Download Epigenetic regulation of methionine adenosyltransferase 1A: A role

HEPATOLOGY, Vol. 57, No. 5, 2013
Epigenetic Regulation of Methionine
Adenosyltransferase 1A: A Role for
MicroRNA-Based Treatment in Liver Cancer?
Yang H, Cho ME, Li TW, Peng H, Ko KS, Mato JM,
et al. MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma. J
Clin Invest. 2013;123:285-298. (Reprinted with
permission.)
Abstract
MicroRNAs (miRNAs) and methionine adenosyltransferase 1A
(MAT1A) are dysregulated in hepatocellular carcinoma (HCC),
and reduced MAT1A expression correlates with worse HCC
prognosis. Expression of miR-664, miR-485-3p, and miR-495,
potential regulatory miRNAs of MAT1A, is increased in HCC.
Knockdown of these miRNAs individually in Hep3B and
HepG2 cells induced MAT1A expression, reduced growth, and
increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of
this trio of miRNAs promoted tumorigenesis and metastasis in
mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and
miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A
induction significantly reduced the antitumorigenic effect of
miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear
level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite occurred
with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p, and miR-495 contributes to
lower MAT1A expression in HCC, and enhanced tumorigenesis
may provide potential targets for HCC therapy.
Comment
Integrity of the hepatic epigenome is a key component of organ homeostasis. Disruption of this integrity
is believed to be a fundamental driver predisposing
many chronic liver diseases to cancer development.1
Consistently, early changes in DNA methylation patterns are observed during malignant transformation preceding allelic imbalances and leading to cancer
progression.2 In line with this, methionine metabolism
and labile methyl groups play crucial roles in hepatocarcinogenesis and are frequently associated with a significant decrease in levels of S-adenosyl-L-methionine
(SAMe), the principal methyl donor in mammals.3
Methionine adenosyltransferase (MAT) is the major
enzyme catalyzing the synthesis of SAMe, thereby
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regulating many biological processes, including proliferation and differentiation.4 MAT activity in mammals
is associated with two gene products, MAT1A and
MAT2A, which display a tissue-specific expression pattern. MAT1A is associated with high SAMe levels and
is exclusively expressed in the adult liver, whereas
MAT2A results in lower SAMe levels and is the main
source of extrahepatic SAMe synthesis. High .levels of
MAT2A are also detected during differentiation of fetal
livers, where its expression is progressively replaced by
MAT1A upon liver maturation.5 Conversely, a switch in
MAT gene expression is observed during liver regeneration and hepatocarcinogenesis, which mimics the fetal
expression pattern and causes re-expression of MAT2A
in place of the liver-specific MAT1A. This oncofetal
switch in MAT gene expression is partly regulated by
HuR/methyl-HuR and AUF1 during dedifferentiation,
development, as well as proliferation and confers to a
growth advantage for tumor cells.6 Decreased MAT1A
expression and subsequent up-regulation of MAT2A is
also observed in hepatoma cell lines, rodent HCCs,
and chronic human liver diseases such as liver cirrhosis
and HCC.3,7 Consistently, MAT1A-deficient mice with
low SAMe levels are prone to liver injury, steatosis,
and tumorigenesis.8 A recent report further indicates
that liver (cancer) stem cells contribute to this phenotype in the MAT1A-deficient animals.9 Interestingly,
protumorigenic effects of MAT1A inhibition are
reversed by blocking of DNA methyltransferases with
5-azacytidine, indicating that DNA methylation is a
key mechanism of hepatocarcinogenesis induced by
SAMe deficiency.10,11 Overall, these observations suggest that MAT1A and MAT2A are important epigenetic regulators whose expression is context-specific
and is dependent on the stage of differentiation in the
corresponding liver cells. Deregulation of MAT signaling is frequently observed during chronic liver disease
progression and malignant transformation, but the
mechanisms behind this tightly controlled regulation
are largely unknown.5 Thus, a more detailed understanding of the MAT/SAMe metabolism and consecutive deregulation of DNA methylation ultimately
leading to carcinogenesis such as that provided by
Yang and colleagues12 contributes significantly to our
understanding of liver cancer and helps to identify
new diagnostic, prognostic, and therapeutic targets.
