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Keywords:

  • hepatocellular carcinoma;
  • cancer stem cell;
  • tumor-initiating cell;
  • EZH2;
  • DZNep

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Recent advances in stem cell biology have identified tumor-initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self-renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self-renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor-initiating HCC cells remains unclear. In this study, we conducted loss-of-function assay of EZH2 using short-hairpin RNA and pharmacological inhibition of EZH2 by an S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep). Both EZH2-knockdown and DZNep treatment impaired cell growth and anchorage-independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)+ tumor-initiating cells. Administration of 5-fluorouracil (5-FU) or DZNep suppressed the tumors by implanted HCC cells in non-obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5-FU predominantly reduced the number of EpCAM+ cells and diminished the self-renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor-initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor-initiating HCC cells.

Although first proposed approximately 50 years ago, the concept of cancer stem cells (CSCs) has drawn renewed attention from many oncologists in recent years.1 According to the concept, tumors consist of a minor component of tumorigenic cells and a major component of non-tumorigenic cells.2 The minor population, termed CSCs or tumor-initiating cells (TICs), organize a cellular hierarchy in a similar fashion to normal stem cell systems and exhibit pronounced tumorigenic activity in xenograft transplantation.3, 4 Recent progress in stem cell biology and technologies has facilitated the identification of TICs in a variety of cancers.5 We previously applied side population (SP) analysis and cell sorting to hepatocellular carcinoma (HCC) cell lines and successfully demonstrated that SP cells in HCC showed TIC-like properties both in culture and in an in vivo transplant model.6 Several surface molecules such as epithelial cell adhesion molecule (EpCAM), CD133, CD90, and CD13 have been reported as specific markers of TICs in HCC cells, although little is known about the molecular machinery operating in these cells.7–10 Accumulating evidence suggests that TICs could play a crucial role, not only in the development, but also in the recurrence of cancer, in part due to the resistance of TICs to anti-cancer therapy.11, 12 Therefore, understanding the molecular machinery operating in TICs is indispensable both for understanding the mechanism of carcinogenesis and for the establishment of novel therapies aimed at the eradication of these cells.

It seems likely that both normal and cancer stem cells share not only a number of surface marker phenotypes but also a variety of molecular mechanisms for self-renewal and differentiation. We and others previously reported that the polycomb-group (PcG) gene product Bmi1 plays a critical role in the self-renewal of a range of somatic stem cells including hepatic stem cells.13, 14 Of note, BMI1 is required for the maintenance of not only leukemic stem cells15 but also cancer stem cells in solid cancers such as HCC.16 PcG complexes are key regulators of epigenetic cellular memory. They establish and maintain cellular identities during embryogenesis, development, and tumorigenesis.17 They have also been implicated in the maintenance of embryonic and somatic stem cells.18 Biochemical and genetic studies have demonstrated that PcG complexes can be functionally separated into at least two distinct complexes; an initiation complex, polycomb repressive complex (PRC) 2, and a maintenance complex, PRC1. Human PRC2 contains three core components: EZH2, EED, and SUZ12. PRC2 possesses catalytic activity specific for trimethylation of histone H3 at lysine 27 (H3K27). In contrast, PRC1 contains four core components; RING1, BMI1, HPH, and CBX, and possesses E3 ubiquitin ligase activity that monoubiquitylates histone H2A at lysine 119. Recently, we demonstrated that loss of Ezh2 function severely impairs the self-renewal capacity of murine hepatic stem/progenitor cells and simultaneously promotes their differentiation towards the hepatocyte lineage.19 However, the role of EZH2 in tumor-initiating HCC cells remains unclear.

In this study, we conducted lentivirus-mediated knockdown of EZH2 and pharmacological disruption of EZH2 to evaluate the role of EZH2 in the maintenance of TICs in HCC. We further examined whether EZH2 depletion could contribute to the eradication of the tumor-initiating HCC cells using sphere culture assays and xenograft transplantation experiments.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mice

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Sankyo Laboratory Co. Ltd., Tsukuba, Japan) were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals.

