Immunopositivity for Histone MacroH2A1 Isoforms Marks Steatosis-Associated Hepatocellular Carcinoma
et al. (2013) Immunopositivity for Histone MacroH2A1 Isoforms Marks Steatosis-Associated
Hepatocellular Carcinoma. PLoS ONE 8(1): e54458. doi:10.1371/journal.pone.0054458
Immunopositivity for Histone MacroH2A1 Isoforms Marks Steatosis-Associated Hepatocellular Carcinoma
Francesca Rappa 0
Azzura Greco 0
Christine Podrini 0
Francesco Cappello 0
Michelangelo Foti 0
Lucie Bourgoin 0
Marion Peyrou 0
Arianna Marino 0
Nunzia Scibetta 0
Roger Williams 0
Gianluigi Mazzoccoli 0
Massimo Federici 0
Valerio Pazienza 0
Manlio Vinciguerra 0
Franco Folli, University of Texas Health Science Center at San Antonio, United States of America
0 1 Department of Experimental Biomedicine and Clinical Neurosciences, Section of Human Anatomy, University of Palermo , Palermo , Italy , 2 Euro-Mediterranean Institute of Science and Technology , Palermo , Italy , 3 Institute of Hepatology, Foundation for Liver Research , London , United Kingdom , 4 Istituto ''Paolo Sotgiu, Libera Universita` degli Studi di Scienze Umane e Tecnologiche, Lugano, Switzerland, 5 Department of Cell Physiology and Metabolism, University of Geneva , Geneva , Switzerland , 6 Department of Systems Medicine, University of Rome Tor Vergata , Rome , Italy , 7 Pathologic Anatomy Unit, Civic Hospital , Palermo , Italy , 8 Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS ''Casa Sollievo della Sofferenza'' Hospital , San Giovanni Rotondo , Italy , 9 Division and Laboratory of Gastroenterology, IRCCS ''Casa Sollievo della Sofferenza'' Hospital , San Giovanni Rotondo , Italy
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. Methods: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fatassociated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. Results: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p,0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (,2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. Conclusions: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.
Competing Interests: Manlio Vinciguerra is a PLOS ONE Editorial Board member. This does not alter the authors adherence to all the PLOS ONE policies on
sharing data and materials.
. These authors contributed equally to this work.
" These authors also contributed equally to this work
Current address: Institute for Liver and Digestive Health, Division of Medicine, University College London (UCL), London, United Kingdom
The current pandemic in obesity/metabolic syndrome is a risk
factor for many types of cancer. The largest increase in cancer risk
(,5 fold) in obese individuals with high body mass index (BMI 35
40) was seen for primary hepatocellular carcinoma (HCC) .
Obesity is accompanied in up to 90% of cases by non-alcoholic
fatty liver disease (NAFLD) . The latter is the consequence of an
imbalance between lipid availability through fatty acid uptake and
de novo lipogenesis, and lipid secretion and disposal via free fatty
acid oxidation, resulting in hepatic accumulation of lipids
(steatosis) . In 10% of the cases NAFLD will progress to a
steatohepatitis (NASH), and in 826% to cirrhosis, with an
increasingly reported percentage of cases with cirrhosis or NAFLD
at an earlier stage developing HCC . Recent studies in rodent
models suggested that high fat in the liver might trigger the
development of HCC through inflammation, activating specific
signalling pathways, growth factors and cytokines [5,6,7,8,9,10].
Alterations in hepatocyte metabolism and proliferation during
steatosis and HCC are triggered by changes in gene transcriptional
patterns. The epigenetic mechanisms involved in HCC associated
with obesity/metabolic syndrome/steatosis have not been
investigated in detail. Nuclear chromatin compaction is regulated at
several levels, allowing transcriptional plasticity : one of these
is the replacement of canonical histones around which DNA is
wrapped (H2A, H2B, H3 and H4) with the incorporation of
histone variants. The histone variant of histone H2A known as
macroH2A1 is believed to act as a strong transcriptional
modulator that can either repress transcription [12,13], but can
also activate a subset of genes in response to as yet undefined
growth signals [14,15]. MacroH2A1 knock out (KO) mice display
hepatic steatosis and derangements in glucose and lipid
metabolism [16,17] and, interestingly, when wild type mice are fed a
methyl-deficient diet, which induce a fatty liver and inflammation,
a total increase in the hepatic content of macroH2A1 is observed
MacroH2A1 is present in 2 isoforms, macroH2A1.1 and
macroH2A1.2, which are generated upon RNA alternative exon
splicing. The expression of both isoforms has been shown to
predict lung cancer recurrence  and, in colon cancer,
macroH2A1.1 inversely correlates with cancer severity and
survival, whereas macroH2A1.2 does not show such correlation
. Recently, it has been shown that splicing of macroH2A1
isoforms regulates cancer cell growth . In A549 lung cancer
cells, HeLa cervical adenocarcinoma cells, IMR90 primary lung
fibroblasts, and MG-63 osteosarcoma cells reduced levels of
macroH2A1.1 compared to macroH2A1.2 were observed .
