EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.


Results
Relationship between EZH1/2 expression and H3K27me3 levels in surgical HCC samples. To understand the relationship between EZH2 expression and H3K27me3 levels in HCC, immunohistochemical analyses were performed in 72 pairs of primary HCC tissues and corresponding non-tumor tissues. In tumorfree tissues, both EZH2 and H3K27me3 were only detected in infiltrated lymphocytes and periportal hepatocytes. In clear contrast, HCC tissues exhibited varying degrees of increased EZH2 expression and H3K27me3 level (Fig. 1A). Based on the percentage of cells strongly expressing these markers (≥ 50% of tumor cells), tumor tissues were divided into either EZH2 low or EZH2 high and either H3K27me3 low or H3K27me3 high .
Regarding EZH2 expression, 42 (58.3%) and 30 (41.7%) of 72 samples were classified as EZH2 low and EZH2 high , respectively. Similarly, 29 (40.3%) and 43 (59.7%) samples were classified as H3K27me3 low and H3K27me3 high , respectively. The Fisher's exact test revealed a significant association between EZH2 expression and H3K27me3 levels (p < 0.01) (Fig. 1B). However, 16 samples out of 43 H3K27me3 high samples showed low EZH2 expression. Because mRNA from 10 of the 16 EZH2 low H3K27me3 high samples was sufficient and of a high enough quality, they were subjected to RT-qPCR. Four out of 10 samples showed an increase in EZH1 levels compared to those in non-tumor tissues (Fig. 1C). It is possible that EZH1 rather than EZH2 was associated with the high levels of H3K27me3 in some EZH2 low H3K27me3 high samples. Among the rest samples, 4 of 6 samples showed a decrease in expression levels of ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a H3K27 demethylase, compared to those in non-tumor tissues (Fig. 1D). These findings suggested that not only EZH1 overexpression but also UTX decreased expression might be attributable to increased H3K27me3 levels in some of the EZH2 low H3K27me3 high cases.
Evaluation of H3K27me3 levels in clinicopathological features. We then analyzed the differences between the clinicopathological features of H3K27me3 low HCCs (n = 29) and H3K27me3 high HCCs (n = 43) ( Table 1). Importantly, H3K27me3 high HCCs were significantly related to stage progression (UICC stage III and IV) (p = 0.04). They also showed a trend for higher serum levels of a-fetoprotein, which is a diagnostic marker of HCC (p = 0.24). We then performed prognostic analyses on these patients, using a Kaplan-Meier survival analysis (Fig. 1E). During the follow-up period (45.6 months), 10 patients died from HCC. The median recurrencefree survival in patients with H3K27me3 low HCC and H3K27me3 high HCC was "not reached" and 25.0 months, respectively (p = 0.02). These results were in agreement with those of previous reports 17 . However, there was no significant difference of PFS between EZH2 low H3K27me3 low and EZH2 high H3K27me3 high patients (Fig. 1F). Collectively, high H3K27me3 levels appeared to be closely associated with HCC prognosis.

Basal levels of EZH1/2 and H3K27me3 and loss-of-function assays in liver cancer cells.
The basal expression of EZH1/2 and H3K27me3 levels was investigated in four liver cancer cell lines: Huh1, Huh7, HepG2, and PLC/PRF/5. Immunofluorescence staining demonstrated a higher expression and co-localization of EZH2 and H3K27me3 in liver cancer cell nuclei. RT-qPCR revealed that, in these cells, EZH2 mRNA expression was significantly higher than that of EZH1 ( Supplementary Fig. S1). Among them, Huh7 and HepG2 cells were subjected to the most of the subsequent experiments.
