.
      • Stratton S.A.
      • Coban Z.
      • Schumacher J.M.
      • Grompe M.
      • Duncan A.W.
      • et al.
      P53 regulates a mitotic transcription program and determines ploidy in normal mouse liver.
      ,
      • Sheahan S.
      • Bellamy C.O.
      • Treanor L.
      • Harrison D.J.
      • Prost S.
      Additive effect of p53, p21 and Rb deletion in triple knockout primary hepatocytes.
      Moreover, disruption of circadian oscillations also leads to hyperpolyploidisation of midlobular and pericentral hepatocytes (zones 2 and 3).
      • Chao H.W.
      • Doi M.
      • Fustin J.M.
      • Chen H.
      • Murase K.
      • Maeda Y.
      • et al.
      Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway.
      A mouse model deficient in all Period genes (Per1-3) displayed transcriptional increases in Mkp1 (mitogen-activated protein kinase phosphatase 1). This phosphatase, in turn, inactivated ERK1/2 during cytokinesis, preventing successful cell division (Fig. 3A).
      • Chao H.W.
      • Doi M.
      • Fustin J.M.
      • Chen H.
      • Murase K.
      • Maeda Y.
      • et al.
      Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway.

      Post-transcriptional control of liver ploidy

      Weaning, the transition from suckling to solid food, causes an increase in insulin levels, which triggers the failure of cytokinesis in rodent hepatocytes. Insulin activates the PI3K/AKT pathway, which is suggested to impair Ras homolog family member A (RhoA) activity.
      • Celton-Morizur S.
      • Merlen G.
      • Couton D.
      • Margall-Ducos G.
      • Desdouets C.
      The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.
      Desdouets and colleagues found that in rat weanlings RhoA and other essential cytokinesis effectors are delocalised upon increased insulin signalling, thereby preventing cleavage furrow formation and ingression.
      • Margall-Ducos G.
      • Celton-Morizur S.
      • Couton D.
      • Bregerie O.
      • Desdouets C.
      Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
      The exact mechanism linking insulin and RhoA impairment is not fully understood (Fig. 3A,B). As in rats, weaning also induces polyploidisation in the mouse liver.
      • Zhang S.
      • Zhou K.
      • Luo X.
      • Li L.
      • Tu H.C.
      • Sehgal A.
      • et al.
      The polyploid state plays a tumor-suppressive role in the liver.
      The centrosome-PIDDosome-p53 signalling axis is part of the E2F transcriptional regulatory circuit and functions as a break on hyperpolyploidisation.
      Another way to repress cytokinesis factors and facilitate hepatocyte polyploidisation involves miR-122, which is mainly expressed in the liver. The mRNA targets that are repressed or degraded by miR-122 include those encoding cytokinesis factors such as RhoA and CUX1 (cut like homeobox 1), a transcriptional activator of centralspindlin components. Consistently, loss of this miR significantly reduces hepatic ploidy.
      • Hsu S.H.
      • Delgado E.R.
      • Otero P.A.
      • Teng K.Y.
      • Kutay H.
      • Meehan K.M.
      • et al.
      MicroRNA-122 regulates polyploidization in the murine liver.
      Several other pathways have been found to negatively regulate polyploidisation, thereby counteracting the effects of insulin, miR-122 and E2Fs. Loss of LKB1 (liver kinase B1), cyclin E1, or CDK1 leads to hyperpolyploidy.
      • Aziz K.
      • Limzerwala J.F.
      • Sturmlechner I.
      • Hurley E.
      • Zhang C.
      • Jeganathan K.B.
      • et al.
      Ccne1 overexpression causes chromosome instability in liver cells and liver tumor development in mice.
      • Miettinen T.P.
      • Pessa H.K.J.
      • Caldez M.J.
      • Fuhrer T.
      • Diril M.K.
      • Sauer U.
      • et al.
      Identification of transcriptional and metabolic programs related to mammalian cell size.
      • Maillet V.
      • Boussetta N.
      • Leclerc J.
      • Fauveau V.
      • Foretz M.
      • Viollet B.
      • et al.
      LKB1 as a gatekeeper of hepatocyte proliferation and genomic integrity during liver regeneration.
      Mice deficient in the E3 ligase S-phase kinase associated protein 2 (SKP2) accumulate its key-target, the CDK inhibitor p27, which results in massive liver polyploidisation.
      • Nakayama K.
      • Nagahama H.
      • Minamishima Y.A.
      • Matsumoto M.
      • Nakamichi I.
      • Kitagawa K.
      • et al.
      Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication.
      Intriguingly, the Hippo pathway also acts through SKP2 to regulate hepatocyte polyploidy. Yes-associated protein (YAP) activation causes AKT phosphorylation, leading to cytoplasmic retention of SKP2 and, therefore, p27 accumulation in the nucleus (Fig. 3B). In addition, degradation of the transcription factor FOXO1/3 (forkhead box O1/3) is deregulated, which together results in hepatic hyperploidy.
      • Zhang S.
      • Chen Q.
      • Liu Q.
      • Li Y.
      • Sun X.
      • Hong L.
      • et al.
      Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2.
      Although work in cell lines suggested that the Hippo pathway acts upstream of p53 to control cell cycle progression of tetraploids in vitro,
      • Ganem N.J.
      • Cornils H.
      • Chiu S.-Y.
      • O’Rourke K.P.
      • Arnaud J.
      • Yimlamai D.
      • et al.
      Cytokinesis failure triggers Hippo tumor suppressor pathway activation.
      in the liver, the increased ploidy observed upon YAP activation was independent of p53.
      • Zhang S.
      • Chen Q.
      • Liu Q.
      • Li Y.
      • Sun X.
      • Hong L.
      • et al.
      Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2.
      Most likely, Hippo and p53 represent parallel mechanisms that act synergistically to limit hepatic ploidy.
      • Zhang S.
      • Chen Q.
      • Liu Q.
      • Li Y.
      • Sun X.
      • Hong L.
      • et al.
      Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2.

      The PIDDosome-p53 axis in hepatocyte polyploidisation

      The PIDDosome is a multiprotein complex that acts as an activating platform for caspase-2 (CASP2).
      • Tinel A.
      • Tschopp J.
      The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress.
      In design, the PIDDosome is similar to the “apoptosome” or “inflammasomes” that are needed to activate caspase-9 or caspase-1, -4 and -5, for the initiation of apoptosis or pyroptosis, respectively.
      • Tinel A.
      • Tschopp J.
      The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress.
      • Bao Q.
      • Shi Y.
      Apoptosome: a platform for the activation of initiator caspases.
      • Zheng D.
      • Liwinski T.
      • Elinav E.
      Inflammasome activation and regulation: toward a better understanding of complex mechanisms.
      The PIDDosome comprises the death domain (DD) containing proteins p53-induced DD protein 1 (PIDD1) and CRADD (also known as RAIDD). CRADD/RAIDD also contains a caspase-recruitment domain, which allows it to interact with and activate an aspartate-specific protease (CASP2). This interaction was initially reported to promote cell death upon DNA damage.
      • Tinel A.
      • Tschopp J.
      The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress.
      Over the last few years, however, we have gained deeper insights into how CASP2 activation is accomplished in normal physiology. First reports by Fava et al. suggested that the PIDDosome becomes activated in transformed or immortalised cells that fail cytokinesis.
      • Fava L.L.
      • Schuler F.
      • Sladky V.
      • Haschka M.D.
      • Soratroi C.
      • Eiterer L.
      • et al.
      The PIDDosome activates p53 in response to supernumerary centrosomes.
      The trigger for PIDDosome activation appeared to be the extra centrosome that accumulates in such cells. Centrosomes consist of a pair of centrioles and the surrounding pericentriolar material; they organise the mitotic spindle apparatus and template cilia formation in multiple differentiated cell types. As with DNA, the centrosome duplicates exactly once per cell cycle to enable the formation of 2 spindle poles for chromosome segregation.
      • Nigg E.A.
      • Holland A.J.
      Once and only once: mechanisms of centriole duplication and their deregulation in disease.
      Accordingly, polyploid cells, such as hepatocytes, accumulate extra centrosomes by incomplete cell division.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      ,
      • Faggioli F.
      • Vezzoni P.
      • Montagna C.
      Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.
      Interestingly, recent work in cultured cells showed that PIDD1 is recruited to the older centriole by the centriolar distal appendage protein ANKRD26 (ankyrin repeat domain 26).
      • Fava L.L.
      • Schuler F.
      • Sladky V.
      • Haschka M.D.
      • Soratroi C.
      • Eiterer L.
      • et al.
      The PIDDosome activates p53 in response to supernumerary centrosomes.
      ,
      • Burigotto M.
      • Mattivi A.
      • Migliorati D.
      • Magnani G.
      • Valentini C.
      • Roccuzzo M.
      • et al.
      Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.
      • Evans L.T.
      • Anglen T.
      • Scott P.
      • Lukasik K.
      • Loncarek J.
      • Holland A.J.
      ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification.
      • Bowler M.
      • Kong D.
      • Sun S.
      • Nanjundappa R.
      • Evans L.
      • Farmer V.
      • et al.
      High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy.
      This localisation is essential for PIDDosome activation in response to excess centrosomes.
      • Fava L.L.
      • Schuler F.
      • Sladky V.
      • Haschka M.D.
      • Soratroi C.
      • Eiterer L.
      • et al.
      The PIDDosome activates p53 in response to supernumerary centrosomes.
      ,
      • Burigotto M.
      • Mattivi A.
      • Migliorati D.
      • Magnani G.
      • Valentini C.
      • Roccuzzo M.
      • et al.
      Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.
      ,
      • Evans L.T.
      • Anglen T.
      • Scott P.
      • Lukasik K.
      • Loncarek J.
      • Holland A.J.
      ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification.
      Once activated, CASP2 proteolytically inactivates the E3 ligase MDM2, which controls protein levels of the tumour suppressor p53.
      • Oliver T.G.
      • Meylan E.
      • Chang G.P.
      • Xue W.
      • Burke J.R.
      • Humpton T.J.
      • et al.
      Caspase-2-Mediated cleavage of Mdm2 creates a p53-induced positive feedback loop.
      Subsequent stabilisation of p53 and the expression of its target genes, most notably p21, results in cell cycle arrest and therefore ensures that polyploid cells with extra centrosomes no longer proliferate.
      • Fava L.L.
      • Schuler F.
      • Sladky V.
      • Haschka M.D.
      • Soratroi C.
      • Eiterer L.
      • et al.
      The PIDDosome activates p53 in response to supernumerary centrosomes.
      This is critical, as centrosome amplification is sufficient to drive tumorigenesis and fuels chromosomal instability (CIN), a common feature of human cancer.
      • Gönczy P.
      Centrosomes and cancer: revisiting a long-standing relationship.
      ,
      • Levine M.S.
      • Bakker B.
      • Boeckx B.
      • Moyett J.
      • Lu J.
      • Vitre B.
      • et al.
      Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals.
      The PIDDosome-p53 axis is critically involved in liver development and regeneration. Remarkably, hepatocytes are mainly diploid until weaning in both wild-type and PIDDosome-deficient mice.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      Nevertheless, 5 days after weaning most hepatocytes fail cytokinesis and become tetraploid, which was paralleled by PIDDosome-dependent p53 accumulation. PIDDosome-deficient mice present considerably increased hepatocyte ploidy levels, mimicking the well-known p53 and p21 loss-of-function phenotypes.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      Together, this confirms the physiological relevance of the PIDDosome-p53 axis during liver development. Strikingly, chromatin immunoprecipitation sequencing analysis revealed Casp2 and Pidd1 as direct targets of E2F1 and E2F8, identifying them as essential effectors of the E2F ploidy control circuit. Consistently, hepatocyte-specific loss of E2F7/8 in mice leads to increased expression of Pidd1 and Casp2, which is no longer detectable upon additional deletion of E2F1 (Fig. 3B).
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      Together, the E2F transcriptional circuit promotes polyploidisation by downregulating the cytokinesis machinery and, at the same time, repressing PIDDosome-p53-dependent growth arrest.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      Inflammatory liver pathologies can directly or indirectly promote hepatocyte polyploidisation.
      PIDD1 and CASP2 are barely detectable in adult hepatocytes, raising the possibility that the reported role for p53 in limiting ploidy during liver regeneration may involve alternative modes of p53 activation. Yet, during regeneration following two-thirds partial hepatectomy, hepatocytes re-express PIDD1 and CASP2 and the lack of a functional PIDDosome-p53-p21 axis resulted in faster regeneration of liver mass.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      The PIDDosome component CRADD/RAIDD is constantly expressed in mouse livers and recent work suggests that it may be preferentially found in zone 2 hepatocytes. More strikingly, CRADD/RAIDD appears to suppress liver regeneration emanating from zone 2 hepatocytes. This is suggested by the results from an elegant targeted transposon-based CRISPR loss-of-function and a trans-activation screen, using selected single guide RNAs for genes preferentially expressed in zone 2 hepatocytes.
      • Wei Y.
      • Wang Y.G.
      • Jia Y.
      • Li L.
      • Yoon J.
      • Zhang S.
      • et al.
      Liver homeostasis is maintained by midlobular zone 2 hepatocytes.
      Together, these observations open up the possibility that breaking the ploidy barrier by chemical inhibition of the PIDDosome may promote liver regeneration in transplantation or other regenerative settings. In support of conservation of mechanism, we confirmed similar dynamics of E2F1, CASP2 and p21 activation, along with increased ploidy, in liver biopsies sequentially taken from patients undergoing the ALPPS procedure (associating liver partition and portal vein ligation for staged hepatectomy).
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.