Reintroduction of macroH2A1.1 suppressed the growth of these
cancer cell lines . Other studies, which did not distinguish
between the isoforms, demonstrated that KO of all macroH2A1
isoforms induced the progression of the melanoma malignant
phenotype both in vitro and in vivo through increased expression of
CDK8 oncogene .
Regulation by macroH2A1 of oncogenes and/or tumor
suppressors expression in hepatocytes could be particularly
relevant for fatty liver-associated HCC, since the activities of
these genes often link mechanistically hepatic steatosis to the onset
of HCC, as we have previously shown for tumor suppressor
phosphatase with tensin homology (PTEN) [23,24,25]. PTEN is
one of the most important tumor suppressors, mutated or deleted
in nearly half of human cancers, including HCC patients  and
changes in its expression have also been shown to regulate hepatic
lipid metabolism and insulin sensitivity [23,24,25,26]. In this study
we explored if an altered expression of macroH2A1 isoforms
might be associated to fatty liver-associated HCC using two robust
mice models for this transition, the dietary high
fat/diethynitrosamine (DEN) diet  and the genetic liver-specific PTEN
knockout (KO) mouse . Furthermore, we examined the expression
of macroH2A1 isoforms in human liver biopsies from patients
where HCC occurred in a background of pure steatosis, in absence
of other liver diseases.
Materials and Methods
Human biopsies: all the procedures followed were in
accordance with the ethical standards of the responsible committees
(institutional and national) on human experimentation and with
the Helsinki Declaration of 1975 (as revised in 2008). Written
informed consents were obtained from all patients at the time of
biopsy and the study was approved by Ethics Committee of the
Civic Hospital, Palermo, Italy.
Mice models: for the PTEN KO model, all the procedures were
in accordance with the Swiss guidelines for animal
experimentation and with ethically written approval by the Geneva
(Switzerland) health head office. For the HF/DEN model, procedures
were in accordance with the Italian national authorities and
written approval was obtained by the Institutional Animal Care of
the University of Rome "Tor Vergata protocol 28/02/
2012 n. 17.
According to an established protocol , in the DEN-induced
HCC model, DEN (25 mg/kg) was injected intraperitoneally into
14 days old mice. After 4 weeks, mice were separated into two
dietary groups and fed either chow or high fat diet until sacrificed
at 36 weeks of age. High fat diet (composed of 59%-fat,
15%protein, 26%-carbohydrates based on caloric content) was
purchased from Research Diet, New Brunswick, NJ, US.
To obtain PTEN liver specific KO mice, Ptenflox/flox mice
(129Ola C57BL6/J F2) were mated to AlbCre transgenic mice
(C57BL6/J background) as previously described , in which
expression of Cre is controlled by the promoter of the
hepatocytespecific gene Albumin. Offspring carrying AlbCre and two copies
of the floxed Pten allele (AlbCrePtenflox/flox), and control Ptenflox/
flox mice were retained for experiments. Mice were sacrificed at 16
weeks for studying the steatotic phenotype and at 52 weeks for
studying the HCC phenotype. PCR analysis of PTEN genotypes
was performed as previously reported .
In both high fat diet DEN-induced HCC and PTEN
KOinduced HCC models, tumors in each liver lobe were counted.
Serum, liver tumor and non-tumor tissue was collected and rapidly
frozen for biochemical and histological analyses (see below).
Human Sample Collection
Formalin-fixed paraffin embedded biopsies were retrospectively
collected from files of the Pathologic Anatomy Unit of the Civico
Hospital, Palermo, Italy. 10 cases were selected of mild mixed
macro- and micro-vesicular steatosis. 10 cases of HCC arising in
macro-vesicular steatosis were also selected. The clinical
characteristics of the patients studied are summarized in Table 1, in
terms of history of either HBV/HCV infection, cirrhosis,
alcoholism and NAFLD score (see next paragraph). Fibrosis
and/or cirrhosis were not observed in the biopsies.