Next, we conducted loss-of-function assays for EZH1 and EZH2 using lentivirus-mediated shRNA in Huh7 and HepG2 cells, successfully achieving stable knockdown of EZH1 and EZH2 using RFP and EGFP as a viral infection marker, respectively. Two shRNAs were made against EZH1 (sh-EZH1-1 and sh-EZH1-2), both markedly repressing EZH1 expression in HCC cells ( Fig. 2A). sh-EZH1-2 was used for subsequent experiments, and sh-EZH2 was used as previously designed and prepared by our group 18 . While EZH2-knockdown remarkably decreased H3K27me3 levels, the decrease caused by EZH1-knockdown was minimal (Fig. 2B). In the limiting dilution and sphere formation assay, EZH2-knockdown pronouncedly inhibited sphere forming abilities of Huh7 www.nature.com/scientificreports/ and HepG2 cells (Fig. 2C-E). Importantly, the double knockdown of EZH1/2 showed an additional inhibitory effect in cell growth and sphere formation in vitro. Subsequently, the profiling of de-repressed genes (fold change > 2.0) after lentiviral knockdown of EZH1 or EZH2 was analyzed in Huh7 cells. The number of genes de-repressed following EZH1 and EZH2 knockdown was 1864 and 1848, respectively (Fig. 2F). The number of overlapped genes was 1267, indicating that approximately 30% of target genes of EZH1 and EZH2 was not necessarily identical. Gene set enrichment analysis (GSEA) demonstrated that EZH1 knockdown cells were significantly enriched for genes involved in mitotic spindle [normalized enrichment score (NES) 1.93, p-value 0, and false discovery rate (FDR) q-value 0], UV response down (NES 1.79, p-value 0, and FDR q-value 0.001), and Hedgehog signaling (NES 1.63, p-value 0, and FDR q-value 0.008). EZH2 knockdown cells were significantly enriched for genes involved in mitotic spindle (NES 1.83, p-value 0, and FDR q-value 0.002), UV response down (NES 1.77, p-value 0, and FDR q-value 0.003), Hedgehog signaling (NES 1.6, p-value 0.003, and FDR q-value 0.010), K-RAS signaling up (NES 1.74, p-value 0, and FDR q-value 0.003) and interferon alpha response (NES 1.63, p-value 0, and FDR q-value 0.009). Taken together, these results show that simultaneous EZH1/2 inhibition is required for sufficient inhibition of cell growth ability and tumorigenic activity.
Pharmacological deletion of EZH1/2 in liver cancer cells. Both the EZH2 inhibitor GSK126 and the EZH1/2 dual inhibitor UNC1999 has been developed elsewhere ( Fig. 3A) 19,20 . Subsequently, we performed loss-of-function assays of EZH1 and EZH2 using these inhibitors. The cell number in HCC cells treated with UNC1999, but not with GSK126, was significantly lower than those in control cells (Fig. 3B, Supplementary  Fig. S2A). Both drugs induced cellular apoptosis in a dose-dependent manner (Fig. 3C, Supplementary Fig. S2B). Of note, UNC1999 exerted a pronounced effect at a low concentration compared with GSK126. Western blotting demonstrated that both GSK126 and UNC1999 apparently reduced H3K27me3 levels in liver cancer cells in a time-and dose-dependent manner (Fig. 3D). Although RIs of H3K27me3 was significantly lower in UNC1999treated cells than those in GSK126-treated cells for 48 h, there was no significant difference in RIs of H3K27me3 between two groups for 96 h.
An increase in H3K27me3 levels in liver cancer cells treated with sorafenib. Next, we examined EZH1/2 expression and H3K27me3 levels in liver cancer cells treated with sorafenib. Unexpectedly, Western blotting showed increased H3K27me3 levels and concomitantly decreased phospho-Akt levels (Fig. 4A). However, only minimal changes were observed in the EZH1/2 protein. An increase in levels of cleaved PARP, a marker of apoptosis, was detected in a dose-dependent manner. Since it was possible that sorafenib treatment caused an ubiquitous downregulation of UTX, a demethylase for H3K27, RT-qPCR demonstrated that UTX expression was rather upregulated in a dose-dependent manner (Fig. 4B). It has been reported that Akt phosphorylates EZH2 at serine 21 (Ser21) and inhibits its enzyme activity for H3K27me3 21 . Consistently, immunocytochemical staining analyses using a phospho-specific antibody, revealed that phosphorylation of EZH2 at Ser21 remarkably decreased in HCC cells treated with sorafenib and regorafenib (Fig. 4C), suggesting augmented enzymatic activity of EZH2 upon sorafenib treatment. Concordant with these findings, Western blotting demonstrated that sorafenib treatment resulted in a decrease in phosphorylated levels of EZH2 (Ser21), but not EZH2 at threonine 311 (Thr311) in Huh7 cells in a dose-dependent manner (Fig. 4D). These findings indicate that phosphorylation of EZH2 might occur in a site-specific manner.
