Accepted Manuscript

Histone methyltransferase G9a promotes liver cancer development by epige- netic silencing of tumor suppressor RARRES3

Lai Wei, David Kung-Chun Chiu, Felice Ho-Ching Tsang, Dicky Cheuk-Ting Law, Carol Lai-Hung Cheng, Sandy Leung-Kuen Au, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Chun-Ming Wong

PII: S0168-8278(17)32051-2 DOI: http://dx.doi.org/10.1016/j.jhep.2017.05.015 Reference: JHEPAT 6540

To appear in: Journal of Hepatology

Received Date: 25 August 2016 Revised Date: 29 April 2017 Accepted Date: 11 May 2017

Please cite this article as: Wei, L., Chiu, D.K-C., Tsang, F.H-C., Law, D.C-T., Cheng, C.L-H., Au, S.L-K., Lee, J.M-F., Wong, C.C-L., Ng, I.O-L., Wong, C-M., Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3, Journal of Hepatology (2017), doi: http://dx.doi.org/ 10.1016/j.jhep.2017.05.015

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3

Lai Wei 1,2,3, David Kung-Chun Chiu 1,2, Felice Ho-Ching Tsang 1,2,3, Dicky Cheuk-Ting Law 1,2,

Carol Lai- Hung Cheng 1, 2 , Sandy Leung-Kuen Au 1,2, Joyce Man-Fong Lee 1,2, Carmen Chak-

Lui Wong 1,2, Irene Oi-Lin Ng 1,2 *, and Chun-Ming Wong 1,2,3 *

1. State Key Laboratory for Liver Research, the University of Hong Kong, Hong Kong

2. Department of Pathology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong

Kong.

3. The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen,

China

* Correspondence address

Correspondence to: Chun-Ming Wong or Irene Oi-Lin Ng, State Key Laboratory for Liver

Research and Department of Pathology, University Pathology Building, The University of Hong

Kong, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail: Irene Oi-Lin Ng ([email protected]) or

Chun-Ming Wong ([email protected])

Keywords: Epigenetics; histone modifications, G9a; gene amplification, miR-1, RARRES3

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.

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.

Author 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.

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. 1 Abstract

2 Background and Aims: Hepatocellular carcinoma (HCC) is a major leading cause of cancer

3 mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an

4 important epigenetics regulator. However, its roles in liver carcinogenesis remain to be

5 investigated.

6 Methods: Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and

7 knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing

8 system. Tumor promoting functions of G9a was studied by both in HCC cell lines and nude mice

9 model. The down-stream targets of G9a were identified by RNA-Seq and confirmed by ChIP

10 assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo.

11 Results: We identified G9a as a frequently up-regulated histone methyltransferase in human

12 HCCs. Up-regulation of G9a was significantly associated with HCC progression and aggressive

13 clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi

14 knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished

15 H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and

16 in vivo models. Mechanistically, we showed that the frequent up-regulation of G9a in human

17 HCCs was attributed to gene copy number gain at 6p21. In addition, we identified

18 miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression

19 on G9a and contributed to its up-regulation in human HCC. Utilizing RNA-sequencing, we

20 identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of

21 RARRES3 contributed to the tumor-promoting function of G9a. 22 Conclusion: Our findings discovered a frequent deregulation of miR-1/G9a/RARRES3 axis in

23 liver carcinogenesis. Our findings also highlighted the pathological significance of G9a and its

24 therapeutic potential in HCC treatment.

25

26 Word count: 274 words

27

28 29 Lay Summary: In this study, we identified G9a histone methylation was frequently up-regulated

30 in human HCC and contribute to epigenetic silencing of tumor suppressor RARRES3 in

31 liver cancer. Targeting G9a may be a novel approach for HCC treatment.

32

33 Graphical abstract

34

35 Graphical abstract: Deregulation of G9a in human HCC. G9a was frequently up-regulated in

36 human HCC and implicated in HCC tumorigenesis and metastasis. The frequent up-regulation of

37 G9a in human HCC was attributed to gene copy number gain at 6p21 and loss of miR-1. The

38 oncogenic function of G9a was at least partially attributed to the epigenetic silencing of tumor

39 suppressor RARRES3.

40 41 Introduction

42 Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide [1]. In 2012,

43 there were 782,000 new HCC cases diagnosed, and 746,000 patients died of HCC. HCC

44 accounts for 9.1% of all cancer deaths, making it the second largest cause of cancer mortality

45 worldwide [1, 2]. The patient survival rate of HCC is extremely poor. This is mainly due to the

46 asymptomatic progression of HCC at the early stage and high metastasis potential at the late 47 stage. Conventional chemotherapies have no significant impact on the overall survival, and only

48 a small fraction of HCC patients are eligible for curative surgical resection [1, 2]). Currently,

49 Sorafenib is the only molecularly targeted drug approved by Food and Drug Administration

50 (FDA) for the treatment of advanced HCC. However, the survival advantage of Sorafenib

51 treatment is only modest [3]. Therefore, a better understanding of the molecular mechanisms

52 underlying liver carcinogenesis is crucial for the development of novel diagnostic methods as

53 well as identification of new therapeutic targets, which may help to improve the patients’

54 survival rates of this deadly disease.

55 Traditionally, cancer was considered as a disease driven by genetic abnormalities. Currently, it is

56 also commonly believed that deregulation of epigenetic components may play an equally

57 important role in human carcinogenesis [4]. The development of HCC follows a multistep

58 process that often initiates from chronic hepatitis B viral (HBV) or C (HCV) infection. The

59 chronic inflammatory microenvironment promotes liver cirrhosis, which may evolve into the

60 pre-malignant dysplastic nodule. These background liver diseases can consequently induce

61 malignant transformation of early HCC and eventually develops into a metastatic outgrowth of

62 advance HCC. This multistep process involves a gradual accumulation of genetic and epigenetic

63 alternations leading to hyperactivation of proto-oncogenes and inactivation of critical tumor 64 suppressor genes to fuel cancer progression [5]. Common epigenetic changes in human cancers

65 include aberrant DNA methylation, altered post-translational histone modifications, disordered

66 chromatin remodeling, and deregulated non-coding RNA expression. Unlike genetic alterations,

67 these epigenetic changes are often reversible, which makes epigenetic therapy an attractive

68 direction for anti-cancer drug development [6]. The profound involvement of aberrant DNA

69 methylation in liver carcinogenesis has already been firmly established in the literature, while the

70 implications of histone modifications in HCC are relatively less well characterized. Nevertheless,

71 histone modifications appear to play important roles in organizing the nuclear architecture, with

72 consequent effects on the regulation of gene transcription [7, 8]. Recently, deregulation of

73 histone modifications has emerged as an important mechanism in cancer development. For

74 instance, histone lysine methyltransferases, EZH2, SUV39H1, and SETDB1 are frequently

75 deregulated in human HCC and are essential for HCC initiation, progression and metastasis [9-

76 11]. In the present study, we showed that histone lysine methyltransferase G9a (also known as

77 euchromatic histone-lysine N-methyltransferase 2, EHMT2) was significantly up-regulated in

78 HCC, as revealed by whole transcriptome sequencing and qRT-PCR analyses. G9a is a SET

79 domain-containing and specifically catalyzes histone 3 lysine 9 di-methylation

80 (H3K9me2), which is a prominent epigenetic mark for transcriptional repression in euchromatin

81 region. G9a-mediated transcriptional repression is essential for cell differentiation and

82 embryogenesis [12-14]. G9a-deficient mice are embryonically lethal due to severe growth

83 retardation [14]. Aberrant installation of H3K9me2 was found to be involved in the pathogenesis

84 of different types of human cancers [15, 16]. However, little is known about the clinical and

85 pathological roles of G9a in human HCC. Given the importance of G9a in mediating histone

86 modification and the fact that it is highly up-regulated in human HCC, we hypothesize that 87 deregulation of G9a may contribute to aberrant epigenetic silencing in HCC. Whether G9a

88 functions as a tumor-promoting gene in HCC or not and the mechanisms by which G9a

89 deregulation promotes hepatocarcinogenesis remain to be clarified. Therefore, a comprehensive

90 investigation into the functional and pathological roles of G9a in human HCC is warranted.