*Assessed according to Ref. 29.
0/6 (UNKNOWN IN 4 CASES)
Figure 1. Protein expression of macroH2A1 isoforms in the liver of HF/DEN mouse model of steatosis and HCC. A. Representative
pictures of trichrome staining in liver samples from mice fed a normal diet (ND) and mice fed a high fat (HF) diet for 36 weeks. B. Histone extracts
from livers of ND, HF and HF/DEN mice were processed for immunoblotting. MacroH2A1.1, macroH2A1.2 and histone H3 (loading control) protein
levels were detected with specific antibodies. Representative immunoblots are shown C. Densitometric quantification of macroH2A1.1, macroH2A1.2
protein levels in the livers of ND, HF and HF/DEN mice. N = 5, * P,0.05, ** P,0.01 versus ND mice.
Figure 2. Protein expression of macroH2A1 isoforms in the liver of the liver-specific PTEN KO mouse model of steatosis and HCC. A.
Representative pictures of trichrome staining in liver samples from PTENflox/flox and PTEN KO mice. B. histone extracts from livers of 16 weeks old
PTENflox/flox, 16 weeks old PTEN KO and from liver tumors of 52 weeks old PTEN KO mice were processed for immunoblotting. MacroH2A1.1,
macroH2A1.2 and histone H3 (loading control) protein levels were detected with specific antibodies. Representative immunoblots are shown. C.
Densitometric quantification of macroH2A1.1, macroH2A1.2 protein levels in the livers of 4 months old PTENflox/flox (N = 5), PTEN KO 16 weeks old
(N = 5) and in the liver tumors of PTEN KO 52 weeks old mice (N = 2), * P,0.05, ** P,0.01 versus PTENflox/flox mice.
Histological Assessment of NAFLD Score
Sections from both mice and human specimens were processed
by haematoxylin and eosin (H&E) and Masson trichrome staining
for histological evaluation of NAFLD score, as previously
described [28,29], in which a semi-quantitative scoring system
that grouped histological features into five broad categories of
Figure 3. Histological features of liver biopsies of patients with steatosis and HCC. A. Representative pictures of hematoxylin and eosin
(H/E) staining. In HCC samples both area with tumor and area of steatosis close to HCC (St/HCC) were examined. Pictures showed the same area
observed with a lower (above) and higher (below) magnification. B. Representative pictures of trichrome staining in samples with steatosis and HCC.
In the latter both area with tumor and St/HCC were examined. Pictures showed the same area observed with a lower (above) and higher (below)
magnification. Trichrome stains showed that collagen (green) was present only in portal space and, in limited amount, in perisinusoidal (Disse) spaces
of lobule in both steatosis and St/HCC samples, in which macro and micro vesicular steatosis is also visible. Collagen was also present in
correspondence of the capsule that delimits HCC (arrow). Bar: 100 mm.
Figure 4. Representative pictures of immunostaining performed for MacroH2A1.1, MacroH2A1.2 and Ki-67 in samples with
steatosis and HCC. In the latter both area with tumor and area of steatosis close to HCC (St/HCC) were examined. All nuclei of tumor cells were
positive for either macroH2A1.1 or macroH2A1.2. Positivity in hepatocytes of steatosis was significantly lower. Pictures showed the same area
observed with a lower (above) and higher (below) magnification. Insets show details of nuclear staining. Bar: 100 mm.
steatosis, inflammation, hepatocellular injury, fibrosis, and
miscellaneous features was performed.
Western Blot Analyses
Cytoplasmic and nuclear protein extraction from nontumorous
liver parenchyma and HCC tissue preparations and
immunoblotting analyses were performed as previously described [23,25].
Histone fraction was enriched using an acid extraction protocol.
Briefly, The snap-frozen tissues were suspended and homogenized
in 200 ml of H-lysis solution (0.2 M sucrose, 3 mM CaCl2, 1 mM
Tris-HCl pH8.0, 0.5 NP40, protease inhibitor cocktail), incubated
on ice for 8 min; centrifuged at 1.3006g, 4uC, for 5 min to
separate supernatant from nuclei fraction (P1). P1 was washed
once with H-wash solution (300 mM NaCl, 5 mg MgCl2, 5 mM
DTT, 0.5% NP40)and lysed for 30 min in 100 ml H-extract
solution (0.5 mM HCl, 10% glycerol, protease inhibitor cocktail),
followed by centrifugation at 13.0006g 4uC, for 5 min. Finally,
TCA precipitation was performed. Antibodies against histone H3
(Activ Motif) were use to normalize protein levels.