Combination treatment with sorafenib and EZH1/2 inhibitor in liver cancer cells. Forced expression of EZH1/2 resulted in an increase in H3K27me3 levels in Huh7 cells ( Supplementary Fig. S3A). The inhibitory effect of sorafenib treatment was diminished in EZH1/2 overexpressed cells compared with control cells (Supplementary Fig. S3B). In clear contrast, proliferation inhibition followed by sorafenib treatment was more effective in EZH1/2 double knockdown cells than those in control cells ( Supplementary Fig. S4). These results implicated that EZH1/2 knockdown sensitized HCC cells to sorafenib treatment. Therefore, we inves-   www.nature.com/scientificreports/ tigated the effect of simultaneous treatment with sorafenib and EZH2 or EZH1/2 inhibitors on the growth of Huh7 cells. The Combination index (CI) was calculated 22 . The results showed a convincing synergism of the combined use of sorafenib and EZH1/2 inhibitor UNC1999 or EZH2-specific inhibitor GSK126 (CI < 1.0) in Huh7 cells (Fig. 5A). Synergism of the combined use of sorafenib and EZH1/2 inhibitor UNC1999, but not GSK126, was observed in HepG2 cells. EZH2 inhibition by GSK126 and UNC1999 successfully canceled abnormal H3K27me3 changes caused by sorafenib (Fig. 5B). The levels of cleaved PARP were enhanced in combined use of sorafenib and GSK126 or UNC1999.  (Fig. 6A,B). The oral administration of sorafenib and/or UNC1999 (i.e., daily administration of 5 mg/kg of sorafenib and/ or 3 days a week administration of 15 mg/kg of UNC1999 for 6 weeks) was started on the days following HCC cells implantation. Subcutaneous tumor growth was significantly suppressed by the concomitant administration of sorafenib and UNC1999, compared to their single administration. Consistent with the results from the in vitro experiments, immunohistochemical analyses of the subcutaneous tumors showed that treatment with sorafenib induced an increase of the H3K27me3 levels, accompanied by a decrease of both the phosphorylated Akt and the phosphorylated EZH2 (Fig. 6C). The combined use of UNC1999 and sorafenib canceled an increase in H3K27me3 levels at least partially. Ki-67 staining revealed that the combined treatment efficiently inhibited cell growth. The Ki-67 labeling index in mock, UNC1999, sorafenib, and combination groups was 47.4%, 11.8%, 7.8%, and 4.7%, respectively. Both CASP3 staining and Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay demonstrated that the combination treatment more clearly induced apoptosis compared with mock or single agent treatment (Fig. 6D). Taken together, these results show that the combined use of EZH1/2 dual inhibitor may enhance the anti-tumor effects of sorafenib by canceling sorafenib-induced activation of EZH2 and additional inhibition of EZH1 (Fig. 7).

Discussion
The PcG proteins are responsible for maintaining the transcriptional repression of target genes through the catalyzation of histone modifications 23 . The PcG gene family members form two major form complexes, PRC1 and PRC2. Both Ezh1 and Ezh2 are the catalytic components of PRC2 and trimethylate H3K27. Although EZH1 partially compensates for EZH2 deficiencies in hematopoietic stem cells, the H3K27me2/3 activity of EZH1 is markedly weaker than that of EZH2 24 . In in vitro assays, the basal expression of EZH1/2 was exactly detected in the HCC cell lines examined. However, the EZH1 expression was significantly lower than that of EZH2. Although EZH2 knockdown caused an apparent decrease in H3K27me3 levels, EZH1 knockdown caused only modest changes in H3K27me3 levels. Consistent with these findings, the inhibition of cell growth and sphere formation was obviously observed in EZH2 knockdown cells compared to EZH1 knockdown cells. Importantly, EZH1/2 double knockdown demonstrated an additional inhibitory effect on cell growth and sphere formation in HCC cells compared to single knockdown. Our clinicopathological analyses of HCC surgical specimens showed a significant association between the expression of EZH2 and H3K27me3 levels, and that the H3K27me3 levels function as a prognostic marker for survival. Interestingly, the aberrant overexpression of EZH1 and decreased expression of UTX were observed in some HCC cases with high levels of H3K27me3 unexpectedly showing low EZH2 expression (EZH2 low H3K27me3 high ). Taken together, the results show that not only EZH2 but also EZH1 plays an important role in the development and progression of HCC through the alteration of H3K27me3 levels.
