Research ArticleHistone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3
Graphical abstract
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide.1 In 2012, there were 782,000 new HCC cases diagnosed, and 746,000 patients died of HCC. HCC accounts for 9.1% of all cancer deaths, making it the second largest cause of cancer mortality worldwide.[1], [2] The patient survival rate of HCC is extremely poor. This is mainly due to the asymptomatic progression of HCC at the early stage and high metastasis potential at the late stage. Conventional chemotherapies have no significant impact on the overall survival, and only a small fraction of HCC patients are eligible for curative surgical resection.[1], [2] Currently, sorafenib is the only molecularly targeted drug approved by Food and Drug Administration (FDA) for the treatment of advanced HCC. However, the survival advantage of sorafenib treatment is only modest.3 Therefore, a better understanding of the molecular mechanisms underlying liver carcinogenesis is crucial for the development of novel diagnostic methods, as well as identifying new therapeutic targets, which may help to improve the patients’ survival rates.
Traditionally, cancer was considered as a disease driven by genetic abnormalities. Currently, it is also commonly believed that deregulation of epigenetic components may play an equally important role in human carcinogenesis.4 The development of HCC follows a multistep process that is often initiated from chronic hepatitis B (HBV) or C (HCV) viral infection. The chronic inflammatory microenvironment promotes liver cirrhosis, which may be evolved in the pre-malignant dysplastic nodule. These background liver diseases can consequently induce malignant transformation of early HCC and eventually develop into a metastatic outgrowth into advanced HCC. This multistep process involves a gradual accumulation of genetic and epigenetic alternations leading to hyperactivation of proto-oncogenes and inactivation of critical tumor suppressor genes to fuel cancer progression.5 Common epigenetic changes in human cancers include aberrant DNA methylation, altered post-translational histone modifications, disordered chromatin remodeling, and deregulated non-coding RNA expression. Unlike genetic alterations, these epigenetic changes are often reversible, which makes epigenetic therapy an attractive direction for anti-cancer drug development.6 The profound involvement of aberrant DNA methylation in liver carcinogenesis has already been firmly established in the literature, while the implications of histone modifications in HCC are relatively less well characterized. Nevertheless, histone modifications appear to play important roles in organizing the nuclear architecture, with consequent effects on the regulation of gene transcription.[7], [8] Recently, deregulation of histone modifications has emerged as an important mechanism in cancer development. For instance, histone lysine methyltransferases, EZH2, SUV39H1, and SETDB1 are frequently deregulated in human HCC and are essential for HCC initiation, progression and metastasis.[9], [10], [11] In the present study, we showed that histone lysine methyltransferase G9a (also known as euchromatic histone-lysine N-methyltransferase 2, EHMT2) was significantly upregulated in HCC, as revealed by whole transcriptome sequencing and qRT-PCR analyses. G9a is a SET domain-containing protein and specifically catalyzes histone 3 lysine 9 di-methylation (H3K9me2), which is a prominent epigenetic marker for transcriptional repression in the euchromatin region. G9a-mediated transcriptional repression is essential for cell differentiation and embryogenesis.[12], [13], [14] G9a-deficient mice are embryonically lethal due to severe growth retardation.14 Aberrant installation of H3K9me2 was found to be involved in the pathogenesis of different types of human cancers.[15], [16] However, little is known about the clinical and pathological roles of G9a in human HCC. Given the importance of G9a in mediating histone modification and the fact that it is highly upregulated in human HCC, we hypothesize that deregulation of G9a may contribute to aberrant epigenetic silencing in HCC. Whether G9a functions as a tumor-promoting gene in HCC or not and the mechanisms by which G9a deregulation promotes hepatocarcinogenesis remain to be clarified. Therefore, a comprehensive investigation into the functional and pathological roles of G9a in human HCC is warranted.
Section snippets
Clinical specimens
The HCC patients involved in this study had surgical resection at Queen Mary Hospital in Hong Kong between 1991 and 2007. The use of clinical specimens has been approval by the Institutional Review Board of the University of Hong Kong and the Hong Kong Hospital Authority.
Establishment of G9a and RARRES3 stable knockdown cell lines
The G9a and RARRES3 stable knockdown cell lines were established by lentivral based stable shRNA overexpression. Non-target control shRNA (shNTC) obtained from Sigma-Aldrich was used as negative control.
Establishment of G9a knockout cell lines
The plasmid pSpCas9
Frequent upregulation of G9a in human HCCs
Our previous expression profiling analyses of human HCCs and their corresponding non-tumorous (NT) livers with TaqMan low density array and whole RNA-Seq revealed that upregulation of epigenetic regulators was a common event.[9], [11] We found that G9a was consistently identified as one of the most significantly upregulated epigenetic regulators. In the RNA-Seq discovery sample set, the mRNA expression of G9a was increased in HCCs by 4.5-fold as compared with their corresponding NT livers (
Discussion
Cancer cells experience intensive epigenetic re-programing. Deregulation of epigenetic regulators that are involved in controlling DNA methylation, histone modifications, and chromatin remodeling may have immense impacts on cellular transformation and evolution of cancer cells. In our previous RNA-Seq profiling study of 591 epigenetic regulators in human HCC, we found that deregulation of epigenetic regulators is a striking feature of HCC.11 In this study, we demonstrated that G9a, a histone
Financial support
The study was supported by National Natural Science Foundation of China General Program (81572446), and Hong Kong Research Grants Council Theme-based Research Scheme (T12-704/16R) and General Research Funds (17115815 and HKU780612M). I.O.L. Ng is Loke Yew Professor in Pathology.
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Authors’ contribution
C.M.W. and L.W. designed the experiments. L.W., D.C., F.T., D.L., C.C., S.A., J. L., and C.C.W. performed the experiments. C.M.W., and L.W. analyzed the data, C.M.W., L.W. and I.N. wrote the manuscript. C.M.W. and I.N. supervised the study.
Acknowledgements
We thank the Core Facility and Center for Genomic sciences of LKS Faculty of Medicine for their technical support. We also thank the Laboratory Animal Unit of the University of Hong Kong for animal holding.
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