Most cells in the liver are polyploid, but the functional role of polyploidy is unknown. Polyploidization occurs through cytokinesis failure and endoreduplication around the time of weaning. To interrogate polyploidy while avoiding irreversible manipulations of essential cell-cycle genes, we developed orthogonal mouse models to transiently and potently alter liver ploidy. Premature weaning, as well as knockdown of E2f8 or Anln, allowed us to toggle between diploid and polyploid states. While there was no detectable impact of ploidy alterations on liver function, metabolism, or regeneration, mice with more polyploid hepatocytes suppressed tumorigenesis and mice with more diploid hepatocytes accelerated tumorigenesis in mutagen- and high-fat-induced models. Mechanistically, the diploid state was more susceptible to Cas9-mediated tumor-suppressor loss but was similarly susceptible to MYC oncogene activation, indicating that polyploidy differentially protected the liver from distinct genomic aberrations. This suggests that polyploidy evolved in part to prevent malignant outcomes of liver injury.

Direct causal connections between polyploidy and tissue function in vivo have not been established because polyploidy is usually not treated as an independent variable. Defects in genes required for cell cycle or cytokinesis (Trp53, Rb, or Cdk1) can dramatically alter ploidy (), but interrogation of mice with these germline mutations cannot distinguish the effects of ploidy from the persistent pleiotropic effects of these mutations. For example, Cdk1 knockout (KO) hepatocytes cannot complete mitosis and are dramatically polyploid (up to 32N or greater). As a consequence, liver-specific Cdk1 KO mice are unable to undergo malignant transformation, but whether this is specifically due to polyploidy or permanent Cdk1 deficiency is unclear (). As a corollary, E2f7/E2f8 double KO livers are almost entirely composed of diploid hepatocytes, but these mice have no physiological or regenerative phenotypes (). Recently, E2f8 KO livers were shown to accelerate tumorigenesis, but it is unknown whether this is due to transcriptional effects of E2f8 deficiency or the diploid state of E2f8 KO hepatocytes (). Similarly,showed that Yap activation promotes polyploidy and cancer, but it is unclear whether the ploidy itself directly affects tumor biology independent of Yap. Due to the lack of appropriate tools and the inability to assess the chronic impact of ploidy change, the role played by polyploidy in diseases involving long-term cell-division cycles remains unclear. Here, we developed multiple methods and in vivo reagents to transiently and reversibly alter ploidy, and found that the polyploid state suppressed liver tumorigenesis by buffering against tumor-suppressor loss.

The influence of polyploidy is likely to be context dependent, mirroring the situation for aneuploidy, which can be tumor suppressive in untransformed cells and oncogenic in cancer cells (). Although the relationship between polyploidy and cancer has been interrogated in cell culture (), it has never been rigorously interrogated in the context of a major organ in vivo. We believe that it is fundamentally important to investigate the role of physiologically programmed polyploidy in one of the largest organs in the body. In this study we specifically focus on the influence of polyploidy on regenerative capacity and transformation risk in the normal liver.

As an argument against increased cancer risk, the polyploid state in hepatocytes has been associated with terminal differentiation and limited proliferation (). Furthermore,demonstrated that tetraploid hepatocyte proliferation/transformation is suppressed by the Hippo pathway. In other studies, 4c and 8c hepatocytes divide efficiently and contribute as much to proliferation, growth, and regeneration as 2c hepatocytes during liver growth and in transplant assays ( Figure S1 A) (). Altogether, it is unclear whether there are substantial differences in the proliferative rates of diploid and polyploid hepatocytes. In theory, polyploids could also be protected from cancer due to increased tumor-suppressor dosage (). Although illuminating, these data from diverse organisms and contexts make it difficult to predict how polyploidy might influence the function of normal liver cells and their risk for transformation.

The diverse functions for polyploidy in other contexts can inform our expectations for polyploidy in hepatocytes. In yeast and plants, polyploidy promotes adaptation to environmental stresses, in some cases through the production of genetically diverse aneuploid daughter cells (). Recently, it was shown that a subset of polyploid hepatocytes undergo reductive cell divisions prone to missegregation, leading to the accumulation of aneuploid cells (). Though the prevalence of aneuploidy in the liver is debated (), aneuploidy may represent a means by which adaptive genetic diversity or premalignancy could evolve. Whether or not polyploidy in the normal liver is a risk for or protective against cancer is unknown. In support of the premalignancy concept, many cancers show evidence for genome-doubling events (). In seminal work,showed that tetraploidy in p53 null mouse mammary epithelial cells is a chromosomally unstable state that predisposes to transformation. Since normal polyploid cells in the liver have intact p53, probably do not proliferate as much, and likely have a much lower risk for genome instability, the relevance to wild-type (WT) tetraploid cells in vivo is uncertain.

