EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers
Ng IO (2013) EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1
in Human Cancers. PLoS ONE 8(6): e68226. doi:10.1371/journal.pone.0068226
EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers
Sandy Leung-Kuen Au 0
Carmen Chak-Lui Wong 0
Joyce Man-Fong Lee 0
Chun-Ming Wong 0
Irene Oi-Lin 0
William B. Coleman, University of North Carolina School of Medicine, United States of America
0 State Key Laboratory for Liver Research and Department of Pathology, Li Ka Shing Faculty of Medicine, the University of Hong Kong , Hong Kong , People's Republic of China
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 EZH2mediated 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 prometastatic role of EZH2 via repressing tumor and metastasis suppressors.
Funding: The study was supported by The University of Hong Kong Seed Funding Program for Basic Research (200811159061 and 201011159045 to
CMW), Hong Kong Research Grants Council General Research Fund (HKU 782411M to CMW) and Hong Kong Research Grants Council Collaborative
Research Fund (HKU 1/06C and HKU 7/CRG/09 to IN). IN is Loke Yew Professor in Pathology. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that co-author Chun-Ming Wong is a PLOS ONE Editorial Board member and this does not alter the authors'
adherence to all the PLOS ONE policies on sharing data and materials. All other authors declare no competing financial interests.
Deregulation of upstream epigenetic regulatory proteins
promotes epigenetic alterations and contributed to aberrant
silencing of tumor suppressor genes in human cancers .
Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of
Polycomb Repressive Complex 2 (PRC2), is one of the most
commonly up-regulated epigenetic regulators in different
human cancers . EZH2 is a histone methyltransferase that
specifically catalyzes histone H3 lysine 27 tri-methylation
(H3K27me3), which in turn acts as a repressive histone
modification to epigenetically control gene transcription [6,7].
Up-regulation of EZH2 plays a crucial role in malignant
progression and was implicated in cancer metastasis . EZH2
functions as an oncogene in different human cancers mainly
through epigenetic silencing of tumor and metastasis
suppressor genes, including E-cadherin , RUNX3 , SLIT2
, DAB2IP , and KLF2 . Recently, we have also
reported that EZH2 epigenetically inactivates expressions of
multiple tumor and metastasis suppressor microRNAs
(miRNAs), such as miR-125b and miR-139 in human
hepatocellular carcinoma (HCC), thereby promotes HCC
tumorigenicity and metastasis . Identifying novel targets
that are silenced by EZH2 will better reveal the molecular roles
Figure 1. Promoter DNA methylation and histone modification of DLC1 in HCC cell lines.
(A) Schematic diagram showing CpG island located at DLC1 locus. DNA methylation status of 45 CpG dinucleotides (BS-1 region
included 35 CpG dinulceotides and BS-2 region included 10 CpG dinucleotides) were subject to bisulfite sequencing analysis. MIHA
showed complete unmethylation, MHCC97L showed partial methylation and SMMC-7721 showed complete methylation. Open
circle represents unmethylated CpG dinucleotides and closed circle represents methylated CpG dinucleotides. Each column
represents a single clone being sequenced. (B) RT-PCR (upper panel) and qRT-PCR (bottom panel) showing detectable DLC1
mRNA expression in MIHA, but not in MHCC97L and SMMC-7721 cells. (C) Chromatin immunoprecipitation (ChIP) assay coupled
with qPCR (qChIP) analysis revealed the relative enrichment of H3K9me3 and H3K27me3 on DLC1 promoter region in MIHA,
MHCC97L and SMMC-7721 cells. Fold of enrichment of ChIP assay was calculated with reference to IgG control after normalized
with the input DNA. Data are represented as mean SEM from three independent experiments.
of EZH2 in cancer metastasis which will be beneficial to the
development of chemotherapies targeting EZH2.