      Polyploidy in liver health and disease

      Physiological polyploidisation is crucial for the function of various cell types, such as megakaryocytes,
      • Mattia G.
      • Vulcano F.
      • Milazzo L.
      • Barca A.
      • Macioce G.
      • Giampaolo A.
      • et al.
      Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release.
      ,
      • Mazzi S.
      • Lordier L.
      • Debili N.
      • Raslova H.
      • Vainchenker W.
      Megakaryocyte and polyploidization.
      trophoblast giant cells,
      • Pandit S.K.
      • Westendorp B.
      • de Bruin A.
      Physiological significance of polyploidization in mammalian cells.
      ,
      • Hu D.
      • Cross J.C.
      Development and function of trophoblast giant cells in the rodent placenta.
      osteoclasts,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • et al.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ,
      • Takegahara N.
      • Kim H.
      • Mizuno H.
      • Sakaue-Sawano A.
      • Miyawaki A.
      • Tomura M.
      • et al.
      Involvement of receptor activator of nuclear factor-κB ligand (RANKL)-induced incomplete cytokinesis in the polyploidization of osteoclasts.
      skeletal muscle,
      • Abmayr S.M.
      • Pavlath G.K.
      Myoblast fusion: lessons from flies and mice.
      or cardiomyocytes.
      • Alkass K.
      • Panula J.
      • Westman M.
      • Wu T Di
      • Guerquin-Kern J.L.
      • Bergmann O.
      No evidence for cardiomyocyte number expansion in preadolescent mice.
      Binucleation was found in keratinocytes, in epithelial cells lining the adult urothelium, and in the lactating mammary gland (Fig. 1A).
      • Wang J.
      • Batourina E.
      • Schneider K.
      • Souza S.
      • Swayne T.
      • Liu C.
      • et al.
      Polyploid superficial cells that maintain the urothelial barrier are produced via incomplete cytokinesis and endoreplication.
      • Gandarillas A.
      • Davies D.
      • Blanchard J.M.
      Normal and c-Myc-promoted human keratinocyte differentiation both occur via a novel cell cycle involving cellular growth and endoreplication.
      • Sanz-Gómez N.
      • de Pedro I.
      • Ortigosa B.
      • Santamaría D.
      • Malumbres M.
      • de Cárcer G.
      • et al.
      Squamous differentiation requires G2/mitosis slippage to avoid apoptosis.
      • Rios A.C.
      • Fu N.Y.
      • Jamieson P.R.
      • Pal B.
      • Whitehead L.
      • Nicholas K.R.
      • et al.
      Essential role for a novel population of binucleated mammary epithelial cells in lactation.
      Aside from beneficial polyploidy in these specialised cell types, several pathogens can induce polyploidisation. Macrophages can become polyploid as macrophage giant cells in response to bacterial infections.
      • Herrtwich L.
      • Nanda I.
      • Evangelou K.
      • Nikolova T.
      • Horn V.
      • Sagar
      • et al.
      DNA damage signaling instructs polyploid macrophage fate in granulomas.
      Viral infections with cytomegalovirus
      • Tang J.
      • Frascaroli G.
      • Lebbink R.J.
      • Ostermann E.
      • Brune W.
      Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability.
      ,
      • Spector D.H.
      Human cytomegalovirus riding the cell cycle.
      or human papilloma virus cause syncytia formation via cell-cell fusion or polyploidisation by cell cycle deregulation.
      • Spector D.H.
      Human cytomegalovirus riding the cell cycle.
      ,
      • Herbein G.
      • Nehme Z.
      Polyploid giant cancer cells, a hallmark of oncoviruses and a new therapeutic challenge.
      Infections with oncogenic viruses, such as human papilloma virus, contribute to the polyploidy seen in cancer. However, the relative contribution of polyploidy to the pathogenesis of virus-driven cancers remains to be clarified.
      Polyploidy is a common feature of tumorigenesis, found in more than one-third of human cancers.
      • Zack T.I.
      • Schumacher S.E.
      • Carter S.L.
      • Cherniack A.D.
      • Saksena G.
      • Tabak B.
      • et al.
      Pan-cancer patterns of somatic copy number alteration.
      • Quinton R.J.
      • DiDomizio A.
      • Vittoria M.A.
      • Ticas C.J.
      • Patel S.
      • Koga Y.
      • et al.
      Whole genome doubling confers unique genetic vulnerabilities on tumor cells.
      • Bielski C.M.
      • Zehir A.
      • Penson A.V.
      • Donoghue M.T.A.
      • Chatila W.
      • Armenia J.
      • et al.
      Genome doubling shapes the evolution and prognosis of advanced cancers.
      Whole genome duplication can cause CIN and is a known driver of karyotype evolution in cancer.
      • Laughney A.M.
      • Elizalde S.
      • Genovese G.
      • Bakhoum S.F.
      Dynamics of tumor heterogeneity derived from clonal karyotypic evolution.
      • Davoli T.
      • de Lange T.
      The causes and consequences of polyploidy in normal development and cancer.
      • Watkins T.B.K.
      • Lim E.L.
      • Petkovic M.
      • Elizalde S.
      • Birkbak N.J.
      • Wilson G.A.
      • et al.
      Pervasive chromosomal instability and karyotype order in tumour evolution.
      • Dewhurst S.M.
      • McGranahan N.
      • Burrell R.A.
      • Rowan A.J.
      • Grönroos E.
      • Endesfelder D.
      • et al.
      Tolerance of whole- genome doubling propagates chromosomal instability and accelerates cancer genome evolution.
      However, in the liver, polyploidy has been suggested to promote the generation and expansion of beneficial karyotypes to cope with stress. This has been investigated using a mouse model for hereditary chronic tyrosinemia caused by deficiency of fumarylacetoacetate hydrolase (Fah-/-). This results in the accumulation of a toxic intermediate that causes cell death. Inhibition or loss of the upstream enzyme, homogentisate 1,2 dioxygenase (Hgd), that generates the toxic intermediate can reverse this effect. Livers of Fah-/-Hgd+/- animals formed regenerative nodules, mainly consisting of a subpopulation of cells with a specific karyotype that had lost the chromosome with the remaining Hgd allele.
      • Duncan A.W.
      • Hanlon Newell A.E.
      • Bi W.
      • Finegold M.J.
      • Olson S.B.
      • Beaudet A.L.
      • et al.
      Aneuploidy as a mechanism for stress-induced liver adaptation.
      ,
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      Wilkinson et al. utilised liver-specific E2F7/8 knock-out to achieve lower ploidy in the Fah-/-Hgd+/- model for chronic liver injury. Lower polyploidy reduced the degree of aneuploidy and impaired successful regeneration, as monitored by animal survival.
      • Wilkinson P.D.
      • Alencastro F.
      • Delgado E.R.
      • Leek M.P.
      • Weirich M.P.
      • Otero P.A.
      • et al.
      Polyploid hepatocytes facilitate adaptation and regeneration to chronic liver injury.
      Whether polyploidy in the liver is associated with aneuploidy is controversial. While studies measuring aneuploidy using fluorescent in situ hybridisation detected a substantial degree of aneuploidy,
      • Duncan A.W.
      • Hanlon Newell A.E.
      • Smith L.
      • Wilson E.M.
      • Olson S.B.
      • Thayer M.J.
      • et al.
      Frequent aneuploidy among normal human hepatocytes.
      ,
      • Duncan A.W.
      • Taylor M.H.
      • Hickey R.D.
      • Hanlon Newell A.E.
      • Lenzi M.L.
      • Olson S.B.
      • et al.
      The ploidy conveyor of mature hepatocytes as a source of genetic variation.
      the results of single cell whole genome sequencing suggest much lower aneuploidy levels.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Knouse K.A.
      • Wu J.
      • Whittaker C.A.
      • Amon A.
      Single cell sequencing reveals low levels of aneuploidy across mammalian tissues.
      ,
      • Knouse K.A.
      • Lopez K.E.
      • Bachofner M.
      • Amon A.
      Chromosome segregation fidelity in epithelia requires tissue architecture.
      However, the degree of chromosome copy number variation clearly increases under proliferative pressure during regeneration and with the degree of polyploidy,
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      yet at overall low levels.
      While the immediate functional advantage of being polyploid is clear for many of the aformentioned cell types, the benefit for liver function and the organism as a whole is less well understood. Recent studies suggest differences between diploid and polyploid hepatocytes in their metabolic capacity and specialisation. Kreutz et al. performed transcriptome analysis on diploid vs. polyploid hepatocytes, which were sorted based on their insulin affinity. Most differentially regulated genes were associated with RNA, protein, and fatty acid metabolism, as well as transport of fatty acids and cholesterol.
      • Kreutz C.
      • MacNelly S.
      • Follo M.
      • Wäldin A.
      • Binninger-Lacour P.
      • Timmer J.
      • et al.
      Hepatocyte ploidy is a diversity factor for liver homeostasis.
      This notion is further supported by RNA-seq of sorted 2n and 4n hepatocyte nuclei showing that genes associated with energy homeostasis and drug metabolism are differentially expressed.
      • Richter M.L.
      • Deligiannis I.K.
      • Danese A.
      • Lleshi E.
      • Coupland P.
      • Vallejos C.A.
      • et al.
      Single-nucleus RNA-seq2 reveals a functional crosstalk between liver zonation and ploidy.
      Nonetheless, E2f7/8 double-deficient mice with mostly diploid hepatocytes have no obvious defects in liver function, hence the true benefit of polyploidy in the liver remains unknown.
      • Pandit S.K.
      • Westendorp B.
      • Nantasanti S.
      • Van Liere E.
      • Tooten P.C.J.
      • Cornelissen P.W.A.
      • et al.
      E2F8 is essential for polyploidization in mammalian cells.
      ,
      • Chen H.Z.
      • Ouseph M.M.
      • Li J.
      • Pécot T.
      • Chokshi V.
      • Kent L.
      • et al.
      Canonical and atypical E2Fs regulate the mammalian endocycle.
      In analogy to other polyploid cell types, hepatocyte polyploidisation could be a way to save energy in periods of stress and/or extensive liver growth. Consistent with this idea, the first wave of cytokinesis failure occurs during weaning, when liver growth coincides with a high metabolic load due to the severe nutritional change.
      • Margall-Ducos G.
      • Celton-Morizur S.
      • Couton D.
      • Bregerie O.
      • Desdouets C.
      Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
      ,
      • Celton-Morizur S.
      • Merlen G.
      • Couton D.
      • Margall-Ducos G.
      • Desdouets C.
      The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.
      Similarly, several mouse models, but also studies with human patient material, suggest that hepatocyte ploidy increases in chronic diseases where proliferation is induced to compensate for ongoing loss of liver tissue.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      ,
      • Chen F.
      • Jimenez R.J.
      • Sharma K.
      • Luu H.Y.
      • Hsu B.Y.
      • Ravindranathan A.
      • et al.
      Broad distribution of hepatocyte proliferation in liver homeostasis and regeneration.
      ,
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      ,
      • Toyoda H.
      • Bregerie O.
      • Vallet A.
      • Nalpas B.
      • Pivert G.
      • Brechot C.
      • et al.
      Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis.
      In contrast to most other tissues, in the liver, hepatocyte polyploidy protects from tumorigenesis by buffering tumour suppressor gene loss.