Polyploid cells and organisms contain more than two homologous sets of chromosomes. Polyploidy is prevalent in plants, fish, and salamanders (), but rare in mammals except in the cells of the heart, marrow, and liver. Surprisingly, up to 90% of rodent and about 50% of human hepatocytes are polyploid, making the liver one of the largest polyploid organs in mammals (). In rodents, liver ploidy dramatically increases around weaning (postnatal day 14 [P14] to P21) and continues to increase with age (). The dominant mechanism for polyploidization is cytokinesis failure that leads to binucleated hepatocytes (). To a lesser extent, hepatocyte endoreduplication also contributes to polyploidy via replication of the nuclear genome in the absence of cell division (). Thus, polyploid hepatocytes (tetraploids, octoploids, etc.) can be bi- or mononuclear. Postnatal liver polyploidization is developmentally regulated, but ploidy is dynamic and can increase with surgery (), fatty liver disease, and oxidative stress (). Given the prevalence and extreme extent to which polyploidy occurs in the liver, it is remarkable that there is so little known about the impact of polyploidy on tissue function or cancer risk.

Given that the mammalian liver is composed of a mixture of polyploid and diploid cells, these results would predict that HCCs more likely originate from diploid cells. If this was true, it would follow that liver tumors are also more frequently diploid rather than polyploid. We found that even tumors arising from siCtrl mice with polyploid cells were predominantly diploid ( Figures 6 B and 6C). In human HCCs examined by flow cytometry, HCCs more often comprised diploid as opposed to polyploid cells (). Altogether, this suggests that human HCCs more likely arise from diploid cells, consistent with the idea that the polyploid state is less compatible with cancer development.

Reduced susceptibility to tumor-suppressor LOH paired with reduced tumorigenesis in more polyploid livers suggested that DEN-induced tumors were dependent on tumor-suppressor loss. Previously, DEN was shown to preferentially select for oncogenic mutations (Hras and Ctnnb1) in studies focused on identifying mutations in these pathways (). We aimed to more broadly and thoroughly map the mutational landscape of these tumors, so we sequenced 242 of the most commonly mutated genes in human and murine HCCs in 50 individual DEN-induced tumors ( Figure 6 A and Table S2 ). We did identify a core group of recurrent, mutually exclusive mutations in oncogenes such as Egfr (Phe254Ile), Hras (Gln61Arg), and Braf (Val637Glu), but a majority of the most commonly mutated genes were bona fide TSGs such as Mll2 (Kmt2d), Brca2, Arid1a, Atm, Apc, and Tsc2. Overall, these data suggest that DEN tumor transformation depends on TSG loss in addition to EGFR-RAS-MAPK pathway activation, further supporting the idea that tumor protection in polyploids is in part mediated through retention of WT tumor-suppressor alleles.

All data in this figure are represented as mean ± SEM. See also Table S2

(D) Model depicting how TSGs could be lost in diploid and polyploid cells. Note that the liver contains both diploid and polyploid cells.

(C) Nucleus sizes of tumors from siRNA-treated mice. The nucleus size was quantified using Photoshop based on H&E staining images. Four mice in each group were quantified, 2 tumors per mouse.

(A) Targeted sequencing on 50 DEN-induced liver tumors. In total, 242 of the most commonly mutated genes in human and murine HCC were sequenced. Genes highlighted in red are oncogenes and those in black are TSGs.

To show that polyploidy protects from cancers that arise predominantly from TSG loss, we used a mouse strain with conditional alleles in important tumor-suppressor genes: P53;Rb1;Rbl2). First, we changed the ploidy of these mice using control and E2f8 siRNAs ( Figure 5 H), after which mice were injected with Ad-Cas9-sgPten as well as AAV-Cre to delete TSG alleles in a mosaic fashion. 2.5 months after viral injection, siCtrl-treated mice exhibited lower tumor burden compared with siE2f8 mice ( Figure 5 I). We also used a second HCC model that is caused by TSG loss. We targeted Pten, P53, and Lkb1 in WT mice. We used Ad-Cas9-gPten and an AAV containing guide strands against P53 and Lkb1 (AAV-KPL) (). This AAV also contains a guide against Kras as well as a KrasHDR template, so this model is not entirely driven by TSG loss. After ploidy was changed with siRNAs ( Figure S6 G), mice were injected with the Ad-Cas9-sgPten and AAV-KPL. Two months later, siAnln-treated mice exhibited the fewest tumors, followed by siCtrl and siE2f8-treated mice ( Figure S6 H). These cancer models both support the hypothesis that polyploidy protects from tumorigenesis caused by TSG loss.