Cell culture and reagents

The human hepatocellular carcinoma cell lines Huh1 and Huh7 were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) containing 10% FCS and 1% penicillin/streptomycin (Invitrogen). For the sphere formation assay, 1,000 cells were plated onto ultra low attachment six-well plates (Corning, Corning, NY). The number of spheres (>100 μm in diameter) was counted on day 14 of culture. For the secondary sphere formation, a single cell suspension derived from original colonies was obtained using a Neurocult chemical dissociation kit (StemCell Technologies, Vancouver, BC). For the pharmacological disruption of EZH2, 3-deazaneplanocin A (DZNep) was chemically synthesized by Chemgenesis Inc. (Tokyo, Japan). Cells were treated with DZNep (1 or 10 μM) or 5-fluorouracil (5-FU) (1 or 10 μM; Sigma-Aldrich, St Louis, MO). Likewise, the cells were treated with a G9a histone methyltransferase (HMT) inhibitor BIX01294 (1 or 10 μM) (Sigma).

Viral production and transduction

Lentiviral vectors (CS-H1-shRNA-EF-1α-EGFP) expressing short hairpin RNA (shRNA) that targets human EZH2 (target sequence: sh-EZH2-1, 5′-GGAAAGAACGGAAATCTTA -3′; sh-EZH2-2, 5′-GGATAGAGAATGTGGGTTT-3′) and luciferase (Luc) were constructed. Flag-tagged mouse Ezh2 cDNA was cloned into a site upstream of IRES-enhanced green fluorescent protein (EGFP) in the pMCs-IG retroviral vector. Recombinant lentiviruses and retroviruses were produced as described elsewhere.13, 14

Cell sorting and analysis

Single-cell suspensions were stained with allophycocyanin (APC)-conjugated anti-EpCAM antibody (Biolegend, San Diego, CA) or APC-conjugated anti-CD133/1 antibody (Miltenyi Biotec, Auburn, CA). After the incubation, 1 μg/ml of propidium iodide was added to eliminate dead cells. Flow cytometirc cell sorting and analyses were performed using FACSAria (BD Biosciences, San Jose, CA). For the transplantation assay, EpCAM+ cells and CD133+ cells were purified with magnetic activated cell sorting (MACS) columns (Miltenyi Biotech).

Xenograft transplantation using NOD/SCID mice

A total of 2 × 106 EpCAM+ or CD133+ Huh7 cells stably expressing shRNA against EZH2 or luciferase were suspended in DMEM and Matrigel (BD) (1:1). The EZH2 knockdown and control cells were implanted into the subcutaneous space on the right and left sides of the backs of recipient NOD/SCID mice, respectively. In the DZNep or 5-FU treatment model, a total of 2 × 106 Huh7 cells were implanted into the subcutaneous space of the backs of NOD/SCID mice. DZNep (1 or 5 mg/Kg) and 5-FU (50 or 100 mg/Kg) was administered intraperitoneally twice per week and every 2 weeks, respectively. Tumor formation and growth were observed weekly. For the analyses of xenograft tumors, subcutaneous tumors were removed and minced in sterile PBS on ice. The small pieces of tumors were put in DMEM containing 5 mg/ml collagenase type II (Roche) and digested. The cell suspension was centrifuged on Ficoll (IBL, Gunma, Japan) to remove dead cells and debris. Harvested cells were subjected to flow cytometric analyses and sphere formation assays. These experiments were performed in accordance with the institutional guidelines for the use of laboratory animals.

Statistical analysis

Data are presented as the mean ± SEM. Statistical differences between 2 groups were analyzed using the Mann-Whitney U test. p values less than 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Stable knockdown of EZH2 in HCC cells