91

92 Results

93 Frequent up-regulation of G9a in human HCCs

94 Our previous expression profiling analyses of human HCCs and their corresponding non-

95 tumorous (NT) livers with TaqMan low density array and whole transcriptome sequencing

96 (RNA-Seq) revealed that up-regulation of epigenetic regulators was a common event [9, 11]. We

97 found that G9a was consistently one of the most significantly up-regulated epigenetic regulators.

98 In the RNA-Seq discovery sample set, the mRNA expression of G9a was increased in HCCs by

99 4.5 folds as compared with their corresponding NT livers (Figure 1A). We validated this initial

100 observation in an expanded sample cohort consisting of 5 normal livers and 92 pairs of HCCs

101 and NT livers by qRT-PCR. As expected, G9a was found to be significantly up-regulated in

102 HCCs (P < 0.0001, Figure 1B). Overexpression of G9a (> 2 folds) was detected in 68% (63/92)

103 of HCCs. In contrast, only 4% (4/92) of HCC cases exhibited down-regulation (< 0.5 fold)

104 (Figure 1C). Since our HCCs are mainly (~80%) associated with HBV infection, to confirm

105 whether G9a up-regulation is a common event across different etiology groups, we analyzed the

106 G9a expression in TCGA (The Cancer Genome Atlas) RNA-Seq dataset, which has mixed

107 etiologies. Consistent with our findings, significant up-regulation of G9a was also detected in 50

108 paired HCCs, with both HCCs and corresponding NT livers available, in TCGA (P < 0.0001, 109 Figure 1D). 60% (30/50) of HCC cases showed at least 2-fold up-regulation, and yet no cases

110 showed down-regulation of G9a (Figure 1E). These findings suggested that up-regulation of G9a

111 was not limited to HBV-associated HCCs but was a general phenomenon in human HCCs. More

112 strikingly, G9a expression was also significantly up-regulated in multiple cancer types available

113 in TCGA, indicating that deregulation of G9a is indeed a common alteration in human cancers

114 (Supplementary figure 1).

115 Clinicopathological significance of G9a up-regulation in human HCCs

116 We investigated G9a expression in different stages of liver carcinogenesis and found that its

117 expression was increased in a step-wise manner. In our qRT-PCR analysis on the expanded HCC

118 cohort, the median G9a expression level increased from normal liver (NL) to chronic hepatitis

119 (CH) and cirrhotic liver (CL), then to early HCC and finally advanced HCC (P < 0.0001, Figure

120 1F). Next, we investigated the clinical implications of G9a up-regulation in human HCCs by

121 correlating G9a mRNA expression changes with the various clinicopathological features. We

122 found that G9a overexpression was significantly associated with the aggressiveness and

123 metastatic features of HCC, in terms of advanced pTNM stage (P = 0.0195), the presence of

124 venous invasion (P = 0.0042) and tumor microsatellite formation (P = 0.0080), and the absence

125 of tumor encapsulation (P = 0.0145) (Figure 1G). Taken together, these findings suggested that

126 up-regulation of G9a had significant pathological implications and might promote cancer

127 metastasis in human HCC.

128 G9a gene copy number gain contributed to G9a mRNA up-regulation in human HCCs

129 G9a was highly up-regulated in human HCCs, yet the underlying mechanisms remain elusive.

130 Based on the published comparative genomic hybridization (CGH) studies, chromosome 6p21,

131 where the G9a gene is located, was frequently amplified in human HCCs (Supplementary Figure 132 2). Therefore, we sought to investigate whether gene copy number gain directly contributed to

133 the frequent up-regulation of G9a in human HCCs. We performed a qPCR-based gene copy

134 number assay in 32 pairs of HCCs and their NT liver counterparts and found that gain of G9a (≥

135 3 copies) was detected in 60% of HCCs. In contrast, all of the NT livers had only 2 copies of

136 G9a gene (Figure 2A and B). Notably, the mRNA expression of G9a was also positively

137 correlated with G9a gene copy number gain in the HCC tumors (Figure 2C). Similar

138 observations were also made from analysis of the G9a gene copy number and mRNA expression

139 data available in TCGA (Figure 2D and 2E). Together, these findings demonstrated that G9a

140 gene copy gain was a common event in HCCs and has a direct impact on the up-regulation of

141 G9a mRNA expression.

142 Loss of miR-1 contributed to G9a up-regulation in human HCC

143 MicroRNA (miRNA) is an important class of post-transcriptional regulators mediating mRNA

144 expression by binding to the 3’ untranslated region (3’UTR) of its target genes. In order to

145 investigate whether miRNAs are involved in regulating G9a expression, we genetically deleted

146 the 3’UTR of G9a in BEL7402 cells by using CRISPR/Cas9 (clustered regularly interspaced

147 short palindromic repeats) genome editing technology (Figure 3A). Successful deletion of G9a-

148 3’UTR was confirmed by Sanger sequencing. Two of the clones, G9a-∆3’UTR#42 and #52,

149 were chosen for further analysis. Strikingly, the expression of G9a at both mRNA and protein

150 levels were significantly augmented upon deletion of G9a-3’UTR (Figure 3B). These

151 observations imply that miRNA may be involved in the post-transcriptional regulation of G9a

152 expression. To identify the potential miRNA binding sites in G9a-3’UTR, in silico analysis was

153 performed using TargetScan and miRANDA miRNA target prediction algorithms

154 (Supplementary figure 3). MiR-1 and miR-613 were suggested as potential miRNAs that could 155 regulate G9a expression. However, miR-613 was not expressed in human HCC nor NT livers

156 (data not shown); thus we focused on studying the impact of miR-1 on G9a expression. MiR-1

157 was predicted to form a thermokinetically stable duplex with G9a-3’UTR and the miR-1 binding

158 sequence was evolutionarily conserved across different species (Figure 3C). The expression of

159 miR-1 was also found to be frequently down-regulated in human HCCs (P = 0.0002, Wilcoxon

160 signed-rank test, Figure 3D). Importantly, miR-1 expression was negatively correlated with the

161 mRNA expression of G9a (R2 = 0.2581, P < 0.0001, Figure 3E). The direct interaction between

162 miR-1 and G9a-3’UTR was experimentally validated by dual-luciferase reporter assay. The wild

163 type (WT) and mutant (Mut) miR-1 binding sequences of G9a-3’UTR were inserted into the 3’

164 end of a firefly luciferase gene (Figure 3F). We found that overexpression of miR-1 precursor

165 significantly repressed luciferase activity associated with WT G9a-3’UTR but not Mut G9a-

166 3’UTR nor the empty vector control (P < 0.001, Figure 3G), which confirms that miR-1 could

167 target 3’UTR of G9a and negatively regulate its expression. Consistent with this finding, we also

168 found that transient overexpression of miR-1 significantly down-regulated endogenous G9a

169 protein expression in HCC cell lines (Figure 3H). Collectively, we demonstrated that miR-1 is a

170 post-transcriptional regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression

171 imposed on G9a and contributed to its up-regulation in human HCC.

172 G9a is the major histone methyltransferase responsible for H3K9 di-methylation in HCC

173 Consistent with our findings in human HCCs, significant up-regulation of G9a was also found in

174 all HCC cell lines when compared with the immortalized normal liver cell line, THLE-3 (Figure

175 4A). To characterize the molecular functions of G9a in HCC, we established G9a stable

176 knockdown models in BEL7402 and SMMC-7721 cell lines by delivering short hairpin RNA

177 (shRNA) targeting the coding sequence of G9a through a lentiviral system. Two independent 178 shRNA sequences (shG9a#69 and #70) were included to avoid possible off-target effects.