      Ploidy in liver injury and inflammation

      Chronic or repetitive liver injury can cause apoptosis and/or necrosis of hepatocytes, creating a proinflammatory microenvironment that can lead to fibrosis and cirrhosis. All of these conditions are accompanied by compensatory hepatocyte proliferation which may ultimately culminate in hepatocellular carcinoma (HCC).
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      ,
      • Arzumanyan A.
      • Reis H.M.G.P.V.
      • Feitelson M.A.
      Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma.
      Several types of chronic liver diseases have been associated with increased hepatocyte polyploidy, consistent with the notion that proliferation under stress conditions promotes alternative cell cycle modes, including failed cytokinesis and endoreduplication. Unlike weaning-induced polyploidisation, during regeneration following partial hepatectomy, hepatocytes tend to switch to endoreduplication rather than cytokinesis failure in mice.
      • Miyaoka Y.
      • Ebato K.
      • Kato H.
      • Arakawa S.
      • Shimizu S.
      • Miyajima A.
      Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.
      This might explain the increase in nuclear polyploidy vs. cellular polyploidy that has been observed upon chronic liver disease.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      ,
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      ,
      • Toyoda H.
      • Bregerie O.
      • Vallet A.
      • Nalpas B.
      • Pivert G.
      • Brechot C.
      • et al.
      Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis.
      Whether the higher frequency of mononucleated polyploids has any direct consequences for the liver is unclear. It seems more likely that the presence of these cells is indicative of compensatory regeneration and proliferation phases in the disease context. Thus, the frequency of these cells could potentially correlate with the severity of the chronic liver disease.
      Polyploidisation in response to injury is also found in other tissues and organisms, such as wound healing in drosophila, or heart regeneration in zebra fish.
      • Cao J.
      • Wang J.
      • Jackman C.P.
      • Cox A.H.
      • Trembley M.A.
      • Balowski J.J.
      • et al.
      Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue.
      ,
      • Losick V.P.
      • Fox D.T.
      • Spradling A.C.
      Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium.
      Similar observations were made in the mammalian urothelium.
      • Wang J.
      • Batourina E.
      • Schneider K.
      • Souza S.
      • Swayne T.
      • Liu C.
      • et al.
      Polyploid superficial cells that maintain the urothelial barrier are produced via incomplete cytokinesis and endoreplication.
      Together, these data support the hypothesis that polyploidisation might be a way to achieve rapid regeneration by saving the time and the energy required for actual cell division in periods of stressful growth, triggered by acute or chronic injury. Interestingly, however, recent data point towards a mutual relationship of polyploidy and fibrosis, suggesting that the situation is actually more complex.
      • Dewhurst M.R.
      • Ow J.R.
      • Zafer G.
      • van Hul N.K.M.
      • Wollmann H.
      • Bisteau X.
      • et al.
      Loss of hepatocyte cell division leads to liver inflammation and fibrosis.

      Viral hepatitis

      A major global liver disease aetiology that often leads to HCC development is viral hepatitis, caused by infection with HBV or HCV.
      • Arzumanyan A.
      • Reis H.M.G.P.V.
      • Feitelson M.A.
      Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma.
      Infection with HBV has been associated with elevated ploidy levels compared to other aetiologies.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      ,
      • Toyoda H.
      • Bregerie O.
      • Vallet A.
      • Nalpas B.
      • Pivert G.
      • Brechot C.
      • et al.
      Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis.
      ,
      • Sagnelli E.
      • Pasquale G.
      • Coppola N.
      • Scarano F.
      • Marrocco C.
      • Scolastico C.
      • et al.
      Influence of chronic coinfection with hepatitis B and C virus on liver histology.
      Toyoda et al. reported a clear correlation between the number of mononuclear polyploids and the severity of HBV- or HCV-driven chronic hepatitis, which was inferred from the degree of fibrosis and the severity of hepatitis. Interestingly, the fraction of binucleated polyploids was higher in patients with HBV than in those with HCV, independent of the severity of the disease.
      • Toyoda H.
      • Bregerie O.
      • Vallet A.
      • Nalpas B.
      • Pivert G.
      • Brechot C.
      • et al.
      Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis.
      This is in line with our own data showing a significantly higher degree of overall polyploidy in HBV-infected livers compared to HCV-infected livers (Fig. 4).
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      Figure thumbnail gr4
      Fig. 4Hepatocyte ploidy in liver health, disease and cancer.
      In physiological conditions (i.e. in the healthy liver) about 40% of human hepatocytes show increased nuclear and cellular polyploidy. Importantly, the levels of hepatocyte ploidy increase with age. In liver disease, increased hepatocyte polyploidy is frequently observed in liver biopsies obtained from patients with viral hepatitis (HBV more than in HCV), and in patients with fatty liver disease (in NASH more than in ALD). In general, increased hepatocyte ploidy has been associated with liver inflammation and liver fibrosis. Consequently, hepatocyte ploidy in patients with liver disease may be used as a prognostic tool for risk stratification of disease progression, and potentially for guiding chemoprevention strategies against HCC. During hepatocarcinogenesis, there is a reduction in cellular and nuclear polyploidy. A standardized assessment of HCC tumour cell ploidy may allow for patient stratification and for optimised and individualised treatment strategies. ALD, alcohol-related liver disease; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis.
      As HCV and HBV are unrelated viruses, they might act through distinct routes to promote polyploidisation. Overexpression of the HCV core protein in different cell lines in vitro has been suggested to increase polyploidy by interfering with the retinoblastoma pathway, but further work is needed to dissect this observation.
      • Machida K.
      • Liu J.-C.
      • McNamara G.
      • Levine A.
      • Duan L.
      • Lai M.M.C.
      Hepatitis C virus causes uncoupling of mitotic checkpoint and chromosomal polyploidy through the Rb pathway.
      While the interrelation of HCV and polyploidy is poorly understood, the HBV protein HBx has been directly implicated in modifying the cell cycle. HBx was found to induce premature DNA replication in G2-phase by deregulation of replication licensing factors and Polo like kinase 1 (PLK1) in an immortalised hepatocyte cell line. This unscheduled DNA replication was accompanied by a high degree of DNA damage and increased polyploidy, the latter was further augmented in the absence of p53.
      • Rakotomalala L.
      • Studach L.
      • Wang W.H.
      • Gregori G.
      • Hullinger R.L.
      • Andrisani O.
      Hepatitis B virus X protein increases the Cdt1-to-geminin ratio inducing DNA re-replication and polyploidy.
      ,
      • Studach L.
      • Wang W.H.
      • Weber G.
      • Tang J.
      • Hullinger R.L.
      • Malbrue R.
      • et al.
      Polo-like kinase 1 activated by the hepatitis B virus X protein attenuates both the DNA damage checkpoint and DNA repair resulting in partial polyploidy.
      Consistently, HBV infection of mice with a humanised liver caused elevated ploidy levels.
      • Ahodantin J.
      • Bou-Nader M.
      • Cordier C.
      • Mégret J.
      • Soussan P.
      • Desdouets C.
      • et al.
      Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation.
      Transgenic mice expressing the HBx protein enter S-phase prematurely and show a delay in G2/M during postnatal liver growth around weaning.
      • Ahodantin J.
      • Bou-Nader M.
      • Cordier C.
      • Mégret J.
      • Soussan P.
      • Desdouets C.
      • et al.
      Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation.
      Moreover, livers of these mice showed persistent activation of p38/MAPK and increased expression of PLK1. p38 signalling clearly increased the fraction of mononucleated tetraploids, yet the consequences and the detailed mechanism of how HBx modifies ploidy remained elusive.
      • Ahodantin J.
      • Bou-Nader M.
      • Cordier C.
      • Mégret J.
      • Soussan P.
      • Desdouets C.
      • et al.
      Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation.
      While alterations in cell cycle progression in the presence of viral proteins can directly increase ploidy, it remains to be explored to what extent these hepatotropic viruses may increase ploidy by promoting hepatocyte fusion during viral entry. Polyploidisation via cell-to-cell fusion has been observed upon infection with human papilloma virus or human cytomegalovirus, with the latter also being a hepatotropic virus.
      • Lizier M.
      • Castelli A.
      • Montagna C.
      • Lucchini F.
      • Vezzoni P.
      • Faggioli F.
      Cell fusion in the liver, revisited.