MicroRNAs (miRNAs) are small, noncoding RNAs
that posttranscriptionally regulate gene expression as a
part of the RNA interference machinery. miRNAs
were first discovered in 1993 in Caenorhabditis elegans.
Since then, miRNA expression has been linked to virtually all known cellular processes, including
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proliferation, differentiation, and apoptosis.13 More
recent studies have demonstrated that miRNAs can act
as disease modifiers and that aberrant regulation of
several miRNAs contributes considerably to cancer initiation, propagation, and progression. Almost every
type of human cancer has been associated with a specific pattern of deregulated miRNA activity, thereby
promoting these molecules to attractive targets for
diagnostic and therapeutic interventions. miRNAs have
also been associated with HCC development and progression by targeting a large number of critical oncogenic features (e.g., differentiation and metastasis) as
well as key molecules involved in hepatocarcinogenesis.14 In liver cancer development, as well as that of
other cancers, two functional subclasses of miRNAs
have been discovered with either tumor-suppressive or
oncogenic activity.15 With the advent of high-throughput technologies, current miRNA profiles are able to
precisely dissect etiological subclasses and histological
or clinical phenotypes in liver cancer.16 Additionally, a
diagnostic and/or prognostic relevance could be attributed to several miRNAs. Although genomic analyses
indicate that almost half of the known miRNAs are
located on cancer-associated regions, the exact regulation of miRNAs during carcinogenesis still remains
elusive.15 However, it seems abundantly clear that
miRNAs not only contribute to epigenetic regulation
during tumor development, but are also tightly regulated by epigenetic alterations such as DNA
methylation.17
The interaction of different epigenetic mechanisms
such as convergence of MATA1 and miRNAs for hepatocarcinogenesis is demonstrated elegantly in the
study by Yang and colleagues.12 As a result of their
investigations, the authors identified three novel miRNAs (miR-664, miR-485-3p, and miR-495) that negatively
regulate
MAT1A
expression
during
hepatocarcinogenesis (Fig. 1). The results of the study
shed further light on how decreased MAT1A levels
contribute to liver cancer development and have several mechanistic, technical, and clinical implications.
The key findings are: (1) a tight interaction of different epigenetic layers is an important driver for the
development and progression of liver cancer; (2) in
silico prediction of molecular targets coupled with experimental validation is a powerful approach to predict new targets in HCC; and (3) miRNA-based
therapies can be effective therapeutic approaches for
HCC.
The basis of the current study is an in silico prediction of the potential regulatory miRNAs of MAT1A, a
commonly used approach. To increase specificity and
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narrow down this query to miRNAs with a high probability of binding to the 30 untranslated region (UTR)
of MAT1A, the authors combined the results from
three different prediction algorithms (TargetScan,
miRSVR, and miRDB), subsequently focusing only on
those miRNAs with a high score and no previous association with hepatocarcinogenesis. Interestingly,
although several targets with known association to
HCCs could be identified, the overall number of identified miRNAs is surprisingly low. Furthermore, only
miR-664 was identified by all three algorithms. This
demonstrates the dilemma of target prediction software—namely, sensitivity and specificity.18 Should the
selection of miRNAs be based on a single algorithm or
a combination of several algorithms? Are the remaining identified miRNAs just noise in the system, or are
we missing essential information? In this context, the
importance of thorough experimental validation in
authentic tumors, as performed in the current study, is
of utmost importance. Consistently, specific binding of
all miRNAs to MAT1A 30 -UTR could be demonstrated, and small interfering RNA–mediated knockdown of all three miRNAs had an additive effect on
MAT1A expression in hepatoma cell lines, which highlights another important aspect of miRNA biology—
namely, redundancy.19 Many of the known miRNAs
are believed to regulate multiple target genes. Similarly,
miRNA-based gene regulation is supposed to overlap
with multiple miRNAs contributing to gene expression
of one target gene. Therefore, the effects of a single
miRNA might only lead to slight changes in the gene
expression of its targets. In this regard, the current
study sets a nice example on the additive effect of multiple miRNAs for the regulation of one gene (i.e.,
MAT1A). This is something to consider in a miRNAbased therapeutic setting.