To investigate the role of EZH2 in HCC cells, we first examined the basal expression of EZH2 in the Huh1 and Huh7 HCC cell lines. Immunocytochemical analyses demonstrated that EZH2, as well as the PRC1 protein BMI1, were highly expressed in the nuclei of both cell lines (Fig. 1a). We next conducted loss-of-function analyses of EZH2 in vitro. We achieved the stable knockdown of EZH2 in Huh1 and Huh7 cells with lentivirus-mediated shRNA against EZH2 using EGFP as a marker for infection. A lentiviral vector expressing shRNA against luciferase was used as a control. Two different shRNAs, sh-EZH2-1 and sh-EZH2-2, both markedly repressed EZH2 protein expression and inhibited the growth of both cell lines (Fig. 1b and 1c). Because knockdown of EZH2 and growth inhibition were more prominent with sh-EZH2-2 than with sh-EZH2-1, we used sh-EZH2-2 for most of the subsequent experiments. Apoptotic cell death, assessed using an anti-CASP3 antibody, was increased by EZH2 knockdown in Huh1 and Huh7 cells approximately fourfold compared to the corresponding control cells (Supporting Information Fig. 1).

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Figure 1. Basal expression and knockdown of EZH2 in HCC cells. (a) Immunocytochemical analyses of EZH2 (green) and BMI1 (red) expression in Huh1 and Huh7 cells. Nuclear DAPI staining (blue) is also shown. Scale bar = 200 μm. (b) Cells transduced with indicated lentiviruses were selected by cell sorting for EGFP expression, and subjected to Western blot analysis using anti-EZH2 and anti-tubulin (loading control) antibodies. (c) Inhibition of proliferation in EZH2 knockdown HCC cells. *Statistically significant (p < 0.05).

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Reduced tumorigenic activity in EZH2-knockdown HCC cells

To examine whether EZH2 knockdown affects the tumorigenic ability of HCC cells, we conducted a soft agar colony formation assay. Reduced colony formation was clearly observed in both cell lines following EZH2 knockdown (Fig. 2a). In addition, we performed a non-adherent sphere assay, a standard assay for evaluating the stem cell activity of both normal stem cells and CSCs. Consistent with the results of the soft agar assay, sphere-forming capacity was significantly impaired in EZH2 knockdown cells compared to the control cells (Fig. 2b and 2c). It has been documented that EpCAM+ cells and CD133+cells function as TICs in HCC cells, including Huh1 and Huh7 cells.7, 8 We then examined the expression of EpCAM in view of EZH2 expression using flow cytometry. Knockdown of EZH2 decreased the EpCAMhigh fraction from 55.5% to 18.7% in Huh1 cells and from 50.4% to 16.0% in Huh7 cells (Fig. 2d). Likewise, knockdown of EZH2 in Huh7 cells decreased the CD133high fraction from 40.8% to 14.3% (Supporting Information Fig. 2). In clear contrast, overexpression of Ezh2 promoted both cell growth and sphere formation in Huh7 cells moderately but significantly (Supporting Information Fig. 3a–3d). Correspondingly, flow cytometric analyses showed an increase in the EpCAMhigh and CD133high fractions (Supporting Information Fig. 3e). Together, these results indicate that EZH2 expression is strongly associated with the frequency of tumor-initiating HCC cells.

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Figure 2. In vitro assays of HCC cells with sh-EZH2-2-induced EZH2 knockdown. (a) Soft agar colony formation at day 21 of culture. (b) Non-adherent sphere formation assay at day 14 of culture. Bright-field (upper panels) and fluorescence (lower panels) images are shown. Scale bar = 100 μm. (c) Number of large spheres generated from 1,000 HCC cells transduced with indicated viruses. *Statistically significant (p < 0.05). (d) Flow cytometric analysis of EZH2-knockdown HCC cells. Flow cytometric profiles in Huh1 and Huh7 cells after the stable knockdown of EZH2. The percentages of EpCAMhigh fraction are shown as the mean values for three independent analyses.