DLC1 was identified as a bona fide tumor suppressor gene
on a recurrently deleted chromosomal region at chromosome
8p21 in HCC . DLC1 is a Rho GTPase-activating protein
(RhoGAP) localized at the focal adhesions [15,16], and is
specific for controlling the activity of RhoA, B, C and CDC42
[17,18]. The RhoGAP activity of DLC1 negatively regulates
these Rho proteins by stimulating their intrinsic GTP hydrolytic
activity, thus converts them from the active GTP-bound state to
the inactive GDP-bound state. The Rho signaling cascade
allows proper control of many biological processes such as cell
proliferation  and cell movement  in normal cells. During
malignancy development, the DLC1/Rho pathway is of
particular importance owing to its regulation on the actin
cytoskeleton related to cancer metastasis. We have shown that
loss of DLC1 in HCC activated RhoA, which subsequently
activated its downstream effector Rho kinase (ROCK) to
remodel the actin cytoskeleton network for cell migration and
invasion [21,22]. Other diverse tumor suppressive roles of
DLC1 include mediation of caspase-3-dependent apoptosis in
HCC model , inhibition of VEGF-dependent angiogenesis in
prostate cancer model , and suppression of clonogenicity in
several types of cancers . Down-regulation of DLC1 is
commonly shown in a wide range of human malignancies 
and its loss of expression is classically associated with
chromosomal deletion or promoter DNA hypermethylation
(Yuan et al 1998; Ng et al 2000; Wong et al 2003; Kim et al
2003). In this present study, we provided the first evidence that
DLC1 is a novel target of repression by EZH2-mediated
H3K27me3. We further showed inactivation of EZH2 by
3Deazaneplanocin A (DZNep) that re-expressed DLC1 and
remarkably abolished cytoskeletal reorganization and inhibited
cell migration in cancer cells. Collectively, our findings suggest
that epigenetic silencing of DLC1 is involved in the
prometastatic function of EZH2 in human cancers.
Materials and Methods
SMMC-7721 cell line (Shanghai Institute of Cell Biology) and
Huh 7 cell line (Japanese Cancer Research Bank) were
maintained in Dulbeccos modified Eagles medium
(DMEM)high glucose. MHCC97L cell line (gift from Prof. Z.Y. Tang of
Fudan University, Shanghai)  and MIHA cell line (Shanghai
Institute of Cell Biology) were maintained in DMEM-high
glucose supplemented with sodium pyruvate. HeLa cell line
(American Type Culture Collection) was maintained in
DMEMlow glucose. CNE2 (American Type Culture Collection) and
HCT116 (gift from Prof. B. Vogelstein, Johns Hopkins
University School of Medicine, Baltimore, MD)  cell lines
were maintained in Roswell Park Memorial Institute 1640
medium. All medium was supplemented with 10% fetal bovine
serum and 100 units/ml penicillin and streptomycin.
Bisulfite modification and methylation analysis
Bisulfite modification of genomic DNA extracted from cell
lines was performed using EZ DNA Methylation-Direct Kit
(ZYMO Research, Orange, CA, USA) according to
manufacturers instruction. Primers used for amplification of
DLC1 promoter after bisulfite modification are:
5Figure 2. EZH2-mediated H3K27me3 was involved in epigenetic repression of DLC1 in HCC and multiple other human
(A) DLC1 was transcriptionally induced upon stable knockdown of EZH2 in MHCC97L cells. (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. (C) DLC1 was transcriptionally repressed in Huh7 cells after transient overexpression of
GFP-EZH2. (D) qChIP analysis revealed a concomitant enrichment of EZH2 at DLC1s promoter locus upon GFP-EZH2
overexpression in Huh7 cells. Data are represented as mean SEM from three independent experiments. (E) EZH2 and
H3K27me3 expression was reduced upon 1M DZNep treatment for 48 hours in MHCC97L cells (left panel). DMSO was used as
mock treatment. Pan H3 and -tubulin were loading control of the immunoblot. DZNep treatment transcriptionally induced DLC1
expression in MHCC97L as indicated by qPCR analysis (right panel). (F) 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 10M DZNep reactivated DLC1
expression. P-values obtained from t-test.