Both GSK126 and UNC1999 inhibit S-Adenosyl methionine (SAM) competitively, thereby inhibiting SAM-dependent methyltransferases, including EZH1/2 25 . Given that the IC50 of EZH1 enzymatic inhibition differs between GSK126 (EZH1; 680 nmol/L and EZH2; 9.9 nmol/L) and UNC1999 (EZH1 45 nmol/L and EZH2 < 10 nmol/L), then UNC1999, but not GSK126, operates as a dual EZH1/2 inhibitor 19,20 . In the present study, UNC1999 inhibited cell growth and apoptosis induction in HCC cells at a low concentration compared with GSK126. Since RNA-seq analyses demonstrated that approximately 30% of the de-repressed genes following EZH1 and EZH2 knockdown are different, dual inhibition of EZH1 and EZH2 might be more potent than EZH2 inhibition alone.
It has been reported that aberrant histone modification is closely involved in drug resistance 26,27 . Enhanced H3K27me3 levels in the gene body of SLFN11, a DNA-damage repair gene, contribute to the resistance for etoposide in a patient-derived xenograft mouse model of small cell lung cancer. The concomitant use of EZH2 inhibitors has been found to prevent the emergence of acquired resistance, thus augmenting the chemotherapeutic efficacy 28 . Moreover, decreased phosphorylation of receptor tyrosine kinase by EZH2 inhibition results in the recovery of sunitinib anti-tumor effects in clear cell renal cell carcinoma 29 . Based on this knowledge, we analyzed the alterations in H3K27me3 level in HCC cells after exposures to sorafenib. Unexpectedly, sorafenib treatment induced an increase in H3K27me3 levels without remarkable changes in EZH1/2 expression.
Recently, there has been increasing evidence that the phosphorylation of various amino acid residue sites in EZH2 affect its methyltransferase activity 30 . These modifications occasionally cause the activation or suppression www.nature.com/scientificreports/ of enzymatic activity due to cell types and conditions. Among them, the phosphorylation of EZH2 at threonine 487 (Thr487) by CDK1/2, at Thr311 by AMPK, and at Ser21 by Akt, all inhibit the methyltransferase activity of EZH2 [31][32][33] . Given that an increase in H3K27me3 levels caused by sorafenib treatment was accompanied by a decrease in the levels of phospho-Akt, we then examined the EZH2 phosphorylation status at Ser21. Immunocytochemical analyses and Western blotting analyses demonstrated a remarkable decrease in the number of phopho-EZH2 (Ser21)-expressing cells. Considering that decreased mRNA expression of histone demethylase UTX was observed in HCC cells treated with sorafenib, these results show that Akt suppression following sorafenib treatment increases H3K27me3 level through decreased EZH2 phosphorylation at Ser21. Co-treatment with sorafenib with either GSK126 or UNC1999 canceled the enhancement in H3K27me3 levels. These drugs synergistically inhibited cell proliferation in vitro and tumor growth in xenograft mouse models. Additionally, immunohistochemical analyses of xenograft tumors in which sorafenib was administrated for 6 weeks, showed similar results to those observed in the Western blotting of cultured cells. Collectively, the results suggest the possibility that the machinery was associated with sorafenib resistance in HCC. Because the hepatocyte-specific EZH1/2 double knockout mice have been generated 34 , HCC mouse model using these mice might be a powerful tool to investigate the role of H3K27me3 in sorafenib treatment of HCC. Irrespective of these findings, the Ser21 phosphorylation of EZH2 by Akt promotes carcinogenesis through methylation of the androgen receptor (AR) or AR-related proteins 35 . Phosphorylated EZH2 at Ser21 was reported to activate STAT via its methylation, and to enhance the tumorigenic potential of glioma stem-like cells 36 . The role of phosphorylated EZH2 in the methylation of non-histone proteins would be further examined.