179 Successful knockdown of G9a was verified at both protein and mRNA levels, and both shRNA

180 sequences achieved more than 70% of knockdown efficiency in BEL7402 and SMMC-7721 cell

181 lines (Figure 4B). In addition, we established the G9a genetically knockout HCC cell models in

182 BEL7402 and SMMC-7721 using CRISPR/Cas9 system to target the exon 7 of G9a and

183 eliminate all isoforms (Figure 4C). Two independent single guide RNA (sgRNA) sequences

184 (sg#3 and sg#4) were used to minimize the possible experimental artifacts. The complete

185 knockout of G9a was confirmed by Western blotting (Figure 4D). We found that the H3K9me2

186 level was substantially diminished upon G9a knockout whereas the levels of H3K9me3,

187 H3K4me3 and H3K27me3 were not affected, suggesting that G9a is the major histone

188 methyltransferase responsible for the transcriptional repressive H3K9me2 in HCC genome

189 (Figure 4E).

190 Knockdown and knockout of G9a suppressed the proliferation and migration of HCC cells

191 in vitro

192 Our RNA-Seq data indicated that the expression level of G9a was positively correlated with the

193 proliferation marker Ki67 in human HCC, suggesting that G9a may play a role in HCC cell

194 proliferation (R2 = 0.7192, P < 0.0001, Figure 5A). We then performed cell proliferation assay

195 and colony formation assay to test the effects of G9a on HCC cell growth. The results showed

196 that knockdown or knockout of G9a significantly inhibited the proliferation of both BEL7402

197 and SMMC-7721 cells (Figure 5B, C, D, and E). We also showed that knockout of G9a induced

198 cell apoptosis and autophagy (Supplementary figure 4), which may contribute to the reduced cell

199 proliferation upon G9a depletion in HCC cells. Besides the effect on cell proliferation, our

200 clinical correlation analysis revealed a positive association between G9a up-regulation and 201 aggressive metastatic features in human HCCs (Figure 1G). It is therefore reasonable to

202 speculate that up-regulation of G9a may also be implicated in HCC metastasis. In order to

203 examine the effect of G9a on HCC cell migration, transwell cell migration assay was performed.

204 Knockdown or knockout of G9a remarkably reduced HCC cell migration rate (Figure 5F and G),

205 which echoed with our observations in the clinicopathological analysis in HCC patients and

206 highlighted the important role of G9a on HCC cell motility and metastasis.

207 Knockdown and knockout of G9a inhibited HCC tumorigenicity and lung metastasis in

208 vivo

209 To consolidate the findings obtained from the above in vitro experiments, we sought to further

210 demonstrate the tumor-promoting role of G9a in HCC using in vivo models. Subcutaneous

211 xenograft model was employed to test the effect of G9a knockdown in HCC tumorigenicity. We

212 found that the size and weight of tumors derived from the shG9a group were significantly

213 decreased as compared with the NTC group (Figure 6A and B). The findings were consistent

214 with both BEL7402 (Figure 6A) and SMMC-7721 (Figure 6B) cell lines. To further verify the

215 tumor-promoting function of G9a, we performed orthotopic liver implantation experiment using

216 G9a knockout BEL7402 HCC cells. We found that knockout of G9a in BEL7402 cells

217 significantly decreased the size of HCC tumors formed in the liver microenvironment as

218 compared with the WT cells (Figure 6C). Importantly, knockout of G9a also attenuated the lung

219 metastasis of HCC cells (Figure 6D). The reduced lung metastasis of HCC cells upon G9a

220 knockout was also observed in tail-vein injection model (Supplementary figure 5). These

221 observations were in accordance with what we observed in the in vitro models. Taken together,

222 these further indicate that G9a functions as a tumor-promoting gene and is crucial for HCC cell

223 growth and metastasis. 224 G9a histone methyltransferase inhibitors suppressed HCC cell proliferation

225 Small molecular inhibitors of histone methyltransferases have recently attracted considerable

226 interests for the development of novel targeted cancer therapy. Our present study showed that

227 G9a as a major histone methyltransferase for H3K9me2 was essential for HCC proliferation and

228 metastasis. These findings prompted us to investigate the therapeutic potential of targeting G9a

229 as a novel approach for HCC treatment. UNC0638 [18] and BIX01294 [19] are specific G9a

230 inhibitors which selectively inhibit the histone methyltransferase activity of G9a, thus

231 modulating the global H3K9me2 level and local histone modification signature to regulate gene

232 expressions. In this study, we showed that the UNC0638 and BIX01294 effectively suppressed

233 HCC cell proliferation with GI50 (50% growth inhibition) at 7.325 µM and 5.225 µM,

234 respectively (Figure 6E and F). UNC0638 and BIX01294 treatment at 5µM substantially

235 inhibited colony formation and induced notable morphological change in HCC cells (Figure 6G).

236 The specificity of G9a inhibition was demonstrated by Western blotting which showed that

237 treatment of UNC0638 and BIX01294 at 5 µM significantly depleted H3K9me2 in HCC cells

238 without affecting H3K4me3 and H3K27me3 levels (Figure 6G and Supplementary figure 6).

239 Since UNC0638 and BIX01294 are not stable in vivo, we used another newly identified G9a

240 inhibitor UNC0642 which has good pharmacokinetic (PK) properties to test the effect of G9a on

241 tumor growth in vivo. We showed that UNC0642 could also specifically inhibited H3K9me2 and

242 significantly reduced tumor volume in vivo (Supplementary figure 7). Taken together, our

243 findings suggested that chemically inhibition of G9a histone methyltransferase activity by small

244 molecular inhibitors selectively reduced H3K9me2 level and consequently resulted in

245 suppression of HCC cell proliferation both in vitro and in vivo. Our findings supported the notion

246 that G9a may be a potential therapeutic target for HCC treatment. 247 Suppression of RARRES3 contributed to the tumor-promoting function of G9a

248 We hypothesize that G9a could exert its tumor-promoting function through epigenetically

249 silencing important tumor suppressor genes in human HCC. To identify possible downstream

250 targets of G9a, we performed RNA-Seq to investigate the transcriptomic changes in G9a stable

251 knockdown HCC cells. To this end, we identified 201 genes that were significantly up-regulated

252 (> 2 folds) upon G9a knockdown in BEL7402 and SMMC-7721 cells (Figure 7A). Among them,

253 16 genes were commonly up-regulated in both two cell lines. We identified RARRES3 (Retinoic

254 acid receptor responder protein 3) as one of the potential targets of G9a in human HCC (Figure

255 7A). The RNA-Seq analysis showed that RARRES3 mRNA expression was increased by 4.24

256 folds in SMMC-7721 cells and 2.93 folds in BEL7402 cells upon stable knockdown of G9a

257 (Figure 7B).

258 RARRES3 was first identified as a tumor suppressor in 1998 and shown to be involved in cell

259 growth and differentiation [20]. Reduced expression of RARRES3 has been observed in several

260 cancer types and re-expression of RARRES3 induced apoptosis and resulted in decreased cell

261 proliferation and migration [21-24]. However, the expression level, functional roles, and

262 regulatory mechanisms of RARRES3 in human HCC are unclear. To this end, we examined the

263 RARRES3 mRNA expression in human HCCs. Consistently, RARRES3 was found to be

264 significantly down-regulated in HCC as revealed by our RNA-Seq data (P < 0.0001, Figure 7C)

265 and TCGA RNA-Seq dataset (P = 0.0041, Supplementary figure 8A). 48% (24/50) of TCGA

266 HCC cases showed at least 2-fold down-regulation (Supplementary figure 8B). In addition, a

267 negative correlation between RARRES3 and G9a was also observed in our human HCC samples

268 (P = 0.0016, R2 = 0.2867, Figure 7C) as well as a panel of HCC cell lines (P = 0.042, R2 =

269 0.2807, Supplementary figure 8C). We then determined the RARRES3 mRNA expression 270 changes in our HCC cell models and found that both knockdown and knockout of G9a

271 significantly up-regulated RARRES3 expression (Figure 7D). Similarly, suppression of G9a

272 histone methyltransferase activity by small molecular inhibitors, UNC0638 and BIX01294,

273 resulted in a remarkable elevation of RARRES3 expression in HCC cell lines (Figure 7E). On

274 the contrary, overexpressing G9a significantly reduced RARRES3 level in HCC cells as

275 demonstrated by our G9a-3’UTR deleted HCC cell model (Supplementary figure 8D). The direct

276 regulation of G9a to RARRES3 was further validated by ChIP assay. We showed that knockout

277 of G9a significantly diminished the binding of G9a as well as the level of H3K9me2 in the

278 promoter region of RARRES3 (Figure 7F). Taken together, these results suggest that G9a

279 negatively regulates the expression of RARRES3 in human HCC.

