EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

<div><p>Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.</p> </div

Figure 5

<div><p><b>Treatment of DZNep inhibited HCC cell migration through disruption of actin cytoskeleton.</b></p> <p>(<b>A</b>) DZNep treatment effectively abolished MHCC97L cell migratory ability. Prior to cell migration assay, cells were treated with 1µM DZNep for 48 hours. Mock and DZNep-treated cells were then subject to cell migration assay using Transwell apparatus. Representative images of three independent experiments were shown. <i>P</i>-values obtained from <i>t</i>-test. (<b>B</b>) DZNep treatment suppressed formation of filopodia (red arrows) and lamellipodia (blue arrows) as illustrated by scanning electron microscopy. Images (2000x magnification) were captured using a Hitachi S-4800 FEG Scanning Electron Microscope. (<b>C</b>) DZNep treatment impaired actin cytoskeleton and caused cell shrinkage in MHCC97L cells. Stress fiber was stained with FITC-conjugated phalloidin and focal adhesions were stained with anti-paxillin antibody. Nuclei were counterstained with DAPI. Mock-treated cells showed organized bundles of stress fibers and well attached paxillin. DZNep-treated cells shrank and lost proper actin cytoskeleton network. Images (100x magnification) were captured by a Leica Q550CW fluorescence microscope (Leica, Wetzler, Germany).</p></div

Figure 4

<div><p><b>DLC1 expression was synergistically restored upon DZNep, 5-Aza-dC</b> and <b>TSA treatment in MHCC97L but not SMMC-7721 cells.</b></p> <p>(<b>A</b>) Combinational epigenetic drug treatment in MHCC97L cells with 10 µM 5-Aza-dC, 0.25 µg/mL TSA and 10µM DZNep induced the most robust DLC1 re-expression than any single or dual epigenetic drugs treatment. DZNep and 5-Aza-dC treatment were performed for 72 hours. TSA was either added to cells alone or with other drugs during the last 24 hours of the treatment. Data are represented as mean ± SEM from three independent experiments. (<b>B</b>) Addition of DZNep in SMMC-7721 cells did not further induce DLC1 re-expression when compared to 5-Aza-dC and TSA dual treatment. Data are represented as mean ± SEM from three independent experiments.</p></div

Figure 2

<div><p><b>EZH2-mediated H3K27me3 was involved in epigenetic repression of DLC1 in HCC</b> and <b>multiple other human cancers.</b></p> <p>(<b>A</b>) DLC1 was transcriptionally induced upon stable knockdown of EZH2 in MHCC97L cells. (<b>B</b>) qChIP analysis confirmed the depletion of H3K27me3 enrichment on DLC1 promoter upon EZH2 knockdown in MHCC97L cells. Data are represented as mean ± SEM from three independent experiments. (<b>C</b>) DLC1 was transcriptionally repressed in Huh7 cells after transient overexpression of GFP-EZH2. (<b>D</b>) qChIP analysis revealed a concomitant enrichment of EZH2 at DLC1’s promoter locus upon GFP-EZH2 overexpression in Huh7 cells. Data are represented as mean ± SEM from three independent experiments. (<b>E</b>) EZH2 and H3K27me3 expression was reduced upon 1µM DZNep treatment for 48 hours in MHCC97L cells (left panel). DMSO was used as mock treatment. Pan H3 and α-tubulin were loading control of the immunoblot. DZNep treatment transcriptionally induced DLC1 expression in MHCC97L as indicated by qPCR analysis (right panel). (<b>F</b>) Different human cancer cells, including the nasopharyngeal carcinoma cell line CNE2, the colorectal carcinoma cell line HCT116 and the cervical adenocarcinoma cell line HeLa were examined for DLC1 re-expression upon DZNep treatment. Treatment of cells with 10µM DZNep reactivated DLC1 expression. <i>P</i>-values obtained from <i>t</i>-test.</p></div

Figure 3

<div><p><b>Inverse correlation between EZH2</b> and <b>DLC1 expressions in human cancers.</b></p> <p>(<b>A</b>) Twenty five-paired HCC samples were examined for EZH2 and DLC1 mRNA expression by qPCR. EZH2 was significantly up-regulated in HCC tumorous (T) tissues than non-tumorous (NT) tissues (left panel). In the same sample cohort, DLC1 was significantly down-regulated in HCC as compared to NT tissues (right panel). <i>P</i> values from Wilcoxon matched pair test. (<b>B</b>) An inverse correlation was observed between EZH2 and DLC1 expression in a subset of HCC samples without DLC1 promoter methylation. Expression level of EZH2 and DLC1 in paired-HCC samples was represented by ΔΔCt (T_{(HPRT Ct –Gene of interest Ct)}-NT_{(HPRT Ct – Gene of interest Ct)}). Linear regression analysis was performed using GraphPad Prism5 (La Jolla, CA, USA). (<b>C</b>) EZH2 upregulation (left panel) and concomitant DLC1 downregulation (right panel) was consistently observed in the tumorous tissues of different cancers, including lung [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068226#B32" target="_blank">32</a>], breast [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068226#B33" target="_blank">33</a>], prostate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068226#B34" target="_blank">34</a>] and ovarian [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068226#B35" target="_blank">35</a>] cancers. Expression data and <i>P</i> values of DLC1 and EZH2 in multiple cancer types was obtained from Oncomine microarray database. Floating bars were shown to illustrate the minimum, median and maximum normalized expression units in non-tumorous (NT) and tumorous (T) tissues.</p></div

Hypoxia inducible factor-1 HIF-1 promotes myeloid-derived suppressor cell accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma

Transcriptome sequencing data HC

PKM2 promoted HCC growth <i>in</i><i>vitro</i> through regulating aerobic glycolysis and ROS levels.