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Impact of EZH2 depletion on tumor-initiating HCC cells

To confirm that EZH2 directly regulates a tumorigenic subpopulation, we purified the EpCAM+ tumor-initiating fraction from Huh1 and Huh7 cells by flow cytometry and conducted a non-adherent sphere assay. The sphere-forming ability of EpCAM+ cells was significantly impaired by EZH2 knockdown compared to that of the control cells (Supporting Information Fig. 4a). The secondary sphere number was also decreased by EZH2 knockdown, indicating that EZH2 plays an important role in the maintenance of self-renewal capability in TICs (Supporting Information Fig. 4b). Real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses of purified EpCAM+ Huh1 and Huh7 cells demonstrated that EZH2 knockdown induced the down-regulation of α-fetoprotein (AFP), a marker of the immature phase of hepatocytes. In clear contrast, various differentiation markers such as albumin (ALB) and cytochrome P450, subfamily 1, polypeptode 2 (CYP1A2), lipid metabolizing enzymes such as apolipoprotein C3 (APOC3), and enzymes involved in gluconeogenesis such as phosphoenolpyruvate carboxykinase (PEPCK) were upregulated to varying extents (Supporting Information Fig. 4c).

We next performed xenograft transplantations of sh-EZH2-2-expressing EpCAM+ cells using NOD/SCID mice (Supporting Information Fig. 4d). Prior to transplantation, the cells were purified by magnetic cell sorting and purity (>90%) was confirmed by flow cytometric analyses of EpCAM expression (data not shown). Importantly, and as expected, the implantation of 2 × 106EZH2-knockdown EpCAM+ cells resulted in delayed tumor development and slower tumor growth compared with sh-Luc expressing control cells (Supporting Information Fig. 4e). Taken together, these results imply that EZH2 depletion impairs the tumorigenicity of tumor-initiating HCC cells partially through the activation of differentiation programs.

Inhibited H3K27 trymethylation by EZH2 knockdown and DZNep treatment

Ezh2 is a histone methyltransferase and catalyzes the addition of methyl groups to H3K27. A S-adenosylhomocysteine hydrolase inhibitor, DZNep, has been reported to inhibit S-adenosylhomocysteine hydrolase and cause the retention of S-adenosylhomocysteine, thereby inhibiting S-adenosyl-L-methionine-dependent methyltransferases including EZH2. Although DZNep is not a specific inhibitor targeting EZH2, it efficiently inhibits EZH2 function.20 DZNep also reportedly depletes EZH2 protein.21 To examine H3K27me3 levels in EZH2-knockdown or DZNep-treated HCC cells, the cells were subjected to Western blotting. As expected, EZH2 knockdown resulted in reduced levels of H3K27me3 in both HCC cells (Fig. 3a). Similarly, DZNep-treated HCC cells showed a significant reduction in levels of EZH2 and H3K27me3 (Fig. 3b).

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Figure 3. Changes in the trimethylated H3K27 level after EZH2 depletion. (a) EZH2-knockdown cells were subjected to Western blot analysis using anti-H3K27 and anti-H3 (loading control) antibodies. (b) Cells treated with DZNep (10 μM) for 48 or 96 hr were subjected to Western blot analysis using anti-EZH2, anti- trimethylated H3K27, and anti-H3 antibodies.

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DZNep inhibits growth and sphere formation of HCC cells

We first examined the effect of 5-FU, a widely used anti-cancer agent, on HCC cells. 5-FU efficiently inhibited the growth of HCC cells in a dose-dependent manner (Supporting Information Fig. 5a). Nonetheless, the effect of 5-FU to suppress non-adherent sphere formation was relatively mild compared with its effect on cell growth (Supporting Information Fig. 5b). Importantly, 5-FU treatment rather increased the EpCAMhigh fraction in both Huh1 (55.9 to 83.5%) and Huh7 (45.3 to 79.1%) cells (Supporting Information Fig. 5c). Likewise, Huh7 cells treated with 5-FU showed an increase in the proportion of the CD133high fraction from 39.0 to 85.4% (Supporting Information Fig. 6a). These findings indicate that tumor-initiating HCC cells were resistant to 5-FU and greatly enriched after 5-FU treatment.

Next, we examined the effect of DZNep on HCC cells in vitro assays. DZNep treatment inhibited growth and non-adherent sphere formation in both cell lines in a dose-dependent manner (Fig. 4a and 4b). Flow cytometric analyses revealed that the DZNep (10 μM) treatment efficiently decreased the EpCAMhigh fraction from 49.0% to 12.5% in Huh1 cells and from 44.4% to 11.6% in Huh7 cells (Fig. 4c). Likewise, the CD133high fraction in Huh7 cells decreased from 37.2% to 9.4% after DZNep (10 μM) treatment (Supporting Information Fig. 6b). These results highlighted that the biological effect of DZNep is quite different from that of 5-FU.