Next, we tested whether TSG LOH is more difficult to achieve in hepatocytes with wholesale genome duplications. To quantitate the dynamics of TSG loss, we engineered an adenovirus carrying Cas9 and a guide strand RNA targeting Pten (Ad-Cas9-sgPten) (the Pten single guide RNA [sgRNA] was validated by), a commonly inactivated TSG in HCC. Fourteen days after intravenous Ad-Cas9-sgPten delivery into mice with different levels of ploidy, Pten LOH was assessed with immunohistochemistry ( Figure 5 D). The frequency of hepatocytes with complete Pten deletion was inversely proportional to the extent of polyploidy ( Figure 5 E). To confirm that the degree of ploidy did not change susceptibility to adenoviral infection, we verified that equal numbers of cells expressed GFP after adenovirus-GFP delivery ( Figure S6 E). To rule out the possibility of a Pten-specific phenomenon, we also assessed Apc, a TSG in the WNT pathway. We injected an adeno-associated virus carrying a guide strand against exon 8 of Apc (AAV-sgApc) into Dox-inducible Cas9 mice (Rosa-rtTA;TRE-Cas9) subjected to siRNA-induced ploidy changes ( Figure 5 F). In vitro, this particular sgApc effectively mutagenized Apc ( Figure S6 F) and had previously been used to generate Apc null hepatocytes (). To quantitate the number of Apc null clones in vivo, we probed for ectopic glutamine synthetase (GS), a specific and sensitive marker of aberrant WNT activation in the liver (). Strikingly, more diploid livers were much more susceptible to Apc LOH than control livers, and more polyploid livers harbored the fewest GScells ( Figure 5 G). These data support the concept that polyploid livers are protected from TSG loss and are not more sensitive to oncogene activation.

To challenge this hypothesis, we first examined the impact of oncogene activation in mice with different levels of polyploidy by overexpressing MYC using a liver-specific driver (LAP-tTA) and a Dox-inducible promoter (TRE-MYC) ( Figure S6 A) (). Prior to inducing MYC overexpression, we gave LAP-tTA;TRE-MYC mice four doses of siRNA to transiently alter ploidy ( Figures 5 B and S6 A). At P30, significant differences in ploidy, but not MYC expression levels, were observed ( Figures S6 B and S6C). Dox withdrawal at P25 leads to transformation of less than 1% of MYC-expressing cells, a dynamic range that allowed us to sensitively quantitate the influence of ploidy on tumor initiation. Nine weeks after induction, tumor burden, liver to body weight ratios, and tumor ploidy were indistinguishable between siRNA-treated ploidy groups ( Figures 5 C and S6 D), showing that ploidy had little impact on MYC oncogene-induced tumorigenesis.

Since the mutagenic activities of DEN were quantitatively similar, it was possible that ploidy differentially influenced tumor-suppressor gene (TSG) loss and proto-oncogene activation. We first performed a theoretical calculation to estimate the risk of TSG loss and oncogene activation in tissues with 10,000 diploid cells versus 5,000 tetraploid cells (which are ∼2-fold larger). Both tissues contain 20,000 sets of chromosomes ( Figure 5 A). Assuming 10% of TSGs are mutated in one massive DNA-damaging event, the rate of loss of heterozygosity (LOH) in a diploid cell is 0.1= 0.01, while for a tetraploid cell it is 0.1= 0.0001. Thus, 100 cells versus 0.5 cells on average achieve LOH in diploid versus tetraploid tissues of the same mass. For simplicity, we can also assume that the oncogene activating mutation rate is also 0.1. The probability that this mutation occurs in a diploid cell is 1 − 0.9≈ 0.19, while in a tetraploid cell it is 1 − 0.9≈ 0.34. Although the probability is higher for tetraploid cells, diploid versus tetraploid tissues have 1,900 and 1,700 cells at risk once total cell numbers are accounted for. This calculation suggests that polyploid tissues are dramatically protected against TSG LOH but are at similar risk for oncogene activation. This model is oversimplified since tumorigenesis involves more subtle and complex genome alterations (), but is still likely to be informative because TSG LOH is an important part of carcinogenesis.

All data in this figure are represented as mean ± SEM. See also Figures S5 and S6

(I) One week after ploidy was altered with siRNAs, the mice were injected with Ad-Cas9-sgPten (10 9 pfu/mouse) and AAV-Cre (2.5 × 10 10 pfu/mouse). Tumor burden was examined 2.5 months later. Liver to body weight ratios are shown on the right.