<p>(A) Two stable PKM2 knockdown clones were generated in MHCC-97L and SMMC-7721 cells. Expression of PKM2, PKM1, and β actin were evaluated by Western Blots. (B) Knockdown of PKM2 by two independent sequences consistently reduced HCC cell proliferation rate by cell counting. (C) Knockdown of PKM2 reduced lactate accumulation in multiple HCC cell lines. (D) Colorimetric assay showed that knockdown of PKM2 reduced the glucose consumption rate of multiple HCC cell lines. (E) Glucose uptake in HCC cells was confirmed with 2-NBDG staining. (F) Knockdown of PKM2 increased ROS accumulation in multiple HCC cells. (G) Knockdown of PKM2 decreased NADPH level in SMMC-7721 cells. Values were normalized to NTC of the according cell lines. *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i><0.001 Student’s <i>t</i> test (n≧3).</p

PKM2 expression in human HCC.

<p>(A) mRNA expression of PKL, PKM1, and PKM2 in HCC and NT tissues. Values = 2^ΔΔCT, ΔΔCT = (CT_PK – CT_HPRT) of HCC - (CT_PK– CT_HPRT) of NT. <i>P</i> values, Wilcoxin signed rank test (B) Waterfall plot shows that, at the mRNA level, PKM2 was up-regulated (HCC/NT2 folds) in 29/60 (48.33%) human HCC samples. (C) Representative pictures of IHC staining with antibody against PKM2 in HCC tissue microarray. PKM2 protein was drastically up-regulated in human HCCs as compared to the paired NT tissues. (D) Mann Whitney test showed that PKM2 over-expression was associated with multiple aggressive clinicopathological features in HCC including the presence of tumor microsatellites, presence of venous invasion, and absence of tumor encapsulation. (E) Over-expression of PKM2 in human HCC was associated with poor prognosis. HCC patients were categorized into two groups: PKM2 over-expression and PKM2 normal/under-expression. PKM2 was considered to be over-expressed when HCC/NT2 folds and was considered to be normal/under-expressed otherwise. HCC patients with PKM2 over-expression had a higher 1-year tumor recurrence rate after surgical resection than HCC patients without PKM2 over-expression, 46.667% Vs 25%. (F) Patients with PKM2 over-expression had lower 5-year overall survival rates after surgical resection. <i>P</i> values were calculated by Kaplan-Meir log rank test.</p

PKM2 promoted HCC growth <i>in</i><i>vivo</i>.

<p>(A) Left: Subcutaneous tumors derived from SMMC-NTC, -shPKM2 cells. Middle: volumes (mm³) of SMMC-NTC and -shPKM2 tumors were measured and plotted against time. Right: mass (g) of SMMC-NTC, -shPKM2 tumors were measured at the end of the experiment. (**<i>P</i><0.01, Student’s t test) (B) Left: subcutaneous tumors derived from MHCC-97L-NTC and -shPKM2 cells. Middle: volumes (mm³) of MHCC-97L-NTC and -shPKM2 tumors were measured and plotted against time. Right: mass (g) of MHCC-97L-NTC and -shPKM2 tumors were measured at the end of the experiment. (C) Left: orthotopic tumors derived from MHCC-97L-NTC and -shPKM2 cells. Right: Tumor volume was measured at the end of the experiment. (D) Left: bioluminescent signals of the lung tissues in the mice orthotopically implanted with luciferase labeled-MHCC-97L-NTC and -shPKM2 cells. Right: mRNA expression of human hexokinase 2 (HK2) in lung tissues of mice orthotopically implanted with luciferase-labeled MHCC-97L-NTC and -shPKM2 cells. Values were normalized to mouse GAPDH. *<i>P</i><0.05, **<i>P</i><0.01,***<i>P</i><0.001, Student’s t test. Scale: 1 cm.</p

Re-expression of miR-122 suppressed HCC growth through modulating aerobic glycolysis.

<p>(A) miR-122 expression in MHCC-97L cells stably expressing miR-122 precursors. MiR-122 expression was normalized to U6 expression and to empty vector (EV) control. (B) Lactate accumulation was reduced in miR-122 over-expressing MHCC-97L cells. (C) Glucose uptake rate was reduced in miR-122 over-expressing MHCC-97L cells. (D) Glucose uptake in MHCC-97L-EV and –miR-122 cells was confirmed with 2-NBDG staining. (E) Glucose uptake in SMMC-EV and –miR-122 cells was confirmed with 2-NBDG staining. (F) Glucose uptake in PLC/PRF/5 cells transfected with LNA-Ctrl and LNA-miR-122. (G) Left: Orthotopic tumors derived from MHCC-97L-EV and -miR-122 subclones. Right: Tumor volume was measured at the end of the experiment. (H) Bioluminescence (left) and H&E staining (right) in lung tissues from mice implanted with MHCC-97L-EV and –miR-122 subclones. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001, Student’s t test or paired t test. Scale: 1 cm.</p