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Figure 4. In vitro assays of HCC cells treated with DZNep. (a) Dose-dependent inhibition of proliferation in DZNep-treated HCC cells. *Statistically significant (p < 0.05). (b) Number of large spheres generated from 1,000 HCC cells at day 14 of culture. *Statistically significant (p < 0.05). (c) Flow cytometric profiles of HCC cells treated with DZNep (1 or 10 μM) for 144 hr. The percentages of EpCAMhigh fraction are shown as the mean values for three independent analyses.

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It has been shown that BIX01294 selectively inhibits G9a HMT activity and the generation of di-methylated H3K9.22 To examine whether G9a inhibitor exhibits similar effect to DZNep, we conducted in vitro assays of Huh7 cells treated with BIX01294. Although basal level of H3K9me2/3 was comparatively high in Huh7 cells, BIX01294 treatment apparently reduced the level of H3K9me2 but not H3K9me3 (Supporting Information Fig. 7a and 7b). BIX01294 inhibited the growth and sphere formation in a dose-dependent manner (Supporting Information Fig. 7c and 7d). Flow cytometric analyses showed that the BIX01294 (10 μM) treatment efficiently decreased the EpCAMhigh fraction as well as the CD133high fraction in Huh7 cells (Supporting Information Fig. 7e). Together, depletion of H3K9me2 by BIX01294 might exert biological effects similar to DZNep.

Effect of DZNep on tumor-initiating Huh7 cells

To evaluate directly the action of DZNep towards TICs, we purified EpCAM+ cells from Huh7 cells by cell sorting and conducted sphere formation assays. DZNep markedly impaired primary sphere formation and even more severely impaired secondary sphere formation (Fig. 5a and 5b). These results indicate that DZNep inhibits self-renewal of tumor-initiating HCC cells. The immunostaining of CASP-3 showed that DZNep treatment induced apoptotis dose-dependently (Fig. 5c). The percentage of apoptotic cells among EpCAM+ Huh7 cells treated with DZNep (10 μM) was approximately eight-fold higher than among control cells (Fig. 5d).

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Figure 5. Effect of DZNep in tumor-initiating EpCAM+ cells. (a) Bright-field images of non-adherent spheres on day 14 of culture. Scale bar = 100 μm. (b) Number of original spheres generated from 1,000 EpCAM+ cells at day 14 of culture and secondary spheres 14 days after replating. *Statistically significant (p < 0.05). (c) Detection of apoptotic cell death by immunostaining of active caspase-3 (CASP3). Scale bar = 200 μm. (d) Quantification of the percentage of apoptotic cells is indicated at the right. *Statistically significant (p < 0.05).

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Subsequently, we determined the ability of DZNep to eradicate TICs using xenograft NOD/SCID mouse models. After the implantation of 2 × 106 Huh7 cells into NOD/SCID mice, and either 5-FU (every 2 weeks) or DZNep (twice a week) was administered intraperitoneally to recipient mice. Tumor initiation and growth were apparently suppressed by both 5-FU and DZNep treatment in a dose-dependent manner. However, DZNep was more effective than 5-FU (Fig. 6a and b). Flow cytometric analyses of xenograft tumors showed that 5-FU treatment subsequently enriched tumor-initiating EpCAMhigh cells as observed in in vitro analyses (Fig. 6c). In clear contrast, DZNep administration resulted in a drastic decrease in tumor-initiating EpCAMhigh cells (Fig. 6c). We next purified EpCAM+ cells derived from xenograft tumors and performed sphere formation assays. EpCAM+ cells treated with DZNep gave rise to significantly fewer spheres in both primary and secondary cultures than those treated with 5-FU (Supporting Information Fig. 8a and 8b).