(H) Representative cellular ploidy distribution of P53 fl/fl ;Rb1 fl/fl ;Rbl2 fl/fl hepatocytes treated with four doses of siRNAs (siCtrl or siE2f8) from P10 to P21, as determined by PI staining and flow cytometry at P24.

(G) GS staining (left panels) 15 days after AAV-sgApc injection. Red arrowheads point to ectopic GS-positive cells that result from homozygous Apc deletion. Note that the normal, non-ectopic GS staining that surrounds central veins was not quantified. Ectopic GS-positive cells per imaging area were quantified (right panel).

(F) Schema of the AAV-sgApc experiment: Dox-inducible Rosa-rtta;TRE-Cas9 mice were given four doses of siRNA to alter ploidy. Five days later, Dox (1 g/L) was given to induce Cas9 expression. Two weeks after the last dose of siRNA, AAV-sgApc (5 × 10 12 pfu/mouse) was retro-orbitally delivered; 15 days later, the Apc LOH was estimated by glutamine synthetase (GS) immunofluorescence.

(E) PTEN staining (left panel) on siAnln, siCtrl, and siE2f8-treated livers 14 days after virus injection. Red arrowheads point to the cells without PTEN, with quantification on the right.

(D) Schema of the Ad-Cas9-sgPten experiment: WT mice were given four doses of siRNA to alter ploidy; 1 week later, adenovirus carrying Cas9 and a Pten sgRNA (Ad-Cas9-sgPten) was injected into these mice (10 9 pfu/mouse); 14 days later, LOH was assessed by PTEN immunohistochemistry.

(C) Tumor burden at P90 (left panel). Liver to body weight ratios, another metric of tumor burden, are shown on the right. N.S., not significant.

(B) Schema of the LAP-MYC experiment: prior to inducing MYC overexpression by Dox withdrawal at P25, LAP-tTA;TRE-MYC mice were given four doses of siRNA to transiently alter ploidy, as described previously. At P30, ploidy distribution and MYC expression levels were measured; At P90, tumor burden was assessed.

To probe underlying mechanisms in the DEN model, we first asked whether ploidy significantly regulated metabolic properties that would influence tumorigenesis. DEN is first bioactivated by CYP450 family enzymes to become α-hydroxylnitrosamine (). Expression of CYP450 enzymes in general and zonation of Cyp2e1 in particular were unchanged in livers with distinct ploidy ( Figures S2 C and S5 A). Elevated levels of reactive oxygen species (ROS) secondary to hepatotoxins are known to accelerate tumor initiation (), but ROS levels were unchanged between siRNA-treated mice and between E2f8 WT/Het/KO mice ( Figures S5 B–S5D). It is also known that following the DEN bioactivation, an ethyldiazonium ion is formed, binds DNA, and causes genotoxic damage (). Although DNA-damage markers such as p-Brca1, p-p53, and p-γH2A.X increased after DEN, the magnitude of induction was similar between groups ( Figures S5 E and S5F). Altogether, livers with altered ploidy did not exhibit differential xenobiotic metabolism, oxidative stress, or DNA-damage responses.

Polyploid livers resulting from transient suppression of Anln were almost completely protected from DEN-induced tumorigenesis, confirming the siRNA results ( Figures 4 G and 4H). We then wanted to exploit this model to analyze the function of polyploidy in a carcinogenesis model caused by another clinically important mechanism. Steatohepatitis represents an increasingly relevant risk factor for hepatocellular carcinoma (HCC) and has been associated with an increase in polyploidy (). To induce long-term fatty liver disease and HCC, we fed mice with a high-fat diet (HFD) after transiently inducing ploidy changes ( Figure 4 I). After 8 months, Rosa and TG-shAnln mice had similarly high levels of steatosis and liver function abnormalities ( Figures 4 J–4L and S4 I), but 50% of control livers while no polyploid livers developed HCC ( Figures 4 M and 4N). In summary, multiple methods of increasing and decreasing ploidy corroborated the fact that higher levels of polyploidy suppressed liver tumors in diverse cancer models.