GTTTTTAGTTAGGATATGGT-3 (forward) and
5ACTTCTTTCTACACATCAAACAC-3 (reverse) for BS-1
region ; and 5-TAGAGTTATTAAGAAAAAGAAGGGA-3
(forward) and 5-AAAACTAAAATATTTCCCCCAC-3 (reverse)
for BS-2 region. PCR product was cloned into TOPO TA
Cloning vector (Invitrogen, Carlsbad, CA) and sequencing of
individual clones was performed by Sanger sequencing.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using EZ ChIP kit (Millipore,
Billerica, MA, USA) as previously described . ChIP-grade
antibody against H3K9me3 (ab8898; Abcam, Cambridge, MA,
USA), H3K27me3 (07-449; Millipore, Billerica, MA, USA) and
EZH2 (#3147; Cell Signaling Technology, Danvers, MA, USA)
were used in the assay. Rabbit IgG (Millipore, Billerica, MA,
USA) or mouse IgG (Millipore, Billerica, MA, USA) was used as
negative control in the assay. Primers spanning -394nt to
-325nt upstream of DLC1 transcription start site were used:
5Figure 3. Inverse correlation between EZH2 and DLC1 expressions in human cancers.
(A) Twenty five-paired HCC samples were examined for EZH2 and DLC1 mRNA expression by qPCR. EZH2 was significantly
upregulated 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). P values from Wilcoxon matched pair test. (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 Ct (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). (C) EZH2 upregulation (left
panel) and concomitant DLC1 downregulation (right panel) was consistently observed in the tumorous tissues of different cancers,
including lung , breast , prostate  and ovarian  cancers. Expression data and P 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.
CACCTC CGCCAAGTAAATGC-3 (forward) and
5CCGAAAAGTCGCCAACTATTG-3 (reverse). Quantification of
antibody-bound region was done by qPCR performed with ABI
Prism 7700 (Applied Biosystems, Carlsbad, CA, USA).
In vitro epigenetic drugs treatment
DZNep (Cayman Chemical, Ann Arbor, MI, USA) was
dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St
Louis, MO, USA). 5-Aza-dC (Sigma Aldrich, St Louis, MO,
USA) was dissolved in 50% acetic acid. TSA (Sigma Aldrich, St
Louis, MO, USA) was dissolved in ethanol. The solvent of the
chemicals was used as mock in the corresponding treatment.
For DZNep treatment, DZNep (1M or 10M) was added to the
culture medium for 48 or 72 hours. For 5-Aza-dC treatment,
5Aza-dC (10 M) was replenished daily for 72 hours. For TSA
Cell migration assay
Prior to cell migration assay, 2x105 MHCC97L cells were
treated with 1M DZNep for 48 hours. Mock and
DZNeptreated cells (1x105) cells were seeded in the upper chamber of
8m-pore size Transwell insert (Millipore, Billerica, MA, USA).
The lower chamber contained conditioned medium collected
from untreated MHCC97L cells to attract cell migration for 18
hours. Migrated cells were fixed with methanol and stained with
crystal violet. Three independent views of the Transwell
membrane were photographed and number of migrated cells
Figure 4. DLC1 expression was synergistically restored upon DZNep, 5-Aza-dC and TSA treatment in MHCC97L but not
(A) Combinational epigenetic drug treatment in MHCC97L cells with 10 M 5-Aza-dC, 0.25 g/mL TSA and 10M 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) 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
Immunofluorescence (IF) microscopy and Scanning
electron microscopy (SEM)
Prior to IF imaging, 2x105 MHCC97L cells were treated with
1M DZNep for 48 hours. After the treatment, mock and
DZNep-treated cells (1x105) cells were trypsinized and seeded
on coverslips in DZNep-free culture medium for 24 hours. Cells
were fixed with 4% paraformaldehyde in PBS and
permeabilized with 0.2% Triton-X-100. Focal adhesions were
stained with anti-paxillin antibody (05-417; Millipore, Billerica,
MA, USA). F-actin was visualized by staining with
FITCconjugated phalloidin (Sigma Aldrich, St Louis, MO, USA).
Nuclei were counterstained with DAPI (Calbiochem, San
Diego, CA, USA). IF images were viewed and captured using a
Leica Q550CW fluorescence microscope (Leica, Wetzler,
Germany). For SEM, mock and DZNep-treated cells were fixed
with 1% osmium tetroxide and 2.5% glutaldehyde, followed by
stepwise ethanol dehydration. After the step of critical point dry,
slides were mounted on silver paste. Images were scanned
and captured under 2,000 magnification using a Hitachi
S-4800 FEG Scanning Electron Microscope.