      NAFLD and NASH

      Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), have become the most common liver diseases in the Western world.
      • Younossi Z.
      • Anstee Q.M.
      • Marietti M.
      • Hardy T.
      • Henry L.
      • Eslam M.
      • et al.
      Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.
      The NAFLD spectrum comprises liver steatosis resulting from hepatocyte triglyceride accumulation that can progress to steatohepatitis (NASH). Importantly, NAFLD/NASH is most often caused by caloric excess and sedentary lifestyle.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      Gentric et al. investigated the ploidy profiles in different NASH models and found increased hepatocyte ploidy in obese mice as well as mice on NASH-inducing diets, such as high-fat or methionine- and choline-deficient diets.
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      Interestingly, hepatocytes in these NAFLD/NASH mouse models were predominantly mononucleated polyploids, suggesting that the cell cycle mode is different from the cytokinesis failure that generates binucleated cells, as seen after weaning.
      • Margall-Ducos G.
      • Celton-Morizur S.
      • Couton D.
      • Bregerie O.
      • Desdouets C.
      Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
      ,
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      Further experiments with hepatocytes from NAFLD/NASH mice showed that oxidative stress, one of the major drivers of NASH, causes endoreduplication cycles by attenuation of CDK1 via ATR kinase signalling.
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      These findings are supported by a recent study exploring the consequences of liver-specific deletion of CDK1 that triggers spontaneous polyploidy.
      • Dewhurst M.R.
      • Ow J.R.
      • Zafer G.
      • van Hul N.K.M.
      • Wollmann H.
      • Bisteau X.
      • et al.
      Loss of hepatocyte cell division leads to liver inflammation and fibrosis.
      Consistent with these experimental findings, increased hepatocyte ploidy was also observed in 2 studies including patients with NASH.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      ,
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      Polyploids can undergo multipolar divisions to generate daughter cells of lower ploidy that are chromosomally instable and become tumour prone.
      As insulin signalling involves AKT activation in hepatocytes,
      • Molinaro A.
      • Becattini B.
      • Mazzoli A.
      • Bleve A.
      • Radici L.
      • Maxvall I.
      • et al.
      Insulin-driven PI3K-AKT signaling in the hepatocyte is mediated by redundant PI3Kα and PI3Kβ activities and is promoted by RAS.
      and hyperinsulinemia with pronounced (hepatic) insulin resistance is a major feature of human NAFLD,
      • Khan R.S.
      • Bril F.
      • Cusi K.
      • Newsome P.N.
      Modulation of insulin resistance in nonalcoholic fatty liver disease.
      it is possible that the hepatic insulin-AKT pathway is also involved in the promotion of hepatocyte polyploidy in NASH.

      Other liver disorders

      Alcohol-related liver disease (ALD) ranges from reversible liver steatosis to severe alcohol-related steatohepatitis (ASH).
      • Seitz H.K.
      • Bataller R.
      • Cortez-Pinto H.
      • Gao B.
      • Gual A.
      • Lackner C.
      • et al.
      Alcoholic liver disease.
      Similar to NAFLD, ALD is driven by oxidative stress, but in the latter case reactive oxygen species production is directly linked to alcohol metabolism by CYP2E1.
      • Butura A.
      • Nilsson K.
      • Morgan K.
      • Morgan T.R.
      • French S.W.
      • Johansson I.
      • et al.
      The impact of CYP2E1 on the development of alcoholic liver disease as studied in a transgenic mouse model.
      ,
      • Leung T.M.
      • Nieto N.
      CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease.
      Hepatocyte ploidy in ALD has not been investigated in detail. However, according to our recent data, the ALD ploidy level tends to be lower than in patients with NASH (Fig. 4).
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      Finally, genetic liver diseases, such as haemochromatosis (leading to hepatic iron overload) or Wilson disease (causing hepatic copper overload) have been associated with increased hepatic polyploidy.
      • Troadec M.B.
      • Courselaud B.
      • Détivaud L.
      • Haziza-Pigeon C.
      • Leroyer P.
      • Brissot P.
      • et al.
      Iron overload promotes Cyclin D1 expression and alters cell cycle in mouse hepatocytes.
      • Madra S.
      • Styles J.
      • Smith A.G.
      Perturbation of hepatocyte nuclear populations induced by iron and polychlorinated biphenyls in C57BL/10ScSn mice during carcinogenesis.
      • Muramatsu Y.
      • Yamada T.
      • Moralejo D.H.
      • Mochizuki H.
      • Sogawa K.
      • Matsumoto K.
      Increased polyploid incidence is associated with abnormal copper accumulation in the liver of LEC mutant rat.
      • Yamada T.
      • Sogawa K.
      • Kim J.K.
      • Izumi K.
      • Suzuki Y.
      • Muramatsu Y.
      • et al.
      Increased polyploidy, delayed mitosis and reduced protein phosphatase-1 activity associated with excess copper in the long evans cinnamon rat.
      Iron overload, a potential risk factor for hepatocarcinogenesis, was found to increase cyclin D1 levels in a mouse model, and in turn promote hepatocyte polyploidy in a chronic overload setting.
      • Troadec M.B.
      • Courselaud B.
      • Détivaud L.
      • Haziza-Pigeon C.
      • Leroyer P.
      • Brissot P.
      • et al.
      Iron overload promotes Cyclin D1 expression and alters cell cycle in mouse hepatocytes.
      Collectively, hepatocytes increase their ploidy during the proliferative stress that underlies various disease conditions. Even though the mechanisms altering the cell cycle mode towards endoreduplication or failed cytokinesis vary between these aetiologies, ploidy increases are observed in liver disorders that cause compensatory liver growth. The role of polyploidy in the interrelation between inflammation, fibrosis, and hepatocarcinogenesis requires further investigation. Intriguingly, a recent study using liver-specific Cdk1 or Ccna2 deficient mice to model high mononuclear hepatic ploidy found that this condition was sufficient to cause DNA damage, liver inflammation and fibrosis, independent of any other trigger. Elevated ALT (alanine aminotransferase) levels, as well as increased cytokine production and the presence of Kupffer cells were observed in animals as young as 2 weeks old.
      • Dewhurst M.R.
      • Ow J.R.
      • Zafer G.
      • van Hul N.K.M.
      • Wollmann H.
      • Bisteau X.
      • et al.
      Loss of hepatocyte cell division leads to liver inflammation and fibrosis.
      In accordance, a second study showed that Cdk1 deficiency in the liver is accompanied by deregulated lipid metabolism, ultimately leading to hepatic steatosis.
      • Ow J.R.
      • Cadez M.J.
      • Zafer G.
      • Foo J.C.
      • Li H.Y.
      • Ghosh S.
      • et al.
      Remodeling of whole-body lipid metabolism and a diabetic-like phenotype caused by loss of CDK1 and hepatocyte division.
      These reports suggest that mononuclear polyploidy, a state often referred to as “pathological polyploidy”, could by itself produce an inflammatory liver disease. Of note, the latter reports used a mouse model deficient in a major cell cycle regulator. Thus, future studies will have to carefully dissect whether mononucleation is a cause or consequence of the described pathologies.
      In general, infection and/or inflammation-triggered compensatory proliferation may eventually be limited by the induction of a cellular senescence programme. Induction of senescence also depends on the p53/p21 axis and can eventually rewire hepatocyte metabolism, leading to steatosis. Accordingly, using senolytic drugs to remove senescent hepatocytes can revert NAFLD-related steatosis,
      • Ogrodnik M.
      • Miwa S.
      • Tchkonia T.
      • Tiniakos D.
      • Wilson C.L.
      • Lahat A.
      • et al.
      Cellular senescence drives age-dependent hepatic steatosis.
      while inhibition of the pro-senescent cytokine transforming growth factor-β facilitated liver regeneration in a model of acute liver injury.
      • Bird T.G.
      • Müller M.
      • Boulter L.
      • Vincent D.F.
      • Ridgway R.A.
      • Lopez-Guadamillas E.
      • et al.
      TGFβ inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.
      Whether high hepatocyte ploidy favours the induction of senescence per se remains to be investigated.
      Mouse models show that elevated hepatocyte polyploidy is protective, while poorly polyploid livers develop spontaneous HCC.