Next, we wanted to generate a more potent and versatile mouse model to increase polyploidy. In addition, our goals were to drive greater levels of polyploidy and to employ a small inhibitory short hairpin RNA (shRNA) distinct from the siAnln used above in order to corroborate on-target effects on Anln. Thus, we employed a doxycycline (Dox)-inducible transgenic mouse expressing an shRNA against Anln (). Mice were generated from embryonic stem cells containing Rosa-rtTA and a GFPshAnln cassette under the control of a tetracycline-responsive promoter element (TRE) ( Figures S4 D and S4E; transgenic design based on Scott Lowe's group []). Anln suppression can be induced by Dox in a temporally specific fashion ( Figure 4 A). Rosa-rtTA alone or Rosa-rtTA;TRE-shAnln (hereafter called Rosa and TG-shAnln) transgenic mice exposed to Dox water from P0 to P20 showed normal growth, development, and liver function ( Figures S4 F–S4H). Anln mRNA levels were suppressed by 50% ( Figure 4 B), which resulted in more polyploid livers at multiple time points after Dox withdrawal ( Figures 4 C–4E). These livers were similar to what was seen with Anln siRNA treatment, but had more profound levels of polyploidization. In addition, GFP protein (and likely Anln shRNA) completely disappeared by 15 days after Dox withdrawal ( Figure 4 F), demonstrating the reversibility of Anln suppression.

All data in this figure are represented as mean ± SEM. See also Figure S4

(N) H&E staining of tumors from Rosa control mice given HFD. Yellow dotted line is the boundary between non-tumor tissue (labeled N) and tumor (labeled T).

(M) Representative gross tumor burden from the HFD experiment. Fifty percent (3 of 6) of Rosa mice had tumors, while no TG-shAnln mice (0 of 11) carried tumor.

(I) Schema for the high-fat diet (HFD)-induced HCC experiment in inducible TG-shAnln mice: Transient Dox treatment mice established ploidy differences. Starting at P42, mice were given ad libitum HFD (60% calories from fat). Tumor burden was examined after 8 months.

(G) Schema for the DEN-induced HCC experiment in inducible shRNA mice: Transient Dox treatment from P0 to P20 in Rosa-rtTA or TG-shAnln mice established ploidy differences. At P35, mice were injected with DEN (100 μg/g). Tumor burden was examined 8 months later.

(D) Representative cellular ploidy distribution of TG-shAnln livers treated with Dox from P0 to P20 was determined by PI staining and flow cytometry at the age of P35 (left panel). On the right is the average ploidy distribution (n = 6 mice in each group).

(C) Representative cellular ploidy distribution of TG-shAnln livers treated with Dox from P0 to P20 was determined by PI staining and flow cytometry at the age of P20 (left panel). On the right is the average ploidy distribution (n = 3 mice in each group).

Given the formal possibility that siRNAs or lipid nanoparticles could have off-target or non-specific effects, we also used Cas9 to generate whole-body E2f8 KO mice ( Figure S4 A), which have predominantly diploid hepatocytes in adulthood ( Figure 3 A). Interestingly, we observed that E2f8 WT, Het, and KO livers had equivalent levels of ploidy at P15, and only diverged in ploidy state by P27 ( Figures 3 A and S4 B). We hypothesized that if ploidy is a specific and essential factor causing tumor suppression in E2f8 KO livers, then mutagenizing and inducing cancer at P15 would not result in differences in liver tumor development, while inducing at P27 would result in large differences. Indeed, DEN given at P15 (25 μg/g) resulted in no cancer differences between the three groups, while DEN at P27 (75 μg/g) caused more liver tumors in E2f8 Het and E2f8 KO mice when compared with E2f8 WT mice ( Figures 3 B–3E and S4 C). These results further demonstrated that the E2f8 gene itself contributed less to cancer development than the ploidy differences resulting from E2f8 deficiency.

All data in this figure are represented as mean ± SEM. N.S., not significant. See also Figure S4

(B) DEN was introduced by intraperitoneal injection in one cohort at P15 (25 μg/g) and in another cohort at P27 (75 μg/g). Gross tumor burden was evaluated 4.5–5 months later. The insets show higher-magnification images that more clearly exhibit tumor burden.

