Another Myc in the Wall: MicroRNA-101 Controls Important Functions in Liver Cancer Formation
2013-10-12

The hepatocellular carcinoma (HCC) field has witnessed major progress in recent years. However, since the introduction of sorafenib into the treatment of advanced disease, no additional compound has been approved for first- or second-line treatment. These failed trials make it increasingly clear that we need to understand the pathophysiology of HCC in the utmost detail to further the development of new drugs and to, perhaps more significant, assign the right patients to the right trials. In this issue of Hepatology, Wang et al.1 give us insight into how complex the mechanisms that control liver cancer formation really are. In their work, they construct a model in which the dysregulation of a microRNA (miRNA; mir-101) changes the expression of a set of important genes that all influence the malignant potential of HCC cells. Moreover, the expression of this miRNA is under the control of several proteins that regulate each other in feedback loops, thus giving small changes in expression levels enormous leverage. Analysis of dys-regulated miRNA expression in cancer cells is increasingly used for classification of tumors and might also have prognostic value in certain dieseases. In HCC, the expression patterns of miRNAs have been used to differentiate tumor tissue from normal liver, subclassify different types of HCC, and make predictions on recurrence after surgery.2 Functionally, miRNAs can have tumor-suppressive or oncogenic functions, depending on the target mRNAs that they regulate. miRNAs are transcribed as long pre-miRNA transcripts that, after processing, become mature miRNAs, which, through binding to partially complementary messenger RNAs, can regulate their stability and translational efficiency. miRNA genes are frequently found in small chromosomal alterations, but can also be dys-regulated by germ-line mutations or epigenetic modulation of their genomic loci. Earlier studies have shown that miRNA-101 is significantly down-regulated in several human HCC cell lines as well as in a set of HCCs, when compared to adjacent normal tissue. These reports also demonstrated that reexpression of miRNA-101 decreases the tumoregenicity of HCC cells in vitro and after xenotransplantation in vivo.3, 4 Wang et al. started their analysis by purifying proteins that associate with enhancer of zeste 2 (EZH2) protein in HCC cells. EZH2 is widely expressed in a large set of human cancers and its overexpression is often correlated with advanced stage and poor prognosis.5 Biochemically, EZH2 acts as the catalytic core protein in polycomb repressor complex 2 (PRC2), which catalyzes the trimethylation of histone 3 lysine 27. EZH2 mediates gene silencing of genes that are involved in fundamental cellular processes, such as cell-fate decision, cell-cycle regulation, senescence, cell differentiation, and cancer.6 Importantly, EZH2 is itself regulated by miRNA-101, and loss of miRNA-101 has been shown to result in overexpression of EZH2.7 Thus, miRNA-101 acts as a tumor suppressor by regulating the expression of EZH2. Using EZH2 in immunoprecipitation experiments, Wang et al. now show that another important regulator of liver cancer formation, the c-myc protein, can bind to EZH2, suggesting that EZH2 and c-myc cooperate in repressing miRNA-101 expression.8 Myc has long been established as an oncogene of central importance for the development of HCCs. Work by Felsher et al. showed that HCC generated in mice become addicted to this oncogene, because ablation of myc in preestablished tumors lead to complete regression.9 A central function of myc, which is critical for its ability to induce tumor formation, is the regulation of miRNAs. The c-myc oncogene is repressing and inducing the expression of several miRNAs. For example, through activation of the mir 17 cluster, c-myc can suppress apoptosis and DNA damage by regulating Bim, E2F, and protein kinase B activity. However, more often, c-myc acts as a repressor of miRNA expression, and widespread repression is often observed in human cancers.10 Based on the finding that EZH2 and c-myc interact in HCC cells, the investigators mapped the expression patterns of miRNAs in cells in which either EZH2 or c-myc had been silenced. They found that several miRNAs (including miRNA-101) seemed to be regulated by both proteins and decided to focus on miRNA-101 for its prominent role in HCC biology. In another twist of complexity, it is known that miRNA-101 is not just repressing the expresion of EZH2, but can itself also be repressed by EZH2, as has been shown in several types of cancer.11 In support of a similar mechanism in HCC cells, the investigators showed that 3-deazaneplanocin A, a pharmacological inhibitor of EZH2, increased the expression of miRNA-101. Mechanistically, the combined regulation of miRNA-101 by EZH2 and c-myc was explained by chromatin Ips, which showed that c-myc accumulates on the miRNA-101 promoter and supports the binding of PRC2. Reduced miRNA-101 expression levels resulted in the induction of several malignant features in HCC cells, including proliferation, migration, and increased angiogenesis, whereas overexpression of the miRNA reduced the malignant potential of these cells. The investigators were able to show that a number of these cellular effects were the result of the miRNA-101 controlled expression of a set of target genes, including Stathmin, c-jun, and C-X-C chemokine receptor type 7. Therefore, Wang et al. demonstrated that miRNA-101 and EZH2 form a negative feedback loop in which miRNA-101 suppresses the expression of EZH2. Importantly, this effect was longstanding, suggesting that perhaps the oncogenic function of EZH2 in HCC cells could be overcome through an miRNA-based therapeutic. Remarkably, the investigators were able to extend their cell-based findings to primary HCC tissues and showed that approximately 30% of the tumors they examined had strongly reduced levels of miRNA-101, but, at the same time, showed increased expression levels of the identifed target genes. This correlation was further analyzed in a subgroup of patients that showed the highest levels of c-myc and EZH2 expression and very low levels of miRNA-101. This group of double-positive patients also had the shortest overall survival, whereas patients expressing low levels of both proteins had the best survival. However, these results have to be taken with a grain of salt because both c-myc and EZH2 are involved in a myriad of different tumor-promoting functions, which make it unlikely that the regulation of miRNA-101 is the singular cause. Can we use the information provided in this article for the improvement of liver cancer treatment? Given the small number of patients included in this analysis, it would certainly be important to extend this study and also to elucidate the ability of miRNA-101 expression to predict response to systemic or ablative treatments. The study also lends more support to epigenetic treatment approaches. Finally, the study underscores the enormous complexity of liver cancer as a tumor disease, but also points of central importance such as the expression of miRNAs, which might help to define new high-impact targets. Nisar P. Malek, M.D. Department of Internal Medicine I, Eberhard Karls University, Tübingen, Germany