      Ploidy and liver cancer

      Chronic liver disease is associated with ongoing hepatocyte injury, inflammation and fibrosis that may ultimately lead to cirrhosis (Kulik L, Gastroenterology 2019). Cirrhosis is an important pre-cancerogenic condition as most cases of HCC arise in cirrhotic livers, while non-cirrhotic HCC is rare – except in patients with HBV infection or with NASH.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      Due to aberrant liver regeneration, hepatocytes accumulate molecular alterations that allow dysplastic cells to develop survival advantages that enable increased proliferative and invasive potential.
      Hepatocyte-derived HCC cells are characterised by somatic DNA mutations and chromosomal aberrations, recently reviewed in detail by Llovet and colleagues.
      • Llovet J.M.
      • Kelley R.K.
      • Villanueva A.
      • Singal A.G.
      • Pikarsky E.
      • Roayaie S.
      • et al.
      Hepatocellular carcinoma.
      HCC tumours can be molecularly characterised as the “proliferation” and the “non-proliferation” class.
      • Zucman-Rossi J.
      • Villanueva A.
      • Nault J.C.
      • Llovet J.M.
      Genetic landscape and biomarkers of hepatocellular carcinoma.
      The “proliferation HCC” subclass is characterised by p53 mutations and CIN, with patients often showing high AFP (alpha-fetoprotein) levels, higher tumour grades and worse clinical outcomes. In contrast, the “non-proliferation HCC” subclass often show mutations in CTNNB1 (beta-catenin) that also promote immune escape (i.e. a low level of immune cell infiltration), and are characterised by a lower tumour grade and better clinical outcomes.
      • de Galarreta M.R.
      • Bresnahan E.
      • Molina-Sánchez P.
      • Lindblad K.E.
      • Maier B.
      • Sia D.
      • et al.
      β-catenin activation promotes immune escape and resistance to anti–PD-1 therapy in hepatocellular carcinoma.
      Even though polyploidy is generally associated with cancer and shown to increase in a variety of diseases that promote tumorigenesis in the liver, several reports demonstrated that the interrelation of hepatocyte polyploidy and hepatocarcinogenesis is quite the opposite. Studies by the Zhu laboratory support the idea that higher polyploidy can protect from HCC in different carcinogen-driven models, which was explained by increasing the copy number of tumour suppressor genes.
      • Zhang S.
      • Zhou K.
      • Luo X.
      • Li L.
      • Tu H.C.
      • Sehgal A.
      • et al.
      The polyploid state plays a tumor-suppressive role in the liver.
      ,
      • Zhang S.
      • Nguyen L.H.
      • Zhou K.
      • Tu H.-C.
      • Sehgal A.
      • Nassour I.
      • et al.
      Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration.
      ,
      • Lin Y.H.
      • Zhang S.
      • Zhu M.
      • Lu T.
      • Chen K.
      • Wen Z.
      • et al.
      Mice with increased numbers of polyploid hepatocytes maintain regenerative capacity but develop fewer hepatocellular carcinomas following chronic liver injury.
      In consequence, polyploid cells need to acquire more loss-of-function events to bypass these safety mechanisms than diploid cells.
      • Zhang S.
      • Zhou K.
      • Luo X.
      • Li L.
      • Tu H.C.
      • Sehgal A.
      • et al.
      The polyploid state plays a tumor-suppressive role in the liver.
      The major role of the liver in detoxification constantly exposes hepatocytes to genotoxic compounds and metabolites. Hence, hepatocytes must have adapted to this condition since the frequency of spontaneous HCC without underlying cirrhosis is rather low.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      Accordingly, mice deficient in E2F7/8 with mostly diploid livers have been found to develop spontaneous HCC at the age of 5 to 6 months.
      • Wilkinson P.D.
      • Delgado E.R.
      • Alencastro F.
      • Leek M.P.
      • Roy N.
      • Weirich M.P.
      • et al.
      The polyploid state restricts hepatocyte proliferation and liver regeneration.
      ,
      • Kent L.N.
      • Rakijas J.B.
      • Pandit S.K.
      • Westendorp B.
      • Chen H.-Z.
      • Huntington J.T.
      • et al.
      E2f8 mediates tumor suppression in postnatal liver development.
      There have been recent advances in our understanding of the role of hepatocyte ploidy in tumorigenesis. Studies have utilised mouse models in which the ploidy of hepatocytes was either reduced by loss of E2F7/8, or increased by premature weaning or the knock-down of the cytokinesis effector anillin.
      • Zhang S.
      • Zhou K.
      • Luo X.
      • Li L.
      • Tu H.C.
      • Sehgal A.
      • et al.
      The polyploid state plays a tumor-suppressive role in the liver.
      ,
      • Zhang S.
      • Nguyen L.H.
      • Zhou K.
      • Tu H.-C.
      • Sehgal A.
      • Nassour I.
      • et al.
      Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration.
      It has been demonstrated that liver tumours arise mostly from poorly polyploid, presumably diploid hepatocytes (Fig. 5). Based on targeted sequencing analyses, the authors attributed these effects to the increased tumour suppressor gene copy numbers in polyploid cells, while in turn the low ploidy hepatocytes were prone to lose tumour suppressor gene copies. While tumour promoting driver mutations were similar in low- and high-ploidy lesions, high polyploidy was clearly protective against loss of heterozygosity of tumour suppressor genes such as p53.
      • Zhang S.
      • Zhou K.
      • Luo X.
      • Li L.
      • Tu H.C.
      • Sehgal A.
      • et al.
      The polyploid state plays a tumor-suppressive role in the liver.
      In accordance with these results, anillin and other cytokinesis effectors are frequently overexpressed in human HCC.
      • Zhang S.
      • Nguyen L.H.
      • Zhou K.
      • Tu H.-C.
      • Sehgal A.
      • Nassour I.
      • et al.
      Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration.
      Figure thumbnail gr5
      Fig. 5HCC develops from low ploidy hepatocytes.
      Hepatocyte polyploidy can function as a buffer against LOH and inactivating mutations of tumour suppressor genes. Hence, the low ploidy fraction of hepatocytes is more likely to transform upon additional tumorigenic hits. Multipolar cell division of polyploid hepatocytes can generate progeny of lower ploidy. This type of erroneous cell division promotes CIN. Inflammatory liver diseases drive proliferation of these cells in a pro-tumorigenic environment that promotes transformation. Consequently, most HCC tumour cells present with a lower degree of cellular and nuclear ploidy than the surrounding liver tissue. CIN, chromosome instability; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity.
      In addition to low polyploidy, ploidy-reducing divisions of highly polyploid hepatocytes have also been proposed to drive tumorigenesis in the liver. This concept has been described in the “ploidy conveyor” model. Polyploid hepatocytes can form multipolar mitotic spindles, which were found to generate daughter cells of reduced ploidy in vitro.
      • Duncan A.W.
      • Taylor M.H.
      • Hickey R.D.
      • Hanlon Newell A.E.
      • Lenzi M.L.
      • Olson S.B.
      • et al.
      The ploidy conveyor of mature hepatocytes as a source of genetic variation.
      Further studies using the Fah-/- mouse model for liver repopulation showed that transplanted polyploids can give rise to regenerative nodules formed by hepatocytes of lower ploidy.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      ,
      • Duncan A.W.
      • Taylor M.H.
      • Hickey R.D.
      • Hanlon Newell A.E.
      • Lenzi M.L.
      • Olson S.B.
      • et al.
      The ploidy conveyor of mature hepatocytes as a source of genetic variation.
      ,
      • Matsumoto T.
      • Wakefield L.
      • Tarlow B.D.
      • Grompe M.
      In Vivo lineage tracing of polyploid hepatocytes reveals extensive proliferation during liver regeneration.
      This reduction in ploidy, supposedly achieved through multipolar mitoses, was accompanied by increased CIN (Fig. 5).
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      Interestingly, after serial transplantations through 3 Fah-/- recipient mice, regenerative nodules consisted mostly of polyploid hepatocytes that had lost their accumulated centrosomes.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      On the one hand, this can prevent the formation of multipolar spindles.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      On the other hand, loss of supernumerary centrosomes will inactivate the PIDDosome-p53 axis that would otherwise reduce the proliferative potential of polyploid hepatocytes, thus alleviating the competitive disadvantage of polyploids.
      • Chen F.
      • Jimenez R.J.
      • Sharma K.
      • Luu H.Y.
      • Hsu B.Y.
      • Ravindranathan A.
      • et al.
      Broad distribution of hepatocyte proliferation in liver homeostasis and regeneration.
      ,
      • Wilkinson P.D.
      • Delgado E.R.
      • Alencastro F.
      • Leek M.P.
      • Roy N.
      • Weirich M.P.
      • et al.
      The polyploid state restricts hepatocyte proliferation and liver regeneration.
      ,
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Fava L.L.
      • Schuler F.
      • Sladky V.
      • Haschka M.D.
      • Soratroi C.
      • Eiterer L.
      • et al.
      The PIDDosome activates p53 in response to supernumerary centrosomes.
      Hepatocyte and HCC tumour cell ploidy may be used for risk stratification, prognosis, and therapeutic strategies in clinics.
      How multipolar divisions of hepatocytes can generate near-euploid, viable daughter cells is not clear. This could either be the result of an unidentified counting mechanism for the individual chromosomes or, more likely, multipolar divisions lead to random chromosome segregation within multiple daughter cells and only those with viable karyotypes survive and repopulate the liver. Another way could be the formation of 2 independent spindles in binucleated hepatocytes for faithful chromosome segregation, however, this is a rarely observed event.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Knouse K.A.
      • Lopez K.E.
      • Bachofner M.
      • Amon A.
      Chromosome segregation fidelity in epithelia requires tissue architecture.
      Answering this question in detail will be essential to understand the role of hepatocyte ploidy reduction in hepatocarcinogenesis.
      Whether mononucleated polyploids that are frequently observed in various liver diseases play a role in hepatocarcinogenesis remains unknown. Interestingly, serial transplantations in the Fah-/- mouse model selected for mononuclear polyploids that were less prone to ploidy-reducing divisions.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      This is reminiscent of chronic liver diseases where mononucleated polyploid hepatocytes occur at a higher frequency, presumably also after compensatory regeneration that requires the cells to cycle several rounds.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      ,
      • Gentric G.
      • Maillet V.
      • Paradis V.
      • Couton D.
      • Hermitte A.L.
      • Panasyuk G.
      • et al.
      Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.
      ,
      • Toyoda H.
      • Bregerie O.
      • Vallet A.
      • Nalpas B.
      • Pivert G.
      • Brechot C.
      • et al.
      Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis.
      Desdouets and colleagues propose that mononuclear polyploidy is associated with HCC in human patients,
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      while a recent study by the Grompe lab using mouse models suggests that the mononuclear state reduces the risk of multipolar divisions, and thus CIN.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      Aside from the “model organism”, one possible explanation for this discrepancy is the proinflammatory disease environment in humans. Nonetheless, the impact of nuclear number on the progression of the disease and eventually HCC development requires further clarification.