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Figure 6. Transplantation and reanalysis of xenograft tumors. (a) A total of 2 × 106 Huh7 cells were transplanted into the subcutaneous space of NOD/SCID mice. Growth of subcutaneous tumors (arrows) was apparently suppressed by 5-FU treatment in a dose-dependent manner 6 weeks after transplantation (left panel). Subcutaneous tumor volume was determined at 6 and 8 weeks after transplantation (right panel). *Statistically significant (p < 0.05). (b) A total of 2 × 106 Huh7 cells were transplanted into NOD/SCID mice. Tumor growth (arrows) was obviously suppressed by DZNep in a dose-dependent manner 6 weeks after the transplantation (left panel). Tumor volume was determined at 6 and 8 weeks after transplantation (right panel). *Statistically significant (p < 0.05). (c) Flow cytometric profiles of xenograft tumor cells treated with 5-FU or DZNep. The expression of EpCAM was assessed in H2K donor tumor cells. The percentages of EpCAMhigh fraction are shown as the mean values for three independent analyses. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Together, these results indicate that DZNep suppresses tumor growth by directly affecting the growth and self-renewal of TICs.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Accumulating evidence implies that the overexpression of EZH2 and deregulation of H3K27 methylation play an important role in a variety of cancers.23 For example, EZH2 expression is highly upregulated in prostate cancer, and frequent genomic loss of microRNA-101 targeting the EZH2 mRNA has been proposed as one mechanism responsible for the upregulation.24 We and others reported that the level of EZH2 expression is closely associated with the progression and prognosis of HCC.25, 26 All of these findings highlight the importance of EZH2 in hepatocarcinogenesis and implicate EZH2 in regulation of the self-renewal capacity of tumor-initiating HCC cells.

In this study, we first conducted the loss-of-function assays in non-purified Huh1 and Huh7 cells. Lentiviral knockdown of EZH2 significantly reduced both anchorage-independent colony formation and sphere formation by Huh1 and Huh7 cells in culture. Importantly, flow cytometric analysis showed a significant decrease in the percentage of EpCAMhigh cells following EZH2 knockdown. Furthermore, EpCAM+ cells purified from Huh1 and Huh7 cells exhibited reduced tumorigenicity in a xenograft transplantation assay when EZH2 was depleted. In clear contrast with the knockdown assays, overexpression of Ezh2 in Huh7 cells enhanced their sphere forming ability and increased the number of EpCAMhigh and CD133high cells. These results implicated that EZH2 directly regulates a tumor-initiating subpopulation.

Of interest, EZH2 knockdown cells also showed reduced expression of AFP, a hepatic stem/progenitor cell marker, and enhanced expression of various differentiation markers of hepatocytes. Analysis of transgenic mice in which Myc expression could be conditionally regulated revealed that multiple HCCs induced by overexpression of Myc lose their neoplastic properties and differentiate into hepatocytes and cholangiocytes upon inactivation of Myc, followed by a reduction in tumor volume and prolonged survival of the hosts.27 Overexpression of hepatocyte nuclear factor 4α, a well-known liver-enriched transcription factor, reportedly impairs the tumorigenic activity of HCC cells by promoting their differentiation.28 Given that the tumorigenicity of TICs is closely associated with their immature state in terms of differentiation, the induction of differentiation programs in TICs is a promising approach for targeting TICs.29, 30

It has recently been reported that the S-adenosylhomocysteine hydrolase inhibitor DZNep depletes cellular levels of PRC2 components including EZH2, SUZ12, and EED and selectively inhibits the trimethylation of H3K27.21 Intriguingly, DZNep more effectively induces apoptosis in transformed cells than normal cells.21 Moreover, DZNep treatment has been demonstrated to be effective in abrogating the self-renewal and tumorigenicity of glioblastoma TICs at levels comparative to EZH2 knockdown.31

As expected, our results showed that HCC cells treated with DZNep showed reduced levels of EZH2 and trimethylated H3K27. To elucidate whether DZNep has an inhibitory effect on tumor-initiating HCC cells, we performed in vitro assays and xenograft transplantation assays. Sphere formation assays showed that DZNep suppressed more severely the formation of spheres originated from HCC cells than did 5-FU treatment. Subsequent analyses for secondary sphere formation after replating showed similar results. These results indicate that DZNep directly affects the growth and self-renewal of tumor-initiating HCC cells. In addition, although both 5-FU and DZNep suppressed the growth of subcutaneous tumors in xenograft transplantation experiments, flow cytometric analyses of xenograft tumors clearly revealed that DZNep significantly reduced the number of tumor-initiating HCC cells, whereas 5-FU treatment inversely enriched these cells. Importantly, the effects of DZNep were augmented dose-dependently. Taken together, DZNep could be of therapeutic value for the eradication of TICs in HCC.