Non-parametric data and continuous parametric data were
analyzed by Mann Whiteny U test and t test, respectively using
the GraphPad Prism version 5.00 (GraphPad Software, San
Diego California USA). Tests were considered significant when
the P value was less than 0.05.
H3K27me3 level is differentially enriched on DLC1
promoter in immortalized normal hepatocyte and HCC
To understand the role of epigenetic deregulation and DLC1
inactivation in HCC, we began with analyzing DLC1 promoter
methylation status by bisulfide sequencing in the immortalized
normal hepatocyte cell line MIHA and two HCC cell lines
SMMC-7721 and MHCC97L. DLC1 promoter was completely
unmethylated in MIHA, heterogeneously and partially
methylated in MHCC97L, and completely methylated in
SMMC-7721 (Figure 1A). The methylation status of DLC1 in
MIHA and SMMC-7721 correlated well with their DLC1
transcriptional level. But in MHCC97L, of which the DLC1
promoter was only partially methylated, the complete silencing
of DLC1 suggested that other epigenetic regulations may be
involved (Figure 1B). We therefore hypothesized that aberrant
histone methylation might contribute to DLC1 silencing in
MHCC97L. Chromatin immunoprecipitation (ChIP) assay was
performed to assess the enrichment of transcriptional
repressive histone modifications, H3K9me3 and H3K27me3,
Figure 5. Treatment of DZNep inhibited HCC cell migration through disruption of actin cytoskeleton.
(A) DZNep treatment effectively abolished MHCC97L cell migratory ability. Prior to cell migration assay, cells were treated with 1M
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. P-values obtained from t-test. (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. (C) 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).
on the DLC1 promoter (Figure 1C). We showed that H3K9me3
and H3K27me3 were not detectable in MIHA and this was
consistent with the transcriptionally active status of the DLC1
gene in this cell line. In contrast, enrichment of both
transcriptional repressive H3K27me3 and H3K9me3 was
detected at the DLC1 promoter of SMMC-7721 and MHCC97L
cells. Interestingly, H3K27me3 was more abundantly enriched
in MHCC97L, as compared with SMMC-7721. This observation
indicates that, in addition to the partial DNA methylation,
H3K27me3 also took part in the epigenetic silencing to
completely transcriptionally inactivate the DLC1 gene in
Suppression of EZH2 induced DLC1 reexpression in
different human cancer cell lines
To provide more evidence that EZH2-mediated H3K27me3
directly regulates DLC1, we investigated whether DLC1
expression could be restored upon the knockdown of EZH2.
HCC cell lines stably expressing non-target control (NTC) or
EZH2 targeting shRNA (shEZH2) were established in our
previous study . Upon stable knockdown of EZH2, DLC1
was transcriptionally induced in MHCC97L (Figure 2A). Loss of
H3K27me3 on the DLC1 promoter was observed in MHCC97L
shEZH2 cells (Figure 2B), indicating that EZH2-mediated
H3K27me3 contributes to the suppression of DLC1 in
MHCC97L. In DLC1-expressing Huh7 cells, transient
overexpression of GFP-EZH2 transcriptionally repressed DLC1
and a concomitant enrichment of EZH2 was detected on the
DLC1 promoter (Figure 2C and D Supplementary Figure S1).