280 Next, we queried whether suppression of RARRES3 contributed to the tumor-promoting

281 function of G9a in HCC. To this end, we found that knockdown of RARRES3 in PLC/PRF/5

282 HCC cell line, which has higher endogenous RARRES3 expression, substantially accelerated

283 cell proliferation (P < 0.0001, Figure 7G). Moreover, stable knockdown of RARRES3 in

284 MHCC-97L also significantly promoted HCC cell migration (Figure 7H). In line with the above

285 findings, BrdU incorporation assay demonstrated that knockdown of RARRES3 remarkably

286 rescued the cell proliferation defects resulted by G9a knockout (Figure 7I). These results

287 collectively suggest that RARRES3 is a downstream target of G9a and the suppression of

288 RARRES3 expression significantly contributes to the tumor-promoting function of G9a in

289 human HCC.

290

291 Discussion 292 Cancer cells experience intensive epigenetic re-programing. Deregulation of epigenetic

293 regulators that are involved in controlling DNA methylation, histone modifications, and

294 chromatin remodeling may have immense impacts on cellular transformation and evolution of

295 cancer cells. In our previous RNA-Seq profiling study of 591 epigenetic regulators in human

296 HCC, we found that deregulation of epigenetic regulators is a striking feature of HCC [11]. In

297 this study, we demonstrated that G9a, a histone methyltransferase, was frequently up-regulated

298 in human HCC. The frequent up-regulation of G9a is a common characteristic of human HCC

299 that can be readily validated in an expand sample cohort consisting of 92 HCC cases as well as in

300 TCGA HCC samples. G9a is a SET domain containing histone methyltransferase specific for

301 H3K9me2 which represents a specific epigenetic mark for transcriptional repression in

302 euchromatin domain. G9a probably is the major enzyme response for this modification, because,

303 as shown in our study, genetic ablation of G9a dramatically abolished H3K9me2 in HCC

304 genome. Emerging lines of evidence have suggested that G9a up-regulation may play a role in

305 human carcinogenesis [25-27]. For instance, over-expression of G9a in ovarian cancer was

306 closely associated with advanced tumor stage and poor survival of the patients. Knockdown of

307 G9a impaired ovarian cancer cell adhesion, migration, and invasion [27]. Consistent with these

308 findings, we found that G9a expression was gradually increased along the multistep liver

309 carcinogenesis, implying that G9a up-regulation might facilitate HCC progression.

310 Clinicopathological analysis showed that G9a overexpression was significantly associated with

311 aggressive features of HCC, which further highlighted the pathological implications of G9a in

312 liver carcinogenesis. Experimentally, we employed two approaches, using RNAi knockdown and

313 CRISPR/Cas9 knockout, resulting in different depletion levels and demonstrated that G9a was

314 indeed indispensable for HCC tumorigenicity and metastasis both in vitro and in vivo. With the 315 above findings, we believe that G9a functions as a tumor-promoting gene to facilitate human

316 HCC proliferation and metastasis. In addition, pharmacological inhibition of G9a elicited a

317 drastic growth suppression effect in HCC cell lines both in vitro and in vivo. Similar observations

318 have also been made in other cancer models including neuroblastoma [28], oral squamous cell

319 carcinoma [29], and head and neck squamous cell carcinoma[30], suggesting that G9a is a

320 promising novel target for cancer epigenetic therapy.

321 In this study, we also investigated the underlying molecular mechanisms that lead to deregulation

322 of G9a in human HCC. Chromosomal aberration is frequently observed in HCC. We previously

323 reported that frequent chromosome gain and amplification at 1q21 contributed to the up-

324 regulation of SETDB1, another histone methyltransferase that responsible for H3K9me3 [11]. In

325 addition to chromosome 1q, chromosome gain of 6p was also reported as one of the most

326 frequent abnormalities in HCC [31, 32]. A recent CGH meta-analysis showed that 22.3% of

327 HCC cases acquired 6p21 amplification in their tumor [33]. Furthermore, the amplification of

328 6p21 was significantly associated with tumor invasiveness [34] and advanced tumor stage [35],

329 supporting the possible oncogenic role of 6p21 amplification in HCC. Several tumor-promoting

330 genes have been identified in this region including cyclin D3 [36], dual-specificity tyrosine-(Y)-

331 phosphorylation-regulated kinase 2 (DYRK2), and protein tyrosine kinase 7 (PTK7) [37] which

332 were implicated in various cancer types. In line with these findings, we also found that the copy

333 number of G9a was frequently gained at chromosome 6p21 in more than 60% of our HCC

334 samples and 43% of TCGA HCC cohort. More importantly, the up-regulation of G9a mRNA

335 was positively correlated with the gene copy number gain. Therefore, our study identified G9a as

336 a tumor-promoting gene with frequent chromosome 6p21 gain and may be implicated in the

337 oncogenic role of 6p amplification in HCC. 338 In addition to chromosomal abnormality, we also investigated the post-transcriptional

339 mechanism that may contribute to the frequent up-regulation of G9a. As an essential post-

340 transcriptional regulator, miRNAs deregulation is associated with the development and

341 progression of various human cancers. For instance, we previously reported that loss of miR-139

342 and miR-125b contributed to over-expression of Rho kinase 2 (ROCK2) and SUV39H1 [10, 38].

343 Recently, we also found that under-expression of miR-29a contributed to frequent up-regulation

344 of SETDB1 in HCC [11]. In this study, we identified miR-1 as an important post-transcriptional

345 regulator that negatively regulates G9a expression in human HCC. Elimination of G9a-3’UTR,

346 which contains miR-1 binding site, led to up-regulation of G9a expression at both mRNA and

347 protein levels as demonstrated by our CRISRP/Cas9-mediated G9a-3’UTR deletion model.

348 Exogenously overexpression of miR-1 reduced the G9a-3’UTR tagged luciferase signal and the

349 endogenous protein level of G9a. The expression of miR-1 was also negatively correlated with

350 that of G9a in clinical HCC specimens. All these findings suggest that loss of miR-1 relieves the

351 post-transcriptionally repression on G9a and contributes to its up-regulation in human HCC,

352 which further underlies the important role of deregulation of miRNA in HCC development.

353 Previous studies have identified several tumor suppressors that were negatively regulated by

354 G9a-mediated epigenetic silencing. In human breast cancer, knockdown of G9a restored E-

355 cadherin expression which resulted in suppression of cell migration in vitro and in vivo [26]. G9a

356 also negatively regulates Ep-CAM to promote lung cancer progression [25]. G9a plays an

357 important role in transcriptional repression in hypoxia adaptation [39, 40]. More recently, G9a

358 has been shown to be a critical modulator of autophagy and metabolic reprogramming [41, 42].