Immunostainings were performed by iVIEW DAB Detection
Kit for Ventana BenchMark XT automated slide stainer on
human biopsies . Primary antibodies for MacroH2A1.1 and
MacroH2A1.2 were generated at the European Molecular Biology
Laboratory (EMBL) and were a courtesy of Prof. Andreas
Ladurner (Ludwig Maximilian University, LMU, Munich,
Germany). Positivity for Ki-67 (Vector VP-K451, DBA ITALIA
S.R.L., Milan, Italy ) was also examined. All primary antibodies
were diluted 1:100. Positive and negative controls were run
concurrently. Immunopositive evaluations were performed in
blind by three expert pathologists (FR, FC and NS) and
percentage of positive nuclei (tumor cells in HCC and hepatocytes
in steatosis) was calculated in ten random high power fields at a
magnification of 400x.
Results are expressed as means 6 S.E. Comparisons were made
by using Students t test. Differences were considered as significant
when P,0.05, P,0.01 or P,0.001, as indicated in the Figures
and Figure Legends.
MacroH2A1.1 and macroH2A1.2 Expression in the Liver
of High Fat Diet fed/DEN Mice
A potent bona fide dietary mouse model of high fat-induced HCC
developed recently was reproduced in this study . Male mice
maintained on HF gained more weight than mice on a normal diet
(ND), developed glucose intolerance and their relative liver weight
and triglycerides were increased (data not shown) . This was
accompanied by increased hepatic steatosis with a mean of
NAFLD score of 1 versus 5, respectively (Fig. 1A). Mice fed with
ND and treated with DEN injection at a low dose of 25 mg/kg did
not display steatosis, they were indistinguishable from animal fed a
ND (data not shown) and therefore they were not retained for further
analyses. At sacrifice, mice injected with DEN and kept on HF
exhibited HCC nodules, as well as augmented levels of
inflamFigure 5. Histograms show statistical results for the evaluation of immunopositivity for macroH2A1.1, macroH2A1.2 and Ki-67 in
samples of steatosis and HCC. In the latter both area with tumor and area of steatosis close to HCC (St/HCC) were examined. Significant
differences (p,0.005) in the percentage of positive nuclei were found for both MacroH2A1.1 and MacroH2A1.2 between St/HCC and steatosis (u),
HCC and steatosis (*) and HCC and St/HCC (). Significant differences (p,0.005) were also present for Ki-67 between HCC and steatosis () and HCC and
matory cytokines IL-6, TNFa and IL-1b mRNAs, while mice
under ND did not (data not shown) .
To determine whether protein expression levels of
macroH2A1.1 and macroH2A1.2 were altered in the context of
steatosis or HCC, a histone extraction protocol was applied to the
livers of mice fed a ND, fed with HF or injected with DEN and fed
with HF (HF/DEN), followed by immunoblotting analysis.
MacroH2A1.1 protein was found weakly expressed in the liver
of ND or HF mice, while macroH2A1.2 expression was
significantly increased in HF-fed mice compared to ND-fed mice
(Fig. 1B). Both macroH2A1.1 and macroH2A1.2 expression levels
were highly enhanced in the HCC tissue of HF/DEN treated
animals (Fig. 1B). Thus, in the HF/DEN dietary model of steatosis
and HCC, both macroH2A1 isoforms were associated with
cancer, whereas macroH2A1.2 is specifically increased in the
presence of fat.
MacroH2A1.1 and macroH2A1.2 Expression in the Liver
of Liver-specific PTEN KO Mice
Hepatocyte-specific PTEN deficiency results in steatosis and
HCC in mice, at 1016 and 7478 weeks of age, respectively .
Expression of adipogenic and lipogenic genes, such as PPARc, is
increased in the liver of hepatocyte-specific PTEN KO mice .