In line with the EZH2 knockdown model, we further showed
that treatment of 3-Deazaneplanocin A (DZNep), a small
molecule EZH2 inhibitor [29,30], significantly reduced the EZH2
protein level and H3K27me3 level and induced DLC1
reexpression in MHCC97L cells (Figure 2E). More importantly,
we found that EZH2-mediated DLC1 epigenetic silencing was
not restricted to HCC. Re-expression of DLC1 upon DZNep
treatment was also observed in different cancer cell lines
including the nasopharyngeal carcinoma cell line CNE2,
colorectal carcinoma cell line HCT116 and cervical
adenocarcinoma cell line HeLa (Figure 2F). The above findings
indicated that epigenetic silencing of DLC1 by EZH2-mediated
H3K27me3 is a common mechanism shared by different
EZH2 and DLC1 expression levels are inversely
correlated in human cancer tissues
To provide clinical relevance of our in vitro observations, we
examined DLC1 mRNA expression in 25-paired primary HCC
samples and correlated the expression level with our previous
EZH2 mRNA expression data . In this cohort of samples,
EZH2 was significantly upregulated while DLC1 was
significantly downregulated in the tumorous tissues than the
non-tumorous tissues (Figure 3A). Interestingly, a significant
inverse correlation between EZH2 and DLC1 was observed in
a subset of HCC samples, in which DLC1 promoter was not
silenced by DNA methylation  (Figure 3B). We also
extended our analysis to other human cancers by retrieving the
microarray gene expression data of lung , breast ,
prostate , and ovarian  cancers from Oncomine
(www.oncomine.org) (Figure 3C). Consistent with the findings
from human HCC, concomitant DLC1 down-regulation and
EZH2 up-regulation was observed in these malignancies,
suggesting the implications of EZH2 up-regulation in silencing
DLC1 expression in different human cancers.
DLC1 is synergistically silenced by H3K27me3 only
when its promoter DNA is partially methylated
Since multiple epigenetic repressive machineries are
intimately linked to establish a less permissive chromatin
environment to suppress gene transcription [36,37], we sought
to demonstrate the combinational effect of different epigenetic
machineries in controlling DLC1 expression. MHCC97L and
SMMC-7721 cells were treated with DZNep together with
5Aza-2deoxycytidine (5-Aza-dC) and Trichostatin A (TSA),
which are well characterized DNA methylation and histone
acetylation inhibitors, respectively. While treatment of DZNep,
5-Aza-dC and TSA individually elevated expression DLC1,
combined treatment of three drugs synergistically restored
DLC1 expression in MHCC97L cells (Figure 4A). However, the
co-treatment in SMMC-7721 cells did not show further increase
in DLC1 expression as compared to 5-Aza-dC alone (Figure
4B), implying that DNA methylation was a predominant
epigenetic mechanism for silencing DLC1 in this cell line.
DZNep treatment disrupts actin cytoskeleton to inhibit
HCC cell migration
DLC1/Rho/ROCK pathway is crucial for regulating proper
cytoskeleton remodeling and cell motility. Our proceeding
findings suggest that EZH2 is involved in suppressing DLC1,
we therefore further tested whether DZNep treatment could
suppress in vitro HCC migration. DZNep treatment at 1M for
48 hours, which showed minimal cytotoxic effect
(Supplementary Figure S2), effectively inhibited cell migration
in MHCC97L cells as demonstrated by Transwell migration
assay (Figure 5A). DZNep treated cells also showed
abolishment of proper actin cytoskeleton network when
examined under scanning electron microscope (Figure 5B) and
immunofluorescence staining (Figure 5C). These cell
morphological alterations induced by treatment with DZNep
highly resembled those of DLC1-induced cell cytoskeletal
collapse and cell shrinkage , implying that DZNep may
inhibit cell migration via interfering DLC1/ROCK pathway and
caused substantial actin cytoskeleton disorganization.
One important pro-metastatic role of EZH2 in cancer is via
epigenetic silencing of tumor and metastasis suppressor genes
. H3K27me3 is the best characterized EZH2-mediated
histone modification in the mammalian epigenetic system. In
the present study, we provide evidence that EZH2-mediated
H3K27me3 is involved in the epigenetic repression of DLC1
during HCC development. Interestingly, from several published
genome-wide data of Polycomb group (PcG) target gene
mapping in human embryonic stem (hES) cells, we also
noticed that DLC1 is a candidate PcG-regulated gene in early
developmental stage (Supplementary Figure S3). In hES cells,
the DLC1 promoter is occupied by SUZ12, which is associated
with EZH2 to form the PRC2 in mediating H3K27me3 and
transcription repression . In addition, the DLC1 promoter is
also marked with H3K27me3  and H3K4me3 , which
constitute the bivalent chromatin signature of PcG targets .