The authors further apply elegant and extensive
knock-out and knock-in experiments in vitro and with
different transplantation models in vivo to confirm a
functional effect on proliferation, apoptosis, and invasion for each miRNA. Although miR-495 had the
most dramatic effects on tumorigenicity, the additive
effect for combinatory targeting of all miRNAs could
be reproduced. Importantly, the authors were able to
prove that the observed effects of the miRNAs are
mediated by modulating MAT1A expression. In the
absence of the 30 -UTR of MAT1A, the effect of the
miRNAs was blunted, thereby directly validating the
used approach and touching on another important
issue: the need for confirmation. The current study
demonstrates the necessity of extensive validations for
miRNA research (both in vitro and in vivo) to obtain
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Fig. 1. MAT1A as a molecular target in hepatocarcinogenesis. The study by Yang and colleagues12 indicates that three regulatory miRNAs
(miR-664, miR-485-3p, and miR-495) are involved in the regulation of MAT1A. (A) High MAT1A expression is detected in the liver under physiological conditions and is associated with high SAMe expression and low levels of the three identified regulatory miRNAs. Disruption of the
MAT1A/SAMe pathway is frequently observed during hepatocarcinogenesis and results in hypomethylation, low levels of H3K27me and Let7, and
induction of Lin28B. (B) Conversely, inhibition of the regulatory miRNAs or direct induction of the MAT1A-SAMe axis leads to tumor regression
and reversal of the molecular alterations, indicating that miRNA-based targeting of this pathway might be a promising therapeutic approach for
hepatocellular carcinoma.
robust data.20 Finally, a mechanistic link involving
DNA methylation, histone modifications, and other
miRNAs (e.g., Let7) could be established, thereby closing the circle of epigenetic regulation. Consistently,
tumors with low miRNA, miR-664, miR-485-3p, and
miR-495 activity showed higher DNA methylation,
increased repressive H3K27me3 levels, lower Let7
expression (via promoter methylation of Lin28B), and
vice versa (Fig. 1). The presented data are convincing;
however, the exact signaling pathways affected by the
loss of MAT1A as well as the corresponding molecular
networks are still largely unknown. It further remains
to be demonstrated if and how this epigenetic interplay contributes to the observed genomic instability
and what role the oncofetal MAT2A as well as other
key characteristics of MAT1A (e.g., sumoylation) play
in this context.21
From a technical point of view, the current study
nicely recapitulates all required steps for effective discovery of regulatory miRNAs. This study also clearly
shows how extensive and time-consuming the study of
miRNAs in cancer research can and should be. During
the last 10 years, studies focusing on miRNAs have
increased almost exponentially.15 As tempting as a sole
computational screen for miRNAs appears, this study
demonstrates that no shortcut exists. An unanswered
but critical question not addressed in the present study
relates to the systemic delivery of miRNA-based therapies for authentic tumors. Although results from recent
studies indicate that systemic administration of antimiRs and miRNA mimics can be performed safely,
more effort is needed before a broad clinical translation is plausible.15 The coming years will determine
whether miRNA-based therapies in liver cancer can
live up to their expectations.
In conclusion, the study by Yang and colleagues12
underlines the critical role of MAT1A and its
miRNA-based epigenetic regulation for hepatocarcinogenesis. This elegant work advances significantly
our current understanding of the pathogenesis of liver
cancer via epigenetic feedback regulation. How and
to what extent the epigenetic interplay of MAT1A,
histone modifications, and miRNAs can be used in a
clinical setting with the plethora of heterogeneous etiological and patient-specific factors, and what role the
cell of origin (e.g., stem cells) during malignant
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transformation plays in this setting remains to be
elucidated.
JENS U. MARQUARDT, M.D.
PETER R. GALLE, M.D.
Department of Internal Medicine I
University Medical Center
Johannes Gutenberg University Mainz
Mainz, Germany
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C 2013 by the American Association for the Study of Liver Diseases.
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DOI 10.1002/hep.26375