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Abstract

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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.

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.)

Comment

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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 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 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.

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Figure 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.

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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 narrow down this query to miRNAs with a high probability of binding to the 3′ 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 3′–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 miRNA–based 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 3′–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 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 anti–miRs 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 transformation plays in this setting remains to be elucidated.

References

  1. Top of page
  2. Abstract
  3. Comment
  4. References