Transcriptional silencing of tumor suppressor genes by DNA methylation is frequently observed in a variety of cancer.32 It has been believed that a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC), inhibits the growth of cancer cells through the reactivation of these tumor suppressor genes, although 5-aza-dC has only shown limited efficacy against solid tumors. PcG-mediated trimethylation on H3K27 reportedly pre-marks genes for de novo methylation in colon cancer cells.33 Although DZNep or EZH2 knockdown is not effective in reactivating genes silenced by DNA methylation, it reactivates developmental genes not silenced by DNA methylation in cancer cells.20, 34 The manner of gene silencing might depend on the gene locus and cell-type. Further analysis is needed to understand the preference for DNA methylation or PcG-mediated histone modifications. Considering that the disruption of EZH2 contributes to the prevention of resilencing after the removal of 5-aza-dC,35 combined use of DZNep and 5-aza-dC might be of therapeutic benefit.

In conclusion, we have successfully demonstrated that both EZH2 knockdown and pharmacological ablation of EZH2 significantly reduced the number and tumorigenic potential of tumor-initiating HCC cells. This effect might be attributed to the impaired self-renewal capability of tumor-initiating HCC cells caused by interference with EZH2.

However, further analysis will definitely be necessary to determine the effort of EZH2 interference in primary tumor-initiating HCC cells. Although the exploration of potential therapies targeting TICs has just begun, compounds targeting PcG proteins such as EZH2 and HMT inhibitors might be of use for the eradication of TICs in HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Kristian Helin (University of Copenhagen) for the anti-EZH2 antibody and Dr. Toshio Kitamura (Tokyo University) for the pMCs-IG retroviral vector, and Drs. Yosuke Osawa and Atsushi Suetsugu (Gifu University) and Dr. Taro Yamashita (Kanazawa University), for valuable discussions.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_26264_sm_SuppTab1.doc45KSupporting Information Table 1. Primer sequences designed for real-time RT-PCR.
IJC_26264_sm_SuppFig1.tif7398KSupporting Information Figure 1. Detection of apoptotic cell death by immunostaining of active caspase-3 (CASP3). Fluorescence images of EGFP (upper panels), activated CASP3 (middle panels) and nuclear DAPI staining (lower panels) in Huh1 (a) and Huh7 (b) cells are shown. Quantification of the percentage of apoptotic cells is indicated at the right. *, Statistically significant (p<0.05). Scale bar = 200 μm.
IJC_26264_sm_SuppFig2.tif3028KSupporting Information Figure 2. Flow cytometric analysis of CD133 expression in EZH2-knockdown Huh7 cells. Flow cytometric profiles in Huh7 cells after the stable knockdown of EZH2. The percentages of CD133high fraction are shown as the mean values for three independent analyses.
IJC_26264_sm_SuppFig3.tif1514KSupporting Information Figure 3. In vitro assays of Ezh2-overexpressed HCC cells. (a) Huh7 cells transduced with Ezh2 were selected by cell sorting for EGFP expression, and subjected to Western blot analysis using anti-Flag, anti-Ezh2 and anti-tubulin (loading control) antibodies. (b) Cell proliferation in Huh7 cells transduced with Ezh2. *, Statistically significant (p<0.05). (c) Non-adherent sphere formation assay at day 14 of culture. Bright-field (upper panels) and fluorescence (lower panels) images are shown. Scale bar = 100 μm. (d) Number of large spheres generated from 1,000 Huh7 cells transduced with indicated viruses. *, Statistically significant (p<0.05). (e) Flow cytometric profiles in Huh7 cells after the stable overexpression of Ezh2. The percentages of EpCAMhigh and CD133high fractions are shown as the mean values for three independent analyses.