      The PIDDosome in liver cancer

      Beside its role in physiological development and regeneration, we also explored the role of the PIDDosome in HCC, as the PIDDosome acts upstream of p53, which is mutated in about 30% of patients with HCC.
      • Totoki Y.
      • Tatsuno K.
      • Covington K.R.
      • Ueda H.
      • Creighton C.J.
      • Kato M.
      • et al.
      Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.
      ,
      • Ahn S.M.
      • Jang S.J.
      • Shim J.H.
      • Kim D.
      • Hong S.M.
      • Sung C.O.
      • et al.
      Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.
      Moreover, the presence of high polyploidy is clearly linked to aneuploidy and CIN, which are both considered hallmarks of cancers in general.
      • Storchova Z.
      • Pellman D.
      From polyploidy to aneuploidy, genome instability and cancer.
      To investigate the impact of the PIDDosome pathway on liver cancer, we used the diethylnitrosamine (DEN) model of HCC in mice. Interestingly, we observed that PIDDosome-deficient mice developed significantly less tumour nodules and reduced overall tumour burden than wild-type mice, contrasting with our expectation that the loss of p53 activation after polyploidisation would favour tumourigenesis.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      Analysis of healthy and tumour tissue revealed lower ploidy in the cancer cells compared to healthy surrounding tissue in both wild-type and PIDDosome-deficient mice.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      As there was no difference in tumour ploidy among the genotypes, it seems likely that the tumours develop from low ploidy cells and therefore higher hepatocyte ploidy hampers tumour development owing to a lower abundance of diploid/tetraploid cells.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      Intriguingly, our data show that high polyploidy is protective even if the tumourigenic event occurs in a mostly diploid stage of the liver. This indicates that increased polyploidy can buffer secondary hits that are acquired later in life. The recent study by the Grompe lab mentioned earlier actually confirms this notion and provides evidence that cell divisions of polyploid hepatocytes leading to reduced ploidy are more error prone and create a pool of low ploidy cells that are more vulnerable to transformation.
      • Matsumoto T.
      • Wakefield L.
      • Peters A.
      • Peto M.
      • Spellman P.
      • Grompe M.
      Proliferative polyploid cells give rise to tumors via ploidy reduction.
      As such, while high ploidy protects, division of polyploid hepatocytes that cause genome size reduction in regenerative settings or chronic inflammatory liver disease may promote malignant disease. This hypothesis is supported by observations made by Lin et al., who report DEN-driven hyperpolyploidisation of centrilobular hepatocytes that foster the formation of premalignant lesions. However, within these preneoplastic lesions, cells were found to be substantially smaller, suggesting that they undergo nuclear- and genome-content reduction during transformation. Blocking DEN-induced hyperpolyploidisation by inhibiting Aurora B kinase reduced tumour number and size, establishing causality.
      • Lin H.
      • Huang Y.S.
      • Fustin J.M.
      • Doi M.
      • Chen H.
      • Lai H.H.
      • et al.
      Hyperpolyploidization of hepatocyte initiates preneoplastic lesion formation in the liver.
      It will be interesting to see if higher basal liver ploidy, as seen in PIDDosome-mutant mice may alter HCC risk in adequate model systems or correlate with HCC penetrance in humans.
      Analysing the “Liver Hepatocellular Carcinoma” TCGA data set containing information on 372 HCC patient samples, we also noted that higher expression of CASP2 and PIDD1 are associated with more severe disease stages and with reduced recurrence-free survival.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      The expression of these PIDDosome components was even higher in patients with mutated p53 and their increased expression was confirmed in primary HCC samples compared to neighbouring non-malignant liver tissue, albeit the p53 status of these patients remained unknown.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      While this indicates that CASP2 or PIDD1 expression may be used as a prognostic marker of disease progression in HCC, this correlation may simply be a consequence of increased cellular proliferation rates that depend on E2F transcription factors that presumably also control the expression of Casp2 and PIDD1 in malignant cells.
      • Sladky V.C.
      • Knapp K.
      • Soratroi C.
      • Heppke J.
      • Eichin F.
      • Rocamora-Reverte L.
      • et al.
      E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
      ,
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

      Ploidy as a prognostic marker in patients with HCC

      Recently, the ploidy state in human HCC has indeed been linked to patient survival. However, in contrast to studies in mouse models, patients with highly polyploid tumours and mutated p53 were found to have significantly decreased progression-free survival.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      Although this observation is in line with observations in other tumour types that high polyploidy fuels CIN and therefore cancer progression and evolution,
      • Dewhurst S.M.
      • McGranahan N.
      • Burrell R.A.
      • Rowan A.J.
      • Grönroos E.
      • Endesfelder D.
      • et al.
      Tolerance of whole- genome doubling propagates chromosomal instability and accelerates cancer genome evolution.
      ,
      • Coward J.
      • Harding A.
      Size does matter: why polyploid tumor cells are critical drug targets in the war on cancer.
      different underlying diseases and patient age make it difficult to dissect the effects attributed to different ploidy levels vs. effects related to liver disease aetiology and p53 status.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      ,
      • van Jaarsveld R.H.
      • Kops G.J.P.L.
      Difference makers: chromosomal instability versus aneuploidy in cancer.
      ,
      • Li T.-N.
      • Wu Y.-J.
      • Tsai H.-W.
      • Sun C.-P.
      • Wu Y.-H.
      • Wu H.-L.
      • et al.
      Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.
      High ploidy in the context of impaired p53 function fuels at least 2 adverse events, (i) proliferation of polyploid cells carrying extra centrosomes and (ii) tolerance to CIN.
      • Watkins T.B.K.
      • Lim E.L.
      • Petkovic M.
      • Elizalde S.
      • Birkbak N.J.
      • Wilson G.A.
      • et al.
      Pervasive chromosomal instability and karyotype order in tumour evolution.
      ,
      • Dewhurst S.M.
      • McGranahan N.
      • Burrell R.A.
      • Rowan A.J.
      • Grönroos E.
      • Endesfelder D.
      • et al.
      Tolerance of whole- genome doubling propagates chromosomal instability and accelerates cancer genome evolution.
      We investigated a large set of HCC specimens from patients with regards to cellular size (specifically cell density), as a measure for tumour cell ploidy, and found that high tumour cell ploidy was a surrogate of better prognosis.
      • Sladky V.C.
      • Knapp K.
      • Szabo T.G.
      • Braun V.Z.
      • Bongiovanni L.
      • Bos H.
      • et al.
      PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
      Yet, while this study could not determine the p53 status of the included patients, the on first sight ‘different’ observations by Bou-Nader et al. and by Sladky et al. may still be compatible. Loss of p53 enables polyploid cancer cells to proliferate in the presence of extra centrosomes, driving evolution of aggressive disease, as observed by Bou-Nader and colleagues.
      • Bou-Nader M.
      • Caruso S.
      • Donne R.
      • Celton-Morizur S.
      • Calderaro J.
      • Gentric G.
      • et al.
      Polyploidy spectrum: a new marker in HCC classification.
      In the presence of functional p53, which reflects still around 70% of all HCC cases analysed, the protective effect may dominate. In our large cohort, this may dilute out the negative effects on survival, imposed by p53 mutation. It will be of interest to investigate if discriminating p53 status on top of tumour cell size will constitute a novel and simple prognostic pair of genetic and morphological markers in HCC. Follow-up studies including larger patient cohorts with detailed genetic information combined with morphometric analyses are needed (i) to confirm the degree of hepatocyte polyploidy as a risk factor for subsequent HCC development, and (ii) to assess if HCC tumour cell density/ploidy is of prognostic value in patients with HCC. Ultimately, all future studies on hepatic ploidy should consider different molecular subtypes of HCC, assess genetic loss/mutations of tumour promoter/suppressor genes, as well as consider age and liver disease aetiologies as potential modulators of hepatocyte ploidy state.

      Perspective

      Despite all the efforts made to understand the role of liver ploidy in health and disease, there are still considerable controversies regarding its clinical relevance. The protective role of hepatocyte ploidy against development of cancer was, however, consistently reported by various groups using different tools and approaches to investigate its impact on liver cancer. This finding may thus be of prognostic value as low ploidy seems to predict HCC progression or recurrence after liver transplantation, especially when combined with the assessment of distinct genetic signatures. In addition, inhibition of molecular mechanisms that restrict ploidy may improve liver regeneration without negatively affecting overall liver function. However, the impact of hepatocyte ploidy on disease progression in different liver pathologies remains to be further explored, especially with regard to ploidy-reducing cell divisions that promote CIN and thus potentially hepatocarcinogenesis. Yet, pharmacological modulation of ploidy regulators, such as the PIDDosome, that facilitate polyploidisation may reduce the risk of liver cancer in patients with viral or metabolic liver disease (i.e. chemoprevention). Such strategies may also help to promote liver regeneration in liver failure, to precondition livers for extensive curative resections, or to allow successful bridging to liver transplantation.

      Abbreviations

      ALD, alcohol-related liver disease; ASH, alcohol-related steatohepatitis; CASP2, caspase-2; CDK, cyclin-dependent kinase; CIN, chromosomal instability; DD, death domain; DEN, diethylnitrosamine; FAH, fumarylacetoacetate hydrolase; HCC, hepatocellular carcinoma; HGD, homogentisate 1,2 dioxygenase; miR, microRNA; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PIDD1, p53-induced death domain protein 1; PLK1, Polo like kinase 1; RhoA, Ras homolog family member A; SKP2, S-phase kinase associated protein 2; YAP, yes-associated protein.

      Financial support

      AV: ERC-AdG “POLICE” (# 787171 ), Ludwig Boltzmann Society . TR: Christian-Doppler Society , Ludwig Boltzmann society . VCS: EMBO Postdoctoral fellowship program ( ALTF 1194-2020 ).

      Authors’ contributions

      VCS, FE, TR, AV wrote and edited the manuscript; FE created the figures; AV coordinated the work.

      Conflict of Interest

      TR received grant support from Abbvie, Boehringer-Ingelheim, Gilead, MSD, Philips Healthcare, Gore; speaking honoraria from Abbvie, Gilead, Gore, Intercept, Roche, MSD; consulting/advisory board fee from Abbvie, Bayer, Boehringer-Ingelheim, Gilead, Intercept, MSD, Siemens; and travel support from Abbvie, Boehringer-Ingelheim, Gilead and Roche. All other authors declare no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We want to thank all members of our teams for fruitful discussion and Lauren Evans for help with editing the manuscript. We apologize to all those whose valuable contribution was not cited due to space constrains. Work in our laboratories is supported by the ERC-AdG “POLICE”, and the Ludwig Boltzmann Society. TR is supported by the Christian-Doppler Society. VCS acknowledges support by the EMBO Postdoctoral fellowship program (ALTF 1194-2020).
      Artwork was created with BioRender.com.