359 Herein, through RNA-Seq analysis, we identified a tumor suppressor RARRES3 (also known as

360 TIG3 and RIG-1) as a novel downstream target of G9a in human HCC. RARRES3 is a 361 phospholipase involved in the production of arachidonic acid and other lipid signaling

362 messengers [43]. The expression of RARRES3 can be induced by retinoid acid to negatively

363 regulate cell proliferation and induce cell differentiation [20]. RARRES3 promoter contains a

364 p53 binding site and its expression can be transactivated by p53 protein [44]. The expression of

365 RARRES3 is reduced in multiple cancers. Loss of RARRES3 has been considered as a key

366 driver of lung metastasis in estrogen receptor negative (ER-) breast cancer by disabling cellular

367 differentiation and facilitating the cancer cells to adhere to lung parenchyma [24]. This could be

368 achieved through modulating the Wnt/β-catenin signaling pathway, thereby influencing the stem

369 cell properties and epithelial-mesenchymal transition of breast cancer cells [45]. However, the

370 mechanism underpinning the transcriptional silencing of RARRES3 in cancers has not been

371 reported. Our present study found that the expression of RARRES3 was frequently

372 downregulated in human HCCs in both our HCC cohort and TCGA sample sets. RARRES3

373 expression was negatively correlated with that of G9a in HCC samples as well as HCC cell lines.

374 Inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout and pharmacological

375 inhibitors consistently relieved the transcriptional silencing of RARRES3 in human HCC cell

376 lines, demonstrating the causal relationship between G9a overexpression and RARRES3 down-

377 regulation. It has been previously reported that ectopic overexpression of RARRES3 resulted in

378 impaired cell proliferation and migration abilities [21, 22, 24, 46]. Consistently, we showed that

379 knockdown of RARRES3 effectively accelerated HCC cell growth and promoted cell migration.

380 Importantly, knockdown of RARRES3 substantially recused the cell proliferation defect of G9a

381 knockout BEL7402 cells. Taken together, our results suggest that G9a negatively regulates

382 RARRES3 and thus contributes to HCC proliferation and metastasis. 383 In summary, we delineated the tumor-promoting function of G9a in HCC. G9a was frequently

384 up-regulated in HCC, partly due to the gene copy number gain and loss of post-transcriptional

385 regulator miR-1. Inactivation of G9a halted HCC cell proliferation and migration in both in vitro

386 and in vivo models. G9a exerts is tumor promoting function at least partially through epigenetic

387 silencing of tumor suppressor RARRES3. Thus, targeting G9a may be a novel approach for HCC

388 treatment.

389

390 Methodology

391 Clinical specimens

392 The HCC patients involved in this study had surgical resection at Queen Mary Hospital in Hong

393 Kong between 1991 and 2007. The use of clinical specimens has been approval by the Institutional

394 Review Board of the University of Hong Kong and the Hong Kong Hospital Authority.

395 Establishment of G9a and RARRES3 stable knockdown cell lines

396 The G9a and RARRES3 stable knockdown cell lines were established by lentivral based stable

397 shRNA overexpression. Non-target control shRNA (shNTC) obtained from Sigma-Aldrich was

398 used as negative control.

399 Establishment of G9a knockout cell lines

400 The plasmid pSpCas9 (BB)-2A-Puro (PX459) (Addgene) was used to simultaneously express

401 wild-type Cas9 and single guide RNA. Two guide RNA sequences

402 GGGTCACTTCTCCTGAACGC (sg#3) and GGTCACTTCTCCTGAACGCC (sg#4) were used

403 in this experiment. These two sgRNAs both target exon 7 of G9a and generate frame-shift indel 404 to disrupt the translation of both G9a-S and G9a-L isoforms. The G9a knockout effect was

405 screened and validated by Western Blotting.

406 ChIP assay

407 The G9a antibody used in CHIP assay was purchased from Abcam (ab40542). The H3K9me2

408 antibody was purchased from Cell Signaling Technology (#9753). The primer sequence to detect

409 G9a and H3K9me2 enrichment in RARRES3 promoter region is RARRES3-ChIP-F:

410 GGGCATCCCCATGGAATGAA and RARRES3-ChIP-R: CATTCGGAGGCAGGGAGATG

411 Orthotopic tumor implantation model

412 For orthotopic tumor implantation model, 2 ×106 luciferase labelled t cells were mixed with 25 µl

413 DMEM-hg/Matrigel (1:1) and injected into the left liver lobule of nude mice. Mice were

414 sacrificed at 6 weeks, livers and lungs were collected. Formation of extra-hepatic metastasis was

415 detected by ex-vivo bioluminescent imaging.

416 Treatment of G9a inhibitors, UNC0638 and BIX01294

417 Small molecule inhibitors of G9a, UNC0638 and BIX01294, were purchased from Cayman

418 Chemical. GI50 of these inhibitors on cell growth was determined in BEL7402 cells upon 48

419 hours of UNC0638 and BIX01294 treatment, respectively.

420 Transcriptome sequencing

421 Transcriptome sequencing (RNA-Seq) was performed G9a stably knockdown (shG9a #69) and

422 non-target control of BEL7402 and SMMC-77721 cells. The sample preparation and data

423 analysis procedures were described previously [11].

424 Statistical analyses 425 Statistical analysis was performed using IBM SPSS version 20 and PRISM 5 software package. P <

426 0.05 was considered as statistically significant.

427 Detailed materials and methods can be found in Supplementary Methodology.

428

429 References

430 [1] Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer

431 incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.

432 International journal of cancer Journal international du cancer 2015;136(5): E359-386.

433 [2] Bruix J, Gores GJ, Mazzaferro V. Hepatocellular carcinoma: clinical frontiers and

434 perspectives. Gut 2014;63(5): 844-855.

435 [3] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in

436 advanced hepatocellular carcinoma. The New England journal of medicine 2008;359(4): 378-

437 390.

438 [4] Momparler RL. Cancer epigenetics. Oncogene 2003;22(42): 6479-6483.

439 [5] Wong CM, Ng IO. Molecular pathogenesis of hepatocellular carcinoma. Liver

440 international : official journal of the International Association for the Study of the Liver

441 2008;28(2): 160-174.

442 [6] Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. Epigenetic protein families: a

443 new frontier for drug discovery. Nature reviews Drug discovery 2012;11(5): 384-400. 444 [7] Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps.

445 Nature reviews Genetics 2007;8(4): 286-298.

446 [8] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293(5532): 1074-1080.

447 [9] Au SL, Wong CC, Lee JM, Fan DN, Tsang FH, Ng IO, et al. Enhancer of zeste homolog

448 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer

449 metastasis. Hepatology 2012;56(2): 622-631.

450 [10] Fan DN, Tsang FH, Tam AH, Au SL, Wong CC, Wei L, et al. Histone lysine

451 methyltransferase, suppressor of variegation 3-9 homolog 1, promotes hepatocellular carcinoma

452 progression and is negatively regulated by microRNA-125b. Hepatology 2013;57(2): 637-647.

453 [11] Wong CM, Wei L, Law CT, Ho DW, Tsang FH, Au SL, et al. Up-regulation of histone

454 methyltransferase SETDB1 by multiple mechanisms in hepatocellular carcinoma promotes

455 cancer metastasis. Hepatology 2016;63(2): 474-487.

456 [12] Feldman N, Gerson A, Fang J, Li E, Zhang Y, Shinkai Y, et al. G9a-mediated irreversible

457 epigenetic inactivation of Oct-3/4 during early embryogenesis. Nature cell biology 2006;8(2):

458 188-194.

459 [13] Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP. Large histone H3 lysine 9

460 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nature

461 genetics 2009;41(2): 246-250.

462 [14] Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, et al. G9a histone

463 methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is

464 essential for early embryogenesis. Genes & development 2002;16(14): 1779-1791. 465 [15] Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, et al. Global levels of

466 histone modifications predict prognosis in different cancers. The American journal of pathology

467 2009;174(5): 1619-1628.

468 [16] Ellinger J, Kahl P, von der Gathen J, Rogenhofer S, Heukamp LC, Gutgemann I, et al.