After establishing that inducible inhibition of Anln and E2f8 alters ploidy without introducing irreversible genetic mutations, we next evaluated the impact of ploidy on tumor development. We injected DEN (75 μg/g × 1 dose) into mice 4 days after the last dose of siCtrl, siAnln, or siE2f8 (siRNA delivery schema in Figure 2 A). Six months later, siE2f8-treated livers with more diploid cells had significantly increased gross tumor burden, microscopic tumor nodules, and liver/body weight ratio than siCtrl livers ( Figures 2 I, 2J, and S3 A–S3C). In contrast, siAnln-treated livers with more polyploid cells showed the opposite. To further exclude the possibility of residual siRNA effects, we also introduced DEN at 14 days, rather than 4 days, after the last siE2f8 injection. Polyploidy again demonstrated a potent tumor-suppressive effect ( Figures 2 K and S3 D). To generate higher levels of polyploidy, we developed two additional and distinct N-acetylgalactosamine (GalNAc) conjugated siRNAs against Anln. The exposed GalNAc moiety mediates siRNA uptake via asialoglycoprotein receptors (ASGPR), which are specifically expressed by hepatocytes. Subcutaneous injections of these siRNAs (3 × 4-mg/kg doses between P8 and P20) resulted in high levels of polyploidy when compared with mice treated with luciferase siRNA (siLuc) ( Figure S3 E). After DEN (75 μg/g at P26), the siAnln mice also exhibited fewer tumors ( Figure S3 F). To avoid the possibility of Anln-specific effects, we also induced liver polyploidy by knocking down Cdk1 with an in vivo siRNA ( Figure S3 G). After siRNA injections, mice were dosed with DEN (75 μg/g). Six months after DEN, siCdk1-treated mice exhibited reduced tumor burden ( Figure S3 H), again supporting the tumor-protective role of polyploidy. Altogether, these findings suggested that the degree of liver polyploidy is anti-correlated with the efficiency of carcinogenesis.

Altered ploidy did not affect overall health, liver mass, body mass, hepatocyte differentiation, CYP450 expression, or proliferation ( Figures S2 A–S2E). Moreover, we challenged these mice to acute regeneration assays such as partial hepatectomy and hepatotoxin treatments. Hepatectomy was performed at 5–6 weeks of age, when ploidy differences were still detected ( Figure S2 F). Liver/body weight ratios after two-thirds partial hepatectomy were not significantly different between different ploidy groups ( Figure S2 G). There were also no differences in necrosis and proliferation after single doses of DEN or carbon tetrachloride ( Figures S2 H and S2I). These results suggested that polyploid and diploid hepatocytes were equivalently able to survive, recover, and proliferate after injuries. These findings are consistent with previous studies in mice with ploidy alterations (), again demonstrating that ploidy state has minimal influences on postnatal liver growth and regeneration, likely because only 2–4 cell-division cycles are required for recovery after these profound, acute injuries.

To inducibly toggle polyploidy without introducing permanent genetic lesions, we used small interfering RNA (siRNA) to transiently knock down genes that affect ploidy. We targeted Anillin (Anln), an actin-binding protein required for cytokinesis () ( Figures S1 F and S1G) and E2f8, a transcription factor required for polyploidization (). In vivo, Anln and E2f8 siRNA versus scramble siRNA (siCtrl) delivered in lipid nanoparticles () from P10 to P20 resulted in significant knockdown of Anln and E2f8 mRNA and protein ( Figures 2 A, 2B , and S1 H). As expected, cellular ploidy was significantly increased after Anln knockdown and decreased after E2f8 knockdown ( Figure 2 C). Ploidy characterization using confocal imaging revealed that siAnln-treated livers had significantly larger cell and nuclear size, while the siE2f8-treated livers showed the opposite ( Figures 2 D and 2E). Since cell and nuclear size correlate with DNA content, this suggested significant ploidy changes. To quantitatively distinguish mono- versus binucleated tetraploids, we integrated flow and confocal imaging data ( Figure 2 F). This revealed that siAnln hepatocytes were 32% mononuclear diploid, 32% mononuclear tetraploid, and 32% binuclear tetraploid. siE2f8 hepatocytes were 64% mononuclear diploid, 17% mononuclear tetraploid, and 16% binuclear tetraploid. Furthermore, fluorescence in situ hybridization (FISH) confirmed the existence of mono- and binuclear tetraploids ( Figures 2 G and 2H). Overall, this indicated that both mononuclear and binuclear tetraploids were increased in siAnln versus control livers and in control versus siE2f8 livers.

All data in this figure are represented as mean ± SEM. See also Figures S2 and S3

(K) Tumor burden in mice treated with DEN at later time points after siRNA delivery. Mice were treated with four doses of siRNAs. Fourteen days after the last siRNA injection, one dose of DEN (100 μg/g) was given, and 7.5 months later, tumor burden was assessed (n = 8 in each group). The liver surface tumors were quantified (right panel).

(H) On the left is the nuclear ploidy of the above siRNA-treated livers, as determined by FISH. Sixty nuclei were analyzed for each mouse. On the right are representative images showing a 2c and a 4c nucleus. Two green and 2 red signals identify a 2c nucleus. Four green and 4 red signals identify a 4c nucleus.