      Supplementary data

      The following is the supplementary data to this article:

      References

        • Fox D.T.
        • Soltis D.E.
        • Soltis P.S.
        • Ashman T.L.
        • Van de Peer Y.
        Polyploidy: a biological force from cells to ecosystems.
        Trends Cell Biol. 2020; 30: 688-694https://doi.org/10.1016/j.tcb.2020.06.006
        • Van de Peer Y.
        • Mizrachi E.
        • Marchal K.
        The evolutionary significance of polyploidy.
        Nat Rev Genet. 2017; 18: 411-424https://doi.org/10.1038/nrg.2017.26
        • Otto S.P.
        • Whitton J.
        Polyploid incidence and evolution.
        Annu Rev Genet. 2000;
        • Albertin W.
        • Marullo P.
        Polyploidy in fungi: evolution after whole-genome duplication.
        Proc R Soc B Biol Sci. 2012; 279: 2497-2509https://doi.org/10.1098/rspb.2012.0434
        • Schmid M.
        • Evans B.J.
        • Bogart J.P.
        Polyploidy in Amphibia.
        Cytogenet Genome Res. 2015; 145: 315-330https://doi.org/10.1159/000431388
        • Gallardo M.H.
        • Kausel G.
        • Jiménez A.
        • Bacquet C.
        • González C.
        • Figueroa J.
        • et al.
        Whole-genome duplications in South American desert rodents (Octodontidae).
        Biol J Linn Soc. 2004; 82: 443-451https://doi.org/10.1111/j.1095-8312.2004.00331.x
        • Svartman M.
        • Stone G.
        • Stanyon R.
        Molecular cytogenetics discards polyploidy in mammals.
        Genomics. 2005; 85: 425-430https://doi.org/10.1016/j.ygeno.2004.12.004
        • Pandit S.K.
        • Westendorp B.
        • de Bruin A.
        Physiological significance of polyploidization in mammalian cells.
        Trends Cell Biol. 2013; 23: 556-566https://doi.org/10.1016/j.tcb.2013.06.002
        • Duncan A.W.
        Aneuploidy, polyploidy and ploidy reversal in the liver.
        Semin Cell Dev Biol. 2013; 24: 347-356https://doi.org/10.1016/J.SEMCDB.2013.01.003
        • Gan P.
        • Patterson M.
        • Sucov H.M.
        Cardiomyocyte polyploidy and implications for heart regeneration.
        Ann Rev Physiol. 2019; : 1-17
        • Ravid K.
        • Lu J.
        • Zimmet J.M.
        • Jones M.R.
        Roads to polyploidy: the megakaryocyte example.
        J Cell Physiol. 2002; 190: 7-20https://doi.org/10.1002/jcp.10035
        • Edgar B.A.
        • Zielke N.
        • Gutierrez C.
        Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.
        Nat Rev Mol Cell Biol. 2014; 15: 197-210https://doi.org/10.1038/nrm3756
        • Donne R.
        • Saroul-Aïnama M.
        • Cordier P.
        • Celton-Morizur S.
        • Desdouets C.
        Polyploidy in liver development, homeostasis and disease.
        Nat Rev Gastroenterol Hepatol. 2020; 17: 391-405https://doi.org/10.1038/s41575-020-0284-x
        • Wang M.-J.
        • Chen F.
        • Lau J.T.Y.
        • Hu Y.-P.
        Hepatocyte polyploidization and its association with pathophysiological processes.
        Cell Death Dis. 2017; 8e2805https://doi.org/10.1038/cddis.2017.167
        • Kudryavtsev B.N.
        • Kudryavtseva M.V.
        • Sakuta G.A.
        • Stein G.I.
        Human hepatocyte polyploidization kinetics in the course of life cycle.
        Virchow Archiv B. 1993; 64
        • Tanami S.
        • Ben-Moshe S.
        • Elkayam A.
        • Mayo A.
        • Bahar Halpern K.
        • Itzkovitz S.
        Dynamic zonation of liver polyploidy.
        Cell Tissue Res. 2017; 368: 405-410https://doi.org/10.1007/s00441-016-2427-5
        • Bou-Nader M.
        • Caruso S.
        • Donne R.
        • Celton-Morizur S.
        • Calderaro J.
        • Gentric G.
        • et al.
        Polyploidy spectrum: a new marker in HCC classification.
        Gut. 2019; (gutjnl-2018-318021)https://doi.org/10.1136/GUTJNL-2018-318021
        • Chen F.
        • Jimenez R.J.
        • Sharma K.
        • Luu H.Y.
        • Hsu B.Y.
        • Ravindranathan A.
        • et al.
        Broad distribution of hepatocyte proliferation in liver homeostasis and regeneration.
        Cell Stem Cell. 2020; 26 (27-33.e4)https://doi.org/10.1016/j.stem.2019.11.001
        • Wei Y.
        • Wang Y.G.
        • Jia Y.
        • Li L.
        • Yoon J.
        • Zhang S.
        • et al.
        Liver homeostasis is maintained by midlobular zone 2 hepatocytes.
        Science (80- ). 2021; (eabb1625): 371https://doi.org/10.1126/science.abb1625
        • He L.
        • Pu W.
        • Liu X.
        • Zhang Z.
        • Han M.
        • Li Y.
        • et al.
        Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.
        Science (80- ). 2021; (eabc4346): 371https://doi.org/10.1126/science.abc4346
        • Wang B.
        • Zhao L.
        • Fish M.
        • Logan C.Y.
        • Nusse R.
        Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver.
        Nature. 2015; 524: 180-185https://doi.org/10.1038/nature14863
        • Sun T.
        • Pikiolek M.
        • Orsini V.
        • Bergling S.
        • Holwerda S.
        • Morelli L.
        • et al.
        AXIN2+ pericentral hepatocytes have limited contributions to liver homeostasis and regeneration.
        Cell Stem Cell. 2020; 26 (97-107.e6)https://doi.org/10.1016/j.stem.2019.10.011
        • Rabes H.M.
        Kinetics of hepatocellular proliferation as a function of the microvascular structure and functional state of the liver.
        Ciba Found Symp. 1977; : 31-53https://doi.org/10.1002/9780470720363.ch3
        • Margall-Ducos G.
        • Celton-Morizur S.
        • Couton D.
        • Bregerie O.
        • Desdouets C.
        Liver tetraploidization is controlled by a new process of incomplete cytokinesis.
        J Cell Sci. 2007; 120: 3633-3639https://doi.org/10.1242/jcs.016907
        • Celton-Morizur S.
        • Merlen G.
        • Couton D.
        • Margall-Ducos G.
        • Desdouets C.
        The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.
        J Clin Invest. 2009; 119: 1880-1887https://doi.org/10.1172/JCI38677
        • Miyaoka Y.
        • Ebato K.
        • Kato H.
        • Arakawa S.
        • Shimizu S.
        • Miyajima A.
        Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.
        Curr Biol. 2012; 22: 1166-1175https://doi.org/10.1016/j.cub.2012.05.016
        • Wilkinson P.D.
        • Delgado E.R.
        • Alencastro F.
        • Leek M.P.
        • Roy N.
        • Weirich M.P.
        • et al.
        The polyploid state restricts hepatocyte proliferation and liver regeneration.
        Hepatology. 2018; (0–3)https://doi.org/10.1002/hep.30286
        • Sladky V.C.
        • Knapp K.
        • Soratroi C.
        • Heppke J.
        • Eichin F.
        • Rocamora-Reverte L.
        • et al.
        E2F-Family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration.
        Dev Cell. 2020; 52 (335-349.e7)https://doi.org/10.1016/j.devcel.2019.12.016
        • Kurinna S.
        • Stratton S.A.
        • Coban Z.
        • Schumacher J.M.
        • Grompe M.
        • Duncan A.W.
        • et al.
        P53 regulates a mitotic transcription program and determines ploidy in normal mouse liver.
        Hepatology. 2013; 57: 2004-2013https://doi.org/10.1002/hep.26233
        • Heinke P.
        • Rost F.
        • Rode J.
        • Welsch T.
        • Alkass K.
        • Feddema J.
        • et al.
        Diploid hepatocytes drive physiological liver renewal in adult humans.
        BioRxiv. 2020;
        • Hsu S.H.
        • Delgado E.R.
        • Otero P.A.
        • Teng K.Y.
        • Kutay H.
        • Meehan K.M.
        • et al.
        MicroRNA-122 regulates polyploidization in the murine liver.
        Hepatology. 2016; https://doi.org/10.1002/hep.28573
        • Zhang S.
        • Chen Q.
        • Liu Q.
        • Li Y.
        • Sun X.
        • Hong L.
        • et al.
        Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2.
        Cancer Cell. 2017; 31 (669-684.e7)https://doi.org/10.1016/J.CCELL.2017.04.004
        • Pandit S.K.
        • Westendorp B.
        • Nantasanti S.
        • Van Liere E.
        • Tooten P.C.J.
        • Cornelissen P.W.A.
        • et al.
        E2F8 is essential for polyploidization in mammalian cells.
        Nat Cell Biol. 2012; 14: 1181-1191https://doi.org/10.1038/ncb2585
        • Chen H.Z.
        • Ouseph M.M.
        • Li J.
        • Pécot T.
        • Chokshi V.
        • Kent L.
        • et al.
        Canonical and atypical E2Fs regulate the mammalian endocycle.
        Nat Cell Biol. 2012; 14: 1192-1202https://doi.org/10.1038/ncb2595
        • Conner E.A.
        • Lemmer E.R.
        • Sánchez A.
        • Factor V.M.
        • Thorgeirsson S.S.
        E2F1 blocks and c-Myc accelerates hepatic ploidy in transgenic mouse models.
        Biochem Biophys Res Commun. 2003; 302: 114-120https://doi.org/10.1016/S0006-291X(03)00125-6
        • Bertoli C.
        • Skotheim J.M.
        • de Bruin R.A.M.
        Control of cell cycle transcription during G1 and S phases.
        Nat Rev Mol Cell Biol. 2013; 14: 518-528https://doi.org/10.1038/nrm3629
        • Trimarchi J.M.
        • Lees J.A.
        Sibling rivalry in the E2F family.
        Nat Rev Mol Cell Biol. 2002; 3: 11-20https://doi.org/10.1038/nrm714
        • Lammens T.
        • Li J.
        • Leone G.
        • De Veylder L.
        Atypical E2Fs: new players in the E2F transcription factor family.
        Trends Cell Biol. 2009; 19: 111-118https://doi.org/10.1016/J.TCB.2009.01.002
        • Di Stefano L.
        • Jensen M.R.
        • Helin K.
        E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes.
        EMBO J. 2003; 22: 6289-6298https://doi.org/10.1093/emboj/cdg613
        • Christensen J.
        • Cloos P.
        • Toftegaard U.
        • Klinkenberg D.
        • Bracken A.P.
        • Trinh E.
        • et al.
        Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription.
        Nucleic Acids Res. 2005; 33: 5458-5470https://doi.org/10.1093/nar/gki855
        • Sheahan S.
        • Bellamy C.O.