469 Global levels of histone modifications predict prostate cancer recurrence. The Prostate

470 2010;70(1): 61-69.

471 [17] Mshvildadze M, Neu J, Shuster J, Theriaque D, Li N, Mai V. Intestinal microbial ecology

472 in premature infants assessed with non-culture-based techniques. J Pediatr 2010;156.

473 [18] Vedadi M, Barsyte-Lovejoy D, Liu F, Rival-Gervier S, Allali-Hassani A, Labrie V, et al.

474 A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nature

475 chemical biology 2011;7(8): 566-574.

476 [19] Kubicek S, O'Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, et al.

477 Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase.

478 Molecular cell 2007;25(3): 473-481.

479 [20] DiSepio D, Ghosn C, Eckert RL, Deucher A, Robinson N, Duvic M, et al. Identification

480 and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene.

481 Proceedings of the National Academy of Sciences of the United States of America 1998;95(25):

482 14811-14815.

483 [21] Tsai FM, Shyu RY, Jiang SY. RIG1 suppresses Ras activation and induces cellular

484 apoptosis at the Golgi apparatus. Cellular signalling 2007;19(5): 989-999. 485 [22] Higuchi E, Chandraratna RA, Hong WK, Lotan R. Induction of TIG3, a putative class II

486 tumor suppressor gene, by retinoic acid in head and neck and lung carcinoma cells and its

487 association with suppression of the transformed phenotype. Oncogene 2003;22(30): 4627-4635.

488 [23] Scharadin TM, Jiang H, Jans R, Rorke EA, Eckert RL. TIG3 tumor suppressor-dependent

489 organelle redistribution and apoptosis in skin cancer cells. PloS one 2011;6(8): e23230.

490 [24] Morales M, Arenas EJ, Urosevic J, Guiu M, Fernandez E, Planet E, et al. RARRES3

491 suppresses breast cancer lung metastasis by regulating adhesion and differentiation. EMBO

492 molecular medicine 2014;6(7): 865-881.

493 [25] Chen MW, Hua KT, Kao HJ, Chi CC, Wei LH, Johansson G, et al. H3K9 histone

494 methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell

495 adhesion molecule Ep-CAM. Cancer research 2010;70(20): 7830-7840.

496 [26] Dong C, Wu Y, Yao J, Wang Y, Yu Y, Rychahou PG, et al. G9a interacts with Snail and

497 is critical for Snail-mediated E-cadherin repression in human breast cancer. The Journal of

498 clinical investigation 2012;122(4): 1469-1486.

499 [27] Hua KT, Wang MY, Chen MW, Wei LH, Chen CK, Ko CH, et al. The H3K9

500 methyltransferase G9a is a marker of aggressive ovarian cancer that promotes peritoneal

501 metastasis. Molecular cancer 2014;13: 189.

502 [28] Lu Z, Tian Y, Salwen HR, Chlenski A, Godley LA, Raj JU, et al. Histone-lysine

503 methyltransferase EHMT2 is involved in proliferation, apoptosis, cell invasion, and DNA

504 methylation of human neuroblastoma cells. Anti-cancer drugs 2013;24(5): 484-493. 505 [29] Ren A, Qiu Y, Cui H, Fu G. Inhibition of H3K9 methyltransferase G9a induces

506 autophagy and apoptosis in oral squamous cell carcinoma. Biochemical and biophysical research

507 communications 2015;459(1): 10-17.

508 [30] Li KC, Hua KT, Lin YS, Su CY, Ko JY, Hsiao M, et al. Inhibition of G9a induces

509 DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma. Molecular

510 cancer 2014;13: 172.

511 [31] Chen YJ, Yeh SH, Chen JT, Wu CC, Hsu MT, Tsai SF, et al. Chromosomal changes and

512 clonality relationship between primary and recurrent hepatocellular carcinoma. Gastroenterology

513 2000;119(2): 431-440.

514 [32] Lin YW, Sheu JC, Huang GT, Lee HS, Chen CH, Wang JT, et al. Chromosomal

515 abnormality in hepatocellular carcinoma by comparative genomic hybridisation in Taiwan.

516 European journal of cancer 1999;35(4): 652-658.

517 [33] Moinzadeh P, Breuhahn K, Stutzer H, Schirmacher P. Chromosome alterations in human

518 hepatocellular carcinomas correlate with aetiology and histological grade--results of an

519 explorative CGH meta-analysis. British journal of cancer 2005;92(5): 935-941.

520 [34] Pang A, Ng IO, Fan ST, Kwong YL. Clinicopathologic significance of genetic alterations

521 in hepatocellular carcinoma. Cancer genetics and cytogenetics 2003;146(1): 8-15.

522 [35] Chochi Y, Kawauchi S, Nakao M, Furuya T, Hashimoto K, Oga A, et al. A copy number

523 gain of the 6p arm is linked with advanced hepatocellular carcinoma: an array-based comparative

524 genomic hybridization study. The Journal of pathology 2009;217(5): 677-684. 525 [36] Sonoki T, Harder L, Horsman DE, Karran L, Taniguchi I, Willis TG, et al. Cyclin D3 is a

526 target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies. Blood 2001;98(9): 2837-2844.

527 [37] Gorringe KL, Boussioutas A, Bowtell DD, Melbourne Gastric Cancer Group PMMAF.

528 Novel regions of chromosomal amplification at 6p21, 5p13, and 12q14 in gastric cancer

529 identified by array comparative genomic hybridization. Genes, & cancer

530 2005;42(3): 247-259.

531 [38] Wong CM, Wong CC, Ng YL, Au SL, Ko FC, Ng IO. Transcriptional repressive H3K9

532 and H3K27 methylations contribute to DNMT1-mediated DNA methylation recovery. PloS one

533 2011;6(2): e16702.

534 [39] Lee SH, Kim J, Kim WH, Lee YM. Hypoxic silencing of tumor suppressor RUNX3 by

535 histone modification in gastric cancer cells. Oncogene 2009;28(2): 184-194.

536 [40] Lee JS, Kim Y, Kim IS, Kim B, Choi HJ, Lee JM, et al. Negative regulation of hypoxic

537 responses via induced Reptin methylation. Molecular cell 2010;39(1): 71-85.

538 [41] Artal-Martinez de Narvajas A, Gomez TS, Zhang JS, Mann AO, Taoda Y, Gorman JA, et

539 al. Epigenetic regulation of autophagy by the methyltransferase G9a. Molecular and cellular

540 biology 2013;33(20): 3983-3993.

541 [42] Ding J, Li T, Wang X, Zhao E, Choi JH, Yang L, et al. The histone H3 methyltransferase

542 G9A epigenetically activates the serine-glycine synthesis pathway to sustain cancer cell survival

543 and proliferation. Cell metabolism 2013;18(6): 896-907.

544 [43] Wang D, Dubois RN. Eicosanoids and cancer. Nature reviews Cancer 2010;10(3): 181-

545 193. 546 [44] Hsu TH, Chu CC, Jiang SY, Hung MW, Ni WC, Lin HE, et al. Expression of the class II

547 tumor suppressor gene RIG1 is directly regulated by p53 tumor suppressor in cancer cell lines.

548 FEBS letters 2012;586(9): 1287-1293.

549 [45] Hsu TH, Jiang SY, Chang WL, Eckert RL, Scharadin TM, Chang TC. Involvement of

550 RARRES3 in the regulation of Wnt acylation and signaling activities in human breast

551 cancer cells. Cell death and differentiation 2015;22(9): 1561.

552 [46] Sturniolo MT, Dashti SR, Deucher A, Rorke EA, Broome AM, Chandraratna RA, et al. A

553 novel tumor suppressor protein promotes keratinocyte terminal differentiation via activation of

554 type I transglutaminase. The Journal of biological chemistry 2003;278(48): 48066-48073.