The transition from steatosis to HCC is accompanied in these
animals by NASH and the appearance of liver adenomas at 4044
weeks of age . Paradoxically, PTEN being a negative regulator
of insulin signalling , liver-specific PTEN KO mice displayed
hepatic insulin hypersensitivity and increased systemic glucose
tolerance . We used this well established genetic model to
study the expression of macroH2A1 isoforms in hepatic steatosis
and HCC. PTENflox/flox mice were crossed to AlbCre transgenic
mice, in which expression of Cre is controlled by the promoter of
the hepatocyte-specific gene albumin. Control PTENflox/flox mice
and PTEN KO obtained by AlbCre-mediated deletion of both
PTEN alleles were retained for experiments. Animals were
sacrificed at 16 and 52 weeks of age for histological and
biochemical analyses. As expected the liver of 16 weeks old
PTEN KO mice showed extensive fat accumulation compared to
16 weeks old PTENflox/flox littermates (mean NAFLD score 4 versus
1, respectively), (Figure 2A). We assessed if the protein expression
levels of macroH2A1.1 and macroH2A1.2 were altered in the
context of steatosis or HCC in liver specific PTEN KO mice.
Immunoblotting analysis on histone extract revealed that
macroH2A1.1 levels were low both in the liver of PTENflox/flox
mice and of 16 weeks old PTEN KO, while macroH2A1.2
expression was greatly enhanced in the liver of 16 weeks old PTEN
KO compared to age-matched PTENflox/flox mice (Fig. 2B). Both
macroH2A1.1 and macroH2A1.2 protein expression levels were
massively increased in the HCC tissue of 52 weeks old PTEN KO
mice (Fig. 2B). Similarly to the HF/DEN model, in the PTEN KO
model of steatosis and HCC both macroH2A1 isoforms associate
with cancer, whereas macroH2A1.2 is specifically upregulated in
the fatty liver.
MacroH2A1.1 and macroH2A1.2 Expression in the Liver
of Steatotic/HCC Patients
Experiments were performed both in human samples with
steatosis and samples with HCC (10 cases per each condition,
Table 1). In the latter, areas with tumor and areas of steatosis close
to HCC (St/HCC) were examined (Fig. 3A, Fig. 3B). Trichrome
stain showed absence of fibrosis in all examined specimens. In
particular, collagen was present only in portal space and, in limited
amount, in perisinusoidal (Disse) spaces of lobule in both steatosis
and St/HCC samples, as well as in the capsule that delimits HCC
(Fig. 3B). Figure 4 shows representative immunostainings. Both
macroH2A1.1 and macroH2A1.2 showed significant differences
(p,0.005) in the percentage of positive nuclei between St/HCC
and steatosis, HCC and steatosis and HCC and St/HCC (100% of
tumor cells positive for either macroH2A1.1 or macroH2A1.2),
when compared to steatosis (,2% of hepatocytes positive for
macroH2A1.1 and macroH2A1.2) (Fig 4, Fig. 5). The St/HCC
areas were highly immunopositive for macroH2A1.1 and
macroH2A1.2, displaying 83% versus 88% of positive nuclei,
respectively (p,0.05) (Fig 4, Fig. 5). Moreover, significant
differences were also present for Ki-67 between HCC and steatosis
and HCC and St/HCC (Fig. 4, Fig. 5). Finally, a number of nuclei
in elements resembling to endothelial (sinusoidal) and
perisinusoidal cells were also found positive both in steatosis and St/HCC
areas (Fig. 4).
In this study we report that the histone variant macroH2A1 and
its two splicing isoforms are strong markers of NAFLD-associated
HCC, pointing to the importance of an epigenetic component in
pathogenesis. One of the most striking epigenetic alterations that
occur at the level of the chromatin is the exchange of the canonical
H2A histone for histone variant macroH2A1, described nearly 20
years ago . MacroH2A1 can play either a positive or negative
role in transcriptional regulation in a context-dependent manner,
and it can control cell cycle and proliferation . The two exon
splicing variants of macroH2A1, macroH2A1.1 and
macroH2A1.2, differ by just 3 aminoacids and differentially bind
NAD metabolites . As referred to earlier, KO mice for both
macroH2A1 isoforms display insulin resistance, hepatic steatosis
and an altered expression of hepatic genes involved in lipid
metabolism (lipoprotein lipase, CD36 and others) [16,17], and
alterations in the expression of macroH2A1.1 and macroH2A1.2
isoforms are associated to the occurrence/survival and/or the
pathogenesis of various human cancers (lung, colon, melanoma)
[19,20,22]. Our data show that both macroH2A1.1 and
macroH2A1.2 protein expression levels are impressively increased
in tumour tissue of human subjects presenting with HCC on a
steatotic background without cirrhosis or fibrosis. The 10 patients
studied with steatosis alone or with HCC show homogeneous and
strongly consistent results. Immunohistochemistry analyses with
specific antibodies showed that 100% of HCC nuclei were positive
for macroH2A1 isoforms compared to surrounding liver
parenchyma or to the liver of steatotic subjects without HCC. This was
observed also in the liver of HCC mice models, either dietary/
carcinogenic (HF/DEN) or genetic (PTEN KO), where HCC
develops on the basis of a pre-existent NAFLD [8,27].