        • Treanor L.
        • Harrison D.J.
        • Prost S.
        Additive effect of p53, p21 and Rb deletion in triple knockout primary hepatocytes.
        Oncogene. 2004; 23: 1489-1497https://doi.org/10.1038/sj.onc.1207280
        • Chao H.W.
        • Doi M.
        • Fustin J.M.
        • Chen H.
        • Murase K.
        • Maeda Y.
        • et al.
        Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway.
        Nat Commun. 2017; 8: 1-14https://doi.org/10.1038/s41467-017-02207-7
        • Zhang S.
        • Zhou K.
        • Luo X.
        • Li L.
        • Tu H.C.
        • Sehgal A.
        • et al.
        The polyploid state plays a tumor-suppressive role in the liver.
        Dev Cell. 2018; 44 (447-459.e5)https://doi.org/10.1016/j.devcel.2018.01.010
        • Aziz K.
        • Limzerwala J.F.
        • Sturmlechner I.
        • Hurley E.
        • Zhang C.
        • Jeganathan K.B.
        • et al.
        Ccne1 overexpression causes chromosome instability in liver cells and liver tumor development in mice.
        Gastroenterology. 2019; 157: 210https://doi.org/10.1053/j.gastro.2019.03.016
        • Miettinen T.P.
        • Pessa H.K.J.
        • Caldez M.J.
        • Fuhrer T.
        • Diril M.K.
        • Sauer U.
        • et al.
        Identification of transcriptional and metabolic programs related to mammalian cell size.
        Curr Biol. 2014; 24: 598-608https://doi.org/10.1016/J.CUB.2014.01.071
        • Maillet V.
        • Boussetta N.
        • Leclerc J.
        • Fauveau V.
        • Foretz M.
        • Viollet B.
        • et al.
        LKB1 as a gatekeeper of hepatocyte proliferation and genomic integrity during liver regeneration.
        Cell Rep. 2018; 22: 1994-2005https://doi.org/10.1016/J.CELREP.2018.01.086
        • Nakayama K.
        • Nagahama H.
        • Minamishima Y.A.
        • Matsumoto M.
        • Nakamichi I.
        • Kitagawa K.
        • et al.
        Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication.
        EMBO J. 2000; 19: 2069-2081https://doi.org/10.1093/emboj/19.9.2069
        • Ganem N.J.
        • Cornils H.
        • Chiu S.-Y.
        • O’Rourke K.P.
        • Arnaud J.
        • Yimlamai D.
        • et al.
        Cytokinesis failure triggers Hippo tumor suppressor pathway activation.
        Cell. 2014; 158: 833-848https://doi.org/10.1016/J.CELL.2014.06.029
        • Tinel A.
        • Tschopp J.
        The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress.
        Science. 2004; 304: 843-846https://doi.org/10.1126/science.1095432
        • Bao Q.
        • Shi Y.
        Apoptosome: a platform for the activation of initiator caspases.
        Cell Death Differ. 2007; 14: 56-65https://doi.org/10.1038/sj.cdd.4402028
        • Zheng D.
        • Liwinski T.
        • Elinav E.
        Inflammasome activation and regulation: toward a better understanding of complex mechanisms.
        Cell Discov. 2020; 6: 1-22https://doi.org/10.1038/s41421-020-0167-x
        • Fava L.L.
        • Schuler F.
        • Sladky V.
        • Haschka M.D.
        • Soratroi C.
        • Eiterer L.
        • et al.
        The PIDDosome activates p53 in response to supernumerary centrosomes.
        Genes Dev. 2017; 31: 34-45https://doi.org/10.1101/gad.289728.116
        • Nigg E.A.
        • Holland A.J.
        Once and only once: mechanisms of centriole duplication and their deregulation in disease.
        Nat Rev Mol Cell Biol. 2018; 19: 297-312https://doi.org/10.1038/nrm.2017.127
        • Sladky V.C.
        • Knapp K.
        • Szabo T.G.
        • Braun V.Z.
        • Bongiovanni L.
        • Bos H.
        • et al.
        PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.
        EMBO Rep. 2020; 21e50893https://doi.org/10.15252/embr.202050893
        • Faggioli F.
        • Vezzoni P.
        • Montagna C.
        Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.
        PloS One. 2011; 6https://doi.org/10.1371/journal.pone.0026080
        • Burigotto M.
        • Mattivi A.
        • Migliorati D.
        • Magnani G.
        • Valentini C.
        • Roccuzzo M.
        • et al.
        Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.
        EMBO J. 2020; : 1-22https://doi.org/10.15252/embj.2020104844
        • Evans L.T.
        • Anglen T.
        • Scott P.
        • Lukasik K.
        • Loncarek J.
        • Holland A.J.
        ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification.
        EMBO J. 2020; : 1-18https://doi.org/10.15252/embj.2020105106
        • Bowler M.
        • Kong D.
        • Sun S.
        • Nanjundappa R.
        • Evans L.
        • Farmer V.
        • et al.
        High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy.
        Nat Commun. 2019; 10 (2019 101): 1-15https://doi.org/10.1038/s41467-018-08216-4
        • Oliver T.G.
        • Meylan E.
        • Chang G.P.
        • Xue W.
        • Burke J.R.
        • Humpton T.J.
        • et al.
        Caspase-2-Mediated cleavage of Mdm2 creates a p53-induced positive feedback loop.
        Mol Cell. 2011; 43: 57-71https://doi.org/10.1016/j.molcel.2011.06.012
        • Gönczy P.
        Centrosomes and cancer: revisiting a long-standing relationship.
        Nat Rev Canc. 2015; 15: 639-652https://doi.org/10.1038/nrc3995
        • Levine M.S.
        • Bakker B.
        • Boeckx B.
        • Moyett J.
        • Lu J.
        • Vitre B.
        • et al.
        Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals.
        Dev Cell. 2017; 40 (e5): 313-322https://doi.org/10.1016/J.DEVCEL.2016.12.022
        • Mattia G.
        • Vulcano F.
        • Milazzo L.
        • Barca A.
        • Macioce G.
        • Giampaolo A.
        • et al.
        Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release.
        Blood. 2002; 99: 888-897https://doi.org/10.1182/blood.V99.3.888
        • Mazzi S.
        • Lordier L.
        • Debili N.
        • Raslova H.
        • Vainchenker W.
        Megakaryocyte and polyploidization.
        Exp Hematol. 2018; 57: 1-13https://doi.org/10.1016/J.EXPHEM.2017.10.001
        • Hu D.
        • Cross J.C.
        Development and function of trophoblast giant cells in the rodent placenta.
        Int J Dev Biol. 2010; 54: 341-354https://doi.org/10.1387/ijdb.082768dh
        • Yagi M.
        • Miyamoto T.
        • Sawatani Y.
        • Iwamoto K.
        • Hosogane N.
        • Fujita N.
        • et al.
        DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
        J Exp Med. 2005; 202: 345-351https://doi.org/10.1084/jem.20050645
        • Takegahara N.
        • Kim H.
        • Mizuno H.
        • Sakaue-Sawano A.
        • Miyawaki A.
        • Tomura M.
        • et al.
        Involvement of receptor activator of nuclear factor-κB ligand (RANKL)-induced incomplete cytokinesis in the polyploidization of osteoclasts.
        J Biol Chem. 2016; 291: 3439-3454https://doi.org/10.1074/jbc.M115.677427
        • Abmayr S.M.
        • Pavlath G.K.
        Myoblast fusion: lessons from flies and mice.
        Development. 2012; 139: 641-656https://doi.org/10.1242/dev.068353
        • Alkass K.
        • Panula J.
        • Westman M.
        • Wu T Di
        • Guerquin-Kern J.L.
        • Bergmann O.
        No evidence for cardiomyocyte number expansion in preadolescent mice.
        Cell. 2015; 163: 1026-1036https://doi.org/10.1016/j.cell.2015.10.035
        • Wang J.
        • Batourina E.
        • Schneider K.
        • Souza S.
        • Swayne T.
        • Liu C.
        • et al.
        Polyploid superficial cells that maintain the urothelial barrier are produced via incomplete cytokinesis and endoreplication.
        Cell Rep. 2018; 25 (464-477.e4)https://doi.org/10.1016/J.CELREP.2018.09.042
        • Gandarillas A.
        • Davies D.
        • Blanchard J.M.
        Normal and c-Myc-promoted human keratinocyte differentiation both occur via a novel cell cycle involving cellular growth and endoreplication.
        Oncogene. 2000; 19: 3278-3289https://doi.org/10.1038/sj.onc.1203630
        • Sanz-Gómez N.
        • de Pedro I.
        • Ortigosa B.
        • Santamaría D.
        • Malumbres M.
        • de Cárcer G.
        • et al.
        Squamous differentiation requires G2/mitosis slippage to avoid apoptosis.
        Cell Death Differ. 2020; 27: 2451-2467https://doi.org/10.1038/s41418-020-0515-2
        • Rios A.C.
        • Fu N.Y.
        • Jamieson P.R.
        • Pal B.
        • Whitehead L.
        • Nicholas K.R.
        • et al.
        Essential role for a novel population of binucleated mammary epithelial cells in lactation.
        Nat Commun. 2016; 7: 11400https://doi.org/10.1038/ncomms11400
        • Herrtwich L.
        • Nanda I.
        • Evangelou K.
        • Nikolova T.
        • Horn V.
        • Sagar
        • et al.
        DNA damage signaling instructs polyploid macrophage fate in granulomas.
        Cell. 2016; 167 (1264-1280.e18)https://doi.org/10.1016/j.cell.2016.09.054
        • Tang J.
        • Frascaroli G.
        • Lebbink R.J.
        • Ostermann E.
        • Brune W.
        Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability.
        Proc Natl Acad Sci USA. 2019; : 201907447https://doi.org/10.1073/pnas.1907447116
        • Spector D.H.
        Human cytomegalovirus riding the cell cycle.
        Med Microbiol Immunol. 2015; 204: 409-419https://doi.org/10.1007/s00430-015-0396-z
        • Herbein G.
        • Nehme Z.
        Polyploid giant cancer cells, a hallmark of oncoviruses and a new therapeutic challenge.
        Front Oncol. 2020; 10: 567116https://doi.org/10.3389/fonc.2020.567116
        • Zack T.I.
        • Schumacher S.E.
        • Carter S.L.
        • Cherniack A.D.
        • Saksena G.
        • Tabak B.
        • et al.
        Pan-cancer patterns of somatic copy number alteration.
        Nat Genet. 2013; 45: 1134-1140https://doi.org/10.1038/ng.2760
        • Quinton R.J.
        • DiDomizio A.
        • Vittoria M.A.
        • Ticas C.J.
        • Patel S.
        • Koga Y.
        • et al.
        Wh