555

556 Figure Legend

557 Figure 1: Frequent up-regulation of G9a in human HCCs. (A) G9a mRNA expression was

558 significantly increased by 4.5 fold in 16 paired HCC samples compared with their non-tumorous

559 (NT) counterpart as revealed by RNA-Seq analysis (P < 0.0001, Wilcoxon signed rank test).

560 Data were presented as FPKM (fragments per kilo base of transcript sequences per aligned

561 million reads). The horizontal lines indicated median expression levels. (B) Significant up-

562 regulation of G9a was validated in an expand sample cohort containing 92 paired HCC and their

563 corresponding NT samples and 5 normal livers (NL) by qRT-PCR. HPRT was used as

564 endogenous control for normalization. G9a expression in primary HCC was increased by 2.7 fold

565 as compared with their adjacent NT samples (P < 0.0001, Wilcoxon signed rank test) and 5.72

566 fold when compared with normal livers (P < 0.0001, Mann-Whitney U-test). (C) Up-regulation

567 of G9a (>2 fold, i.e. log2 (HCC/NT > 1)) was found in 68% (63/92) of primary HCC samples. 568 Only 4 HCC samples showed down-regulation of G9a. (D) Significant up-regulation of G9a was

569 observed in 50 pairs of HCC samples available in TCGA RNA-Seq dataset (P < 0.0001,

570 Wilcoxon signed rank test). The expression of G9a was increased by 1.97 fold in HCC as

571 compared with NT livers. Data were presented as RSEM (RNA-Seq expression estimation by

572 Expectation-Maximization) normalized count. (E) Up-regulation of G9a (>2 fold) was found in

573 60% (30/50) of TCGA HCC samples. (F) The expression of G9a increased step-wisely along

574 with HCC disease progression. G9a mRNA expression level was progressively increased from

575 normal liver without background disease (NL) (N = 6; median = 0.950) to chronic hepatitis (CH)

576 (N = 36; median = 1.043) and cirrhotic liver (CL) (N = 48; median =1.289), then to early HCC

577 (N = 31; median = 2.458) and advanced HCC (N =56; median = 3.856) (P < 0.0001, Kruskal-

578 Wallis test). (G) G9a overexpression was significantly associated with advanced pTNM stage (P

579 = 0.0195, t-test), the presence of venous invasion (P = 0.0042), tumor microsatellite (P = 0.0080)

580 and absence of tumor encapsulation (P = 0.0145). The qRT-PCR data are presenting as ∆∆Ct (i.e.

581 log2 fold change HCC/NT liver).

582 Figure 2: Gene copy number gain contributed to the frequent up-regulation of G9a. (A) and

583 (B) Gene copy number gain (≥ 3 copies) of G9a was detected in 60% of primary HCC (N = 32)

584 as revealed by TaqMan gene copy number assay. (C) G9a mRNA expression was positively

585 correlated with G9a gene copy number alternation (P < 0.0001, R2 = 0.2384, linear regression).

586 (D) TCGA gene copy number data showed that G9a gene copy number gain and amplification

587 was detected in 43% of all its HCC samples (N = 254). (E) G9a gene copy number gain was

588 positively correlated with increased G9a mRNA expression in TCGA HCC sample cohort (P <

589 0.0001, One-Way ANOVA). 590 Figure 3: Loss of miR-1 contributed to G9a up-regulation in human HCC. (A) Two PX459

591 plasmids with different single guide RNAs (sgRNAs) targeting the genome sequence at the

592 beginning and the end of G9a-3’UTR were employed to create double-strand break

593 simultaneously and then lead to complete deletion of G9a 3’UTR via non-homologous end

594 joining repair mechanism. Genome sequence of two G9a-3’UTR deleted cell clones BEL7402-

595 G9a-Δ3UTR-#42 and -#52 showed that the 322 bp G9a-3’UTR was completely deleted. The

596 underlined sequences were the expected editing sites of CRISPR/Cas9 system. (B) After deletion

597 of G9a-3’UTR, the expression of G9a was significantly increased in both mRNA and protein

598 levels. (C) RNA hybrid predicted a thermokinetically stable duplex to be formed between miR-1

599 and G9a-3’UTR. The miR-1 binding site on G9a-3’UTR is evolutionarily conserved across

600 different species. (D) The expression of miR-1 was analyzed by qRT-PCR in 32 pairs of human

601 HCCs. miR-1 was significantly down-regulated in human HCC when compared with their

602 corresponding NT counterpart (P = 0.0002, Wilcoxon signed-rank test). (E) Expression of miR-1

603 was negatively correlated with G9a expression in human HCC (R2 = 0.2581, P < 0.0001, linear

604 regression). (F) Wild type and mutant (Mut) miR-1 binding site of G9a 3’UTR were cloned into

605 pmiRGLO vector. (G) miR-1 significantly repressed luciferase activity of WT G9a 3’UTR but

606 not in the Mut G9a-3’UTR nor the empty vector control. (H) Exogenously overexpression of

607 miR-1 precuorsor repressed endogenous G9a protein expression in HCC cell lines. *** P <0.001

608 Figure 4: G9a is responsible for H3K9me2 in HCC. (A) Overexpression of G9a was found in

609 HCC cell lines including HepG2, Hep3B, SMMC-7721, BEL7402 and MHCC97L when

610 compared with immortalized normal liver cell line THLE-3. Both the short form (G9a-s, 140

611 KDa) and long form (G9a-L, 165 KDa) of G9a can be detected in Western blot. The relative G9a

612 protein expression in HCC cell line was determined with Image J and compared to THLE-3. (B) 613 G9a stable knockdown cell model was established in BEL7402 and SMMC-7721 cells by

614 lentiviral delivery of shRNA sequences targeting G9a coding region. Two shRNA sequences

615 (shG9a #69 and #70) were used. Knockdown efficiency was verified at both protein level by

616 Western blotting (upper panel) and mRNA level by qRT-PCR (lower panel). NTC: non-target

617 control. (C) The single guide RNAs were designed to target the exon 7 of G9a to disrupt protein

618 translation of both G9a isoforms. (D) The G9a protein was completely depleted as shown by

619 Western blotting. (E) Knockout of G9a in both BEL7402 and SMMC-7721 cell lines effectively

620 decreased H3K9me2 level without affecting H3K9me3, H3K4me3 and H3K27me3 levels.

621 Figure 5: Knockdown and knockout of G9a suppressed HCC proliferation and migration

622 in vitro. (A) RNA sequencing data in 16 paired HCC samples showed that G9a mRNA

623 expression was positively correlated with proliferation marker Ki67 in HCCs and NT livers (R2 =

624 0.7192, P < 0.0001, linear regression). (B) and (C) Knockdown (shG9a #69 and #70) and

625 knockout (sg#3 and #4) of G9a in both BEL7402 and SMMC-7721 cell lines significantly

626 reduced HCC cell proliferation when compared with their non-target control or wild type cells.

627 (D) and (E) Knockdown and knockout of G9a in both BEL7402 and SMMC-7721 cell lines

628 significantly suppressed HCC cell colony formation ability. The cell colonies formed were

629 counted as average colony numbers in three wells of each 6-well plate. Each sample was tested

630 in triplicate. (F) and (G) Knockdown and knockout of G9a in both BEL7402 and SMMC-7721

631 cell lines significantly suppressed HCC cell migration as shown by transwell cell migration assay.

632 The migrated cells were counted as average cell numbers in three random areas in each transwell

633 membrane. Each sample was tested in triplicate. ** P < 0.01, *** P < 0.001, t-test.