Independently of the causes underlying steatosis, an increase in
macroH2A1.1 and macroH2A1.2 is strongly associated to HCC
development in these experimental models. mRNA levels for
macroH2A1.1 and macroH2A1.2 in the animal models and in
liver biopsies from patients were variable and did not reflect the
differences observed in the protein levels found in steatosis and
HCC (data not shown). This is consistent with a previous study,
indicating that, differently from most tissues analyzed to date, in
the liver the mRNA splicing that generate the two isoforms of
macroH2A1 is not mirrored by changes in the protein levels .
We also found an interesting discrepancy between the NAFLD of
the mouse models and that of the patients studied. In mice
macroH2A1.2 expression is significantly increased in steatosis,
whereas macroH2A1.1 is not; in human liver, mild content of fat
alone was not associated to an increase of the isoforms (,2% of
positive nuclei for either isoform). However, in the steatotic areas
of the liver proximal to the HCC tissue, high immunopositivity for
both macroH2A1.1 and macroH2A1.2 was present, with a slightly
greater number of positive hepatocytes for macroH2A1.2 (88%)
versus macroH2A1.1 (83%). This would be consistent with an
involvement of macroH2A1.1 and/or macroH2A1.2 in the
pathogenic progression of steatotic liver to malignant HCC in
One limitation of the mouse studies is that the antibodies used
for detection of the macroH2A1 isoforms cannot be used in
immunohistochemistry in this species. Consequently,
immunoblotting data alone cannot distinguish between variations in
macroH2A1.2 expression in the total liver histone extracts, if these
are dependent from increases in hepatocyte expression or from
other cell types, as non-hepatocyte cell types are stained in human
liver immunohistochemistry (Fig. 4). Differences between HCC
mouse models and HCC patients are multiple: mice do not
develop HCC spontaneously, hence we manoeuvred to induce the
disease either using an injection of DEN combined with a high fat
diet, or by liver specific ablation of tumour suppressor PTEN, as
previously described [8,27]. There are also conflicting issues about
the role of genes involved in hepatocarcinogenesis (i.e., MET,
NFkB, Stat3, Jnk, Shp2, and b-catenin) modelled in mice, which have
arisen recently . In any case, an increase of macroH2A1.2
observed only during fat accumulation may have metabolic
implications. In this respect, the property of macroH2A1.1 in
binding with very tight affinity NAD-derived metabolites,
differently from macroH2A1.2 , is intriguing. NAD-derived
metabolites such as O-acetyl-ADP ribose (OAADPR) is generated
by the enzymatic reaction catalyzed by SIRT1, a NAD-dependent
protein deacetylase, whose activation is considered protective
against cellular metabolic and oxidative stresses, and against aging
[36,37]. Of note, liver-specific SIRT1 transgenic mice are
protected against metabolic syndrome-associated HCC .
MacroH2A1.1 apparently suppresses growth of lung cancer cells
and adenocarcinoma cells in a manner dependent on its ability to
bind NAD-derived metabolites . The presence of a
metabolitebinding function in a chromatin component opens new potential
connections between gene expression and lipid metabolism in the
liver. Macro domains could also represent a novel tool for studying
NAD metabolites and may be an attractive drug target .
We thank Dr. Wing Kin Syn for critical reading of the manuscript. We are
grateful to Prof. Tak W. Mak (University Health Network, Toronto,
Canada) for providing us with the Ptenflox/flox mice.
Conceived and designed the experiments: FC M. Foti GM M. Federici VP
MV. Performed the experiments: FR AG CP LB MP AM NS. Analyzed
the data: FC M. Foti GM M. Federici VP MV. Contributed reagents/
materials/analysis tools: FC M. Foti NS M. Federici. Wrote the paper: RW
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