Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3

Highlights

• G9a was frequently upregulated in human HCC and associated with HCC aggressiveness.

• G9a promoted HCC growth and metastasis both in vitro and in vivo.

• Upregulation of G9a in HCC was attributed to gene amplification and loss of miR-1.

• G9a epigenetically silenced the expression of tumor suppressor gene RARRES3 in HCC.

• Targeting G9a by small molecular inhibitors suppressed HCC growth.

Background & Aims

Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an important epigenetics regulator however, its role in liver carcinogenesis remains to be investigated.

Methods

Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing system. Tumor-promoting functions of G9a was studied in both HCC cell lines and nude mice model. The downstream targets of G9a were identified by RNA-Seq and confirmed by ChIP assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo.

Results

We identified G9a as a frequently upregulated histone methyltransferase in human HCCs. Upregulation of G9a was significantly associated with HCC progression and aggressive clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and in vivo models. Mechanistically, we showed that the frequent upregulation of G9a in human HCCs was attributed to gene copy number gain at chromosome 6p21. In addition, we identified miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression on G9a and contributed to its upregulation in human HCC. Utilizing RNA sequencing, we identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of RARRES3 contributed to the tumor-promoting function of G9a.

Conclusion

This study shows a frequent deregulation of miR-1/G9a/RARRES3 axis in liver carcinogenesis, highlighting the pathological significance of G9a and its therapeutic potential in HCC treatment.

Lay summary: In this study, we identified G9a histone methyltransferase was frequently upregulated in human HCC and contributes to epigenetic silencing of tumor suppressor gene RARRES3 in liver cancer. Targeting G9a may be a novel approach for HCC treatment.

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.