634 Figure 6: Knockdown and knockout of G9a inhibited HCC tumorigenicity and metastasis

635 in vivo. (A) and (B) G9a stable knockdown (shG9a) and its non-target control BEL7402 or 636 SMMC-7721 cells were injected into BALB/c nude mice subcutaneously. Tumor size was

637 monitored every 3 days and weighed after harvest. G9a knockdown significantly suppressed

638 HCC tumor growth. (C) and (D) Knockout of G9a significantly inhibited HCC tumorigenicity

639 and lung metastasis in vivo as demonstrated by orthotopic tumor implantation experiment in

640 nude mice. (E) The GI50 of G9a small molecule inhibitors UNC0638 (GI50 = 7.325µM) and

641 BIX01294 (GI50 = 5.225 µM) were determined in BEL7402 cells. (F) UNC0638 and BIX01294

642 effectively inhibited HCC cell growth at the concentration of 5µM after 48 hours of treatment.

643 The morphology of the cells was also altered upon treatment. (G) Treatment of UNC0638 and

644 BIX01294 at 5µM in BEL7402 cell specifically attenuated G9a-mediated H3K9me2 level

645 without affecting other histone modificaitons. * P < 0.05, ** P < 0.01, *** P < 0.001, t-test.

646 Figure 7: Suppression of Retinoic Acid Receptor Responder Protein 3 (RARRES3)

647 contributed to oncogenic function of G9a. (A) The transcriptome profiles of G9a stable

648 knockdown and non-target control BEL7402 and SMMC-7721 cell lines were introgerrated by

649 RNA-Seq and subjected to differential analysis. After filtering the low exprssion

650 genes (with FPKM < 1), there are 140 and 77 genes up-regulated (> 2 fold) in BEL7402 and

651 SMMC-7721, respectively. Among them, 16 genes were commonly up-regulated in both cell

652 lines. RARRES3 was one of the most up-regulated genes as shown in the heat map diagram. (B)

653 Aligned RNA-sequencing reads visualized through USCS genome browser revealed the

654 expression levels of G9a and RARRES3 in HCC cell lines. The data showed that G9a was

655 successfully knocked down in both BEL7402 and SMMC-7721 cells. RARRES3 mRNA

656 expression was increased by 4.24 fold in SMMC-7721 cells and 2.93 fold in BEL7402 cells upon

657 G9a knockdown. (C) Expression level of RARRES3 in 16 pairs of HBV associated human HCC

658 samples determined by RNA-Seq. Data were presented in FPKM. The scatter plot showed the 659 significant down-regulation of RARRES3 in primary HCC (P < 0.0001, Wilcoxon signed rand

660 test). Median expression level was indicated by horizontal lines. The mRNA expression of

661 RARRES3 in 16 paired HCC samples was negatively correlated with mRNA expression of G9a.

662 (R2 = 0.2867, P = 0.0016, linear regression). (D) Knockdown and knockout of G9a significantly

663 up-regulated RARRES3 mRNA expression in both BEL7402 and SMMC-7721 cell lines

664 compared with non-target control and wild type cells. (E) The mRNA expression of RARRES3

665 was significantly up-regulated upon treatment of G9a specific small molecule inhibitors,

666 UNC0638 and BIX01294, in both BEL7402 and SMMC-7721 cell lines. (F) Knockout of G9a

667 significantly diminished the G9a binding and H3K9me2 level at the promoter region of

668 RARRES3 as determined by ChIP assay. (G) Knockdown of RARRES3 in PLC cells which has

669 higher level of endogenous RARRES3 increased its proliferation rate. (H) Stable knockdown of

670 RARRES3 by shRNA in MHCC97L increased cell migration rate. (I) Inactivation of RARRES3

671 by siRNA in BEL7402-G9a (-/-) cell lines recused the fast cell proliferating phenotype as

672 demonstrated by BrdU incorporation assay. ** P < 0.01, *** P < 0.001, t-test.

673 Supplementary Figure 1: Frequent up-regulation of G9a in multiple cancers. Significant up-

674 regulation of G9a was also observed in multiple cancer types including bladder urothelial

675 carcinoma (BLCA), breast invasive carcinoma (BRCA), colorectal adenocarcinoma

676 (COADREAD), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell

677 carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma

678 (LIHC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) (Wilcoxon

679 signed ranked test). The mRNA expression data of G9a was retrieved from TCGA RNA-Seq

680 datasets. Each type of cancer contains paired tumors and their corresponding non-tumor

681 counterpart. 682 Supplementary Figure 2: Chromosome 6p21 was frequently gained or amplified in human

683 HCC. According to oncogenomic database for HCC (OncoDB.HCC), the frequent gain or

684 amplification of chromosome 6p21, where G9a is located, was observed in all CGH studies. (N =

685 14)

686 Supplementary Figure 3: In silico analysis of G9a 3’UTR using miRANDA and TargetScan.

687 MiR-1 and miR-613 were commonly predicated by both miRANDA and TargetScan as the 688 potential post-transcriptional regulators of G9a.

689 Supplementary Figure 4: Depletion of G9a induces cell apoptosis and autophagy. (A)

690 Knockout of G9a induced cell apoptosis in SMMC7721 cells upon 48 hours of starvation. (B)

691 Knockout of G9a induced autophagy in SMMC7721 cells. *** P < 0.001, t-test.

692 Supplementary Figure 5: knockout of G9a inhibited HCC metastasis in vivo. G9a knockout

693 (Ga9 sg#3) and wild type (WT) BEL7402 cells were injected into BALB/c nude mice by tail-

694 vein injection. The mice were dissected 4 weeks later and the formation of lung metastasis was

695 detected by ex-vivo bioluminescent imaging. The result showed that knockout of G9a

696 significantly inhibited HCC lung metastasis in vivo.

697 Supplementary Figure 6: Treatment of G9a inhibitors specifically suppressed H3K9me2.

698 HCC cell cells were treated with at 0.5µM and 1.0µM UNC0638 and BIX01294. The change of

699 global H3K9me2, H3K4me3 and H3K27me3 levels were detected with Western blot.

700 Supplementary Figure 7: Inhibition of G9a by small molecule inhibitor UNC0642

701 suppressed tumor growth in vivo. (A) Chemical structure of UNC0642 which is a newly

702 identified G9a specific inhibitor with good pharmacokinetic (PK) properties and is suitable for in

703 vivo study. (B) Treatment of UNC0642 at 5µM in BEL7402 and SMMC7721 cells specifically 704 attenuated G9a-mediated H3K9me2 level without affecting other histone modificaitons. (C)

705 Nude mice bearing subcutaneous tumour were treated with UNC0642 daily by intraperitoneal

706 injection at 5mg/kg. The tumour volume was monitored daily and the mice were dissected after

707 23 days of tumour growth and 17 days of UNC0642 treatment. * P < 0.05, t-test

708 Supplementary Figure 8: Frequent down-regulation of RARRES3 in human HCCs. (A)

709 Significant down-regulation of RARRES3 was validated in 50 pairs of HCC samples from 710 TCGA RNA-Seq dataset (P = 0.0041, Wilcoxon signed rank test). Data were presented as RSEM

711 normalized count. (B) Down-regulation of RARRES3 (< 0.5 fold) was found in 48% (24/50) of

712 TCGA HCC samples. (C) The mRNA expression of RARRES3 in HCC cell lines (n = 15) was

713 negatively correlated with mRNA expression of G9a (R2 = 0.2807, P = 0.0042, linear regression).

714 (D) Overexpressing G9a significantly reduced RARRES3 level in HCC cells as demonstrated in

715 G9a 3’UTR deleted BEL7402 cell model. ***P < 0.001, t-test.

716

717

718

719

720

721

722

723

724

725

726

727

728

729

730 731 Highlights

732

733 • G9a was frequently up-regulated in human HCC and associated with HCC 734 aggressiveness. 735 • G9a promoted HCC growth and metastasis both in vitro and in vivo. 736 • Up-regulation of G9a in HCC was attributed to gene amplification and loss of miR-1. 737 • G9a epigenetically silenced the expression of tumor suppressor gene RARRES in HCC. 738 • Targeting G9a by small molecular inhibitors suppressed HCC growth. 739

740

741