In conclusion, our study demonstrated that the combined use of sorafenib and EZH1/2 inhibitors exerted pronounced anti-tumor effects. Considering that the loss of PRC2 function was reported to cause RAS-MAPK signaling activation in peripheral nerve sheath tumors accompanied by NF1 mutations 37 , combined therapy with EZH1/2 inhibitors and MKIs might be reasonable. Given the many ongoing clinical trials of EZH2 and EZH1/2 inhibitors in a variety of cancers including HCC 38 , the combined use of epigenetic therapeutic agents, such as UNC1999, may prove useful to treat patients with advanced HCC.

Materials and methods
Reagents. Cell culture and sphere formation assay. Human HCC cell lines (Huh7: CVCL_0336, HuH1: CVCL-2956, and PLC/PRF/5: CVCL_0485) and hepatoblastoma-derived cell line (HepG2: CVCL_0027) were obtained from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum, 2 μM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and all experiments were performed with mycoplasma-free cells. For the sphere formation assay, 1000 cells were plated onto ultra-low attachment 6-well plates (Corning, Corning, NY). The number of spheres (> 100 μm in diameter) was counted following 14 days of culture.
In the limiting dilution assays, cells ranging from 5 to 50 cells/well were seeded into ultra-low attachment 96 well plates (Thermo Fisher Scientific, Waltham, MA). After 7 days of culture, the percentage of wells without spheres (loge, Y-axis) for each cell plating density was calculated and plotted against the number of cells plated per well (X-axis). To calculate the sphere formation ability, the regression lines were plotted. These analyses were performed using the Extreme Limiting Dilution Analysis software (http:// bioinf. wehi. edu. au/ softw are/ elda/).

Xenograft transplantation.
Six-week-old non-obese diabetic/severe combined immunodeficiency (NOD/SCID) male mice (Sankyo Laboratory Co. Ltd., Tsukuba, Japan) were bred and maintained according to our institutional guidelines for the use of laboratory animals. A total of 2 × 10 6 Huh7 cells were implanted into the subcutaneous space of the mice's backs. Sorafenib (5 mg/kg) was administrated daily by oral gavage, and UNC1999 (15 mg/kg) was administrated intraperitoneally 3 days a week for 6 weeks (n = 5 for each group). The treatment with sorafenib and/or UNC1999 was started the day after the cell transplantation. Subcutaneous tumors were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry with anti-EZH2, anti-phospho-EZH2 (Ser21), anti-phospho-Akt, anti-H3K27me3, anti-CASP3, and anti-Ki67 (DAKO, Carpinteria, CA) antibodies. TUNEL assay was also performed for the detection of apoptosis.
Patients and surgical specimens. Surgical specimens were collected from 72 patients who underwent surgical resection for HCC at the Chiba University hospital between August 2009 and August 2015. A total of 72 pairs of tumor and tumor-free liver tissue were histologically examined. Paraffin embedded tumor sections and the surrounding tumor-free tissues were examined by H&E staining and immunohistochemistry with anti-EZH2 and anti-H3K27me3 antibodies. To investigate the mechanisms underlying the discrepancy between EZH2 expression and H3K27me3 levels, mRNA expression of EZH1 and UTX in EZH2 low H3K27me3 high tumors was analyzed by RT-qPCR.
Statistical analysis. Data are presented as the mean ± standard error of the mean (SEM www.nature.com/scientificreports/ Ethics statement. All animal procedures were performed according to Chiba University guidelines for the use of laboratory animals and approved by the review board for animal experiments of Chiba University. Our in vivo study was carried out in accordance with ARRIVE guidelines (https:// arriv eguid elines. org). All patients provided informed consent and this study was approved by the Research Ethics Committees of the Graduate School of Medicine, Chiba University (approval number: 3024). We confirm that all procedures were performed in accordance with the relevant guidelines and regulations of the Declaration of Helsinki.