This bivalent chromatin signature, involving the
H3K27me3silencing and H3K4me3-activating histone modifications, allows
genes of developmental importance and genes required for
stemness maintenance to be poised in a ready-to-transcribe
state until differentiation [41,42]. DLC1 is essential for
embryonic development  and possibly has a role in lineage
specification . DLC1 knockout mice is embryonic lethal .
However, DLC1 is ubiquitously transcribed in adult tissues ,
implying that PcG silencing effect has been relieved in
differentiated somatic cells. Upon oncogenesis, it is possible
that DLC1 regains its embryonic PcG marking as a result of
EZH2 up-regulation. At the molecular level, we demonstrated
that DLC1 was co-regulated by EZH2-mediated H3K27me3,
DNA methylation and histone deacetylation in HCC cells.
However, the co-regulation between the three epigenetic
mechanisms on DLC1 was only observed when its promoter
was partially methylated (such as in MHCC97L cells). Once
DLC1 promoter was densely methylated (such as in
SMMC-7721 cells), H3K27me3 was absent and DNA
methylation was the key repressive mechanism with modest
participation of histone deacetylation. This observation may
reflect the crosstalk of EZH2 silencing and DNA methylation in
an earlier stage of epigenetic repression of tumor suppressor
genes before the eventual replacement of H3K27me3 silencing
by the stable DNA methylation machinery [45,46]. Indeed, our
findings that DLC1 is a PcG-regulated target and its
subsequent epigenetic silencing during malignancy progression
are also in line with the recently proposed model on PcG
marking of genes in ES cells are predisposed to de novo
cancer specific DNA methylation [45,46].
Pharmacologic targeting of deregulated epigenetic proteins
emerges as an attractive approach in cancer therapy [47,48].
DZNep has been shown to eradicate tumor-initiating HCC cells
in nude mice implanted with HCC , induce apoptosis in
breast cancer cells  and target human acute myeloid
leukemia in mice model when used in combination with
panobinostat . However, the metastasis suppressive effect
of DZNep has never been evaluated. Our findings indicate that
DZNep inhibit EZH2 expression and may further function as a
potent metastasis inhibitor through disrupting actin
cytoskeleton organization. A detailed pharmacological
characterization of DZNep to suppress HCC tumor growth and
progression in vivo is still warranted to investigate the
therapeutic potential of DZNep as cancer treatment regimen.
We have previously showed that EHZ2 promotes HCC
metastasis in part through epigenetic repression of multiple
Rho/ROCK signaling-targeting miRNAs . For example,
miR-139 that targets ROCK2  was frequently
downregulated in human HCC by EZH2-mediated H3K27me3 .
In this study, we additionally demonstrated that DLC1, the key
up-stream regulator of Rho/ROCK pathway, is also silenced by
EZH2. Altogether, our findings unravel a tight and multilayered
regulation of DLC1/Rho/ROCK signaling by EZH2, which may
underpin a critical epigenetic driven event in promoting cancer
Figure S1. Transient overexpression of GFP-EZH2, as
confirmed by immunoblotting (left panal) and qPCR (right
panal), augmented the EZH2 and H3K27me3 levels in Huh-7
cells. Anti-EZH2 antibody (#3147, Cell Signaling Technology,
Danvers, MA, USA) detected both the endogenous and
GFPEZH2 fusion protein in the immunoblot. Protein and RNA were
harvested six-day post transfection.
Figure S3. Publicly available ChIP-sequencing and
ChIP-onchip database was analyzed to provide clues of DLC1
chromatin status in human embryonic stem cell. DLC1 locus
was found to be marked by H3K27me3 (Zhao et al., 2007) and
H3K4me3 (Pan et al., 2007) in independent studies.
Furthermore, DLC1 promoter was also found to be bound by
SUZ12 (Lee et al., 2006), which is a core component of the
Polycomb Repressive Complex 2.
Conceived and designed the experiments: SA CMW IN.
Performed the experiments: SA CCLW JL. Analyzed the data:
SA CMW. Contributed reagents/materials/analysis tools: CMW
IN. Wrote the manuscript: SA CMW IN.
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