(F) Representative cellular ploidy distribution of siRNA-treated livers (upper panel). The lower panel shows the distribution of mono- and binucleated tetraploid hepatocytes. Calculations were performed as follows: % of mononucleated tetraploid cells = (% tetraploid cells by fluorescence-activated cell sorting) minus (% binucleated cells by confocal imaging) (images from Figure 2 D, n = 3 images were counted for each mouse, 2 mice for each group).

(E) Cross-sectional cell and nucleus size measurements. Each group includes 12 analyzed image fields in total from 4 individual mice. For nucleus size quantification, each data point is one nucleus; for cell size quantification, each data point is an average of the cell sizes from one image, and 3 images were taken from each mouse.

(C) Average cellular ploidy distribution within livers, as determined by PI staining and flow cytometry (n = 6 mice in each group were analyzed).

(B) mRNA knockdown after Anln and E2f8 siRNA treatments. qRT-PCR was performed on the liver 4 days after the last siRNA dose.

(A) Schema for the siRNA experiment: Anln, E2f8, and scramble siRNAs (siCtrl) were encapsulated into lipid nanoparticles and injected into WT C3H strain mice starting at P10. A total of four injections (two intraperitoneal and two retro-orbital) were performed twice per week. Between P24 and P26, hepatocytes were dissociated for ploidy analysis or mice were injected with DEN (75 μg/g). Tumor burden was examined 6 months later.

To answer these questions, we reasoned that the transient, reversible control of ploidy state would represent a fundamental advance to enable the elucidation of ploidy functions. Since the predominant mechanism of widespread hepatocyte polyploidization is cytokinesis failure, a phenomenon temporally associated with weaning in rats (), we asked whether differential weaning times could transiently influence ploidy in mice. By weaning WT mice at P13 (premature weaning) or P21 (normal weaning) ( Figure 1 A), we found that prematurely weaned mice had significantly more binucleated hepatocytes ( Figure 1 B) and increased cellular ploidy ( Figures 1 C and S1 B) at 19 days of age, but not at 2.5 months ( Figure S1 C). To induce liver tumors when ploidy states were divergent, we gave a single intraperitoneal dose (25 μg/g) of the agent diethylnitrosamine (DEN) to both cohorts at P19. Notably, the expression of cytochrome P450 and other differentiation genes was not significantly different between premature and normal weaning mice ( Figures S1 D and S1E). Five months later, prematurely weaned mice with greater ploidy at P19 exhibited significantly reduced gross and microscopic tumor burden ( Figures 1 D and 1E), suggesting that polyploidy could exert tumor-suppressive effects. Since weaning is confounded by factors other than ploidy, we also used additional methods to control ploidy state.

All data in this figure are represented as mean ± SEM. See also Figure S1

(E) Microscopic tumor nodules are circled by yellow dashed lines. The right panel shows nodule quantification (tumors from one section of an entire lobe were quantified per mouse, n = 10 and n = 9 for normal and early weaning groups).

(C) Ploidy distribution was analyzed by flow cytometry. Representative histograms show DNA content as stained by propidium iodide (PI). Compiled data from the two groups are shown on the right (n = 8 mice in each group).

(B) DAPI and CTNNB1 immunofluorescence staining identified nuclei and cell membranes (left panel). Representative mononucleated and binucleated hepatocytes are circled by the yellow dashed lines. The percentages of binucleated hepatocytes are quantified on the right (4 images were analyzed and averaged per mouse, n = 3 mice per group).

(A) WT mice were weaned at P13 and P21 to induce differences in polyploidization. Diethylnitrosamine (DEN) was injected to both cohorts at P19 to induce hepatocellular carcinoma (HCC) formation.

Discussion

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et al. DNA ploidy pattern in human chronic liver diseases and hepatic nodular lesions. Flow cytometric analysis on echo-guided needle liver biopsy. Fujimoto et al., 1991 Fujimoto J.

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Yamanaka N.

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Mitsunobu M. Flow cytometric DNA analysis of hepatocellular carcinoma. Nagasue et al., 1993 Nagasue N.