IJC_26264_sm_SuppFig4.tif1116KSupporting Information Figure 4. Impact of EZH2 knockdown in tumor-initiating HCC cells. (a) Number of large spheres generated from 1,000 EpCAM+ HCC cells transduced with indicated viruses. *, Statistically significant (p<0.05). (b) Number of secondary spheres at day 14 after replating. *, Statistically significant (p<0.05). (c) Real-time RT-PCR analyses of various differentiation markers of hepatocytes in EpCAM+ cells. The relative fold increase was calculated between the EZH2-knockdown cells and control cells. Abbreviations: AFP, α-fetoprotein; ALB, albumin; CYP, cytochrome P450; APOC3, apolipoprotein C3; PEPCK, phosphoenolpyruvate carboxykinase. (d) Transplantation of EZH2-knockdown EpCAM+ cells. A total of 2x106 EZH2-knockdown EpCAM+ Huh1 cells (left 3 mice) and EpCAM+ Huh7 cells (right 2 mice) were transplanted into the subcutaneous space of NOD/SCID mice. EZH2-knockdown cells developed apparently smaller tumors in the right subcutaneous space (arrows) than did control cells in the left space (arrowheads) 6 weeks after the transplantation. (e) Tumor volume was determined at 4 and 6 weeks after transplantation. *, Statistically significant (p<0.05).
IJC_26264_sm_SuppFig5.tif991KSupporting Information Figure 5. In vitro assays of HCC cells treated with 5-FU. (a) Dose-dependent inhibition of proliferation in 5-FU-treated HCC cells. *, Statistically significant (p<0.05). (b) Number of large spheres generated from 1,000 HCC cells at day 14 of culture. *, Statistically significant (p<0.05). (c) Flow cytometric profiles of HCC cells treated with 5-FU (10μg/ml) for 144 hours. The percentages of EpCAMhigh fraction are shown as the mean values for three independent analyses.
IJC_26264_sm_SuppFig6.tif530KSupporting Information Figure 6. Flow cytometric analysis of CD133 expression in 5-FU- or DZNep-treated Huh7 cells. (a) Flow cytometric profiles of Huh7 cells treated with 5-FU (10μg/ml) for 144 hours. The percentage of CD133high fraction is shown as the mean value for three independent analyses. (b) Flow cytometric profiles of Huh7 cells treated with DZNep (1 or 10μM) for 144 hours. The percentages of CD133high fraction are shown as the mean values for three independent analyses.
IJC_26264_sm_SuppFig7.tif1523KSupporting Information Figure 7. In vitro assays of HCC cells treated with BIX01294. (a) Immunocytochemical analyses of H3K9me2/3 (red) in Huh7 cells. Nuclear DAPI staining (blue) is also shown. Scale bar = 200 μm. (b) Huh7 cells treated with BIX01294 for 96 hours were subjected to Western blot analysis using anti-H3K9me2, anti-H3K9me3 and anti-H3 (loading control) antibodies. (c) Dose-dependent inhibition of proliferation in Huh7 cells treated with BIX01294 (1 or 10μM). *, Statistically significant (p<0.05). (d) Number of large spheres generated from 1,000 Huh7 cells treated with BIX01294 (1 or 10μM). *, Statistically significant (p<0.05). (e) Flow cytometric profiles of Huh7 cells treated with BIX01294 (10μM) for 144 hours. The percentages of EpCAMhigh and CD133high fractions are shown as the mean values for three independent analyses.
IJC_26264_sm_SuppFig8.tif392KSupporting Information Figure 8. Sphere formation assays of xenograft tumor cells. (a) Number of large spheres derived from 1,000 EpCAM+ tumor cells on day 14 of culture. *, Statistically significant (p<0.05). (b) Number of secondary spheres 14 days after replating. *, Statistically significant (p<0.05).
IJC_26264_sm_SuppInfo.doc72KSupporting Information

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