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Nakamura T. Lack of intratumoral heterogeneity in DNA ploidy pattern of hepatocellular carcinoma. Here, we devised multiple in vivo methods to alter liver ploidy in a reversible fashion, such that long-term consequences of such changes could be assessed and compared. In agreement with previous studies (), we found that polyploidy had little impact on acute injury or liver regeneration, but chronically, the polyploid state demonstrated tumor-suppressive functions in multiple cancer models. In general, the extent of ploidy increase or decrease in different siRNA, genetically engineered mouse models, and inducible transgenic models appeared to correlate with tumor number and burden. Moreover, we believe that this protection is due in large part to buffering against TSG loss rather than limiting proliferation after oncogenic insults. As shown in Figure 6 D, cells within predominantly diploid tissues lose their tumor suppressors through classical LOH mechanisms. In contrast, polyploid hepatocytes harbor up to 16 alleles for each TSG, thereby buffering against a second or even third hit. Consistent with this, DEN tumors were most often diploid, suggesting a diploid origin. This is consistent with human data showing that HCCs are predominantly diploid (). We also showed that MYC-induced oncogenesis in the liver was agnostic to ploidy states. It is possible that the strong oncogenic effect of MYC overcame tumor-protective effects of ploidy, but the effects of other oncogenes may be more influenced by polyploidy.

Davoli and de Lange, 2012 Davoli T.

de Lange T. Telomere-driven tetraploidization occurs in human cells undergoing crisis and promotes transformation of mouse cells. Fujiwara et al., 2005 Fujiwara T.

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Pellman D. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Zhang et al., 2017a Zhang S.

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et al. Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2. Ben-David et al., 2014 Ben-David U.

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Maimon A.

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Carlos Biancotti J. Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells. Sheltzer et al., 2017 Sheltzer J.M.

Ko J.H.

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Habibe Burgos N.C.

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Amon A. Single-chromosome gains commonly function as tumor suppressors. Silk et al., 2013 Silk A.D.

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Weaver B.A. Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors. It is also important to note that we have only altered ploidy in the benign liver and not within established tumors; thus it is possible that increasing ploidy within cancer cells would cause genome-destabilizing effects documented by others (). This would be in line with aneuploidy studies, which have shown that aneuploidy is either tumor suppressive or oncogenic depending on context (). It is important to note here that our approaches did not allow us to distinguish euploidy and aneuploidy within polyploid hepatocytes. Polyploidy will probably also demonstrate context dependency, but it is important to underscore that the extensive polyploidy found in the normal liver is an extremely important context to fully understand. Our findings suggest that in the setting of normal hepatocytes, TSG buffering could be an important tumor-suppressive function of polyploidy.

Lee et al., 2004 Lee J.S.

Chu I.S.

Mikaelyan A.

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Thorgeirsson S.S. Application of comparative functional genomics to identify best-fit mouse models to study human cancer. Westcott et al., 2015 Westcott P.M.

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et al. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. We believed that it was essential to use mouse models to interrogate the role of ploidy in cancer because it is the most rigorous way to examine tumor initiation. For example, in vivo and in vitro human HCCs, which are already transformed, cannot be used to study ploidy's role in the transition state between normal and malignant cells. We also used cancer models that were the most reflective of human disease states. The DEN model generates cancers that are not dependent on singular, artificially strong genetic drivers such as Yap or Akt. Using expression profiling, Thorgeirsson’s group showed that DEN-induced mouse HCCs were more similar to poor-survival human HCCs than other mouse cancer models (). Also, Balmain's group recently argued for the use of chemically induced mouse cancer models because they more accurately reflect the diverse genomics of human cancers (). Interestingly, Trp53 mutations were not detected in the DEN tumors, suggesting that there might be a role for Trp53 in the development of diploid tumors. It would be interesting to test the polyploidy protection hypothesis in a Trp53 null background. Nevertheless, the tumors in our study harbored a wide diversity of oncogenic and tumor-suppressor mutations that reflect the biology of human HCCs.

Liu and Wu, 2010 Liu Y.

Wu F. Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. The DEN model may resemble the types of liver cancers caused by acute exposure to mutagens such as aflatoxin. Although HCC in the United States is commonly caused by chronic etiologies (hepatitis B and C viruses, alcohol, non-alcoholic steatohepatitis), acute liver injury and mutagenesis by aflatoxin is a common problem in tropical and subtropical climates in Africa, Asia, and South America (). It is conceivable that polyploidy in part evolved to mitigate the cancer risk associated with these types of acutely acting liver mutagens, which frequently induce lethal cancers during the reproductive years. We believe it essential to understand polyploidy in the context of acute injury events because these may be the types of massive toxic events that mammalian livers have evolved to buffer against.

Despite the importance of acute liver injuries, it is possible that polyploidy might have different implications for chronic injury. The HFD model ( Figure 4 ) involves chronic nutritional stress that can contribute to cirrhosis and HCC in patients. Our data suggest that in this chronic context, polyploidy is protective against HCC development. Work on this and other liver cancer models will help to determine whether polyploidy in the liver is generally tumor suppressive or is dependent on factors such as the nature of the genetic driver lesion or environmental injury.