Regulation of Tumor Angiogenesis by EZH2
Chunhua Lu,1,20Hee Dong Han,1,20Lingegowda S. Mangala,1,20Rouba Ali-Fehmi,8Christopher S. Newton,9
Laurent Ozbun,9Guillermo N. Armaiz-Pena,1Wei Hu,1Rebecca L. Stone,1Adnan Munkarah,10Murali K. Ravoori,2
Mian M.K. Shahzad,1,11Jeong-Won Lee,1,12Edna Mora,1,13Robert R. Langley,3Amy R. Carroll,1Koji Matsuo,1
Whitney A. Spannuth,1Rosemarie Schmandt,1Nicholas B. Jennings,1Blake W. Goodman,1Robert B. Jaffe,14
Alpa M. Nick,1Hye Sun Kim,1,15Eylem Ozturk Guven,16Ya-Huey Chen,17Long-Yuan Li,17,18Ming-Chuan Hsu,4
Robert L. Coleman,1,5George A. Calin,5,6Emir B. Denkbas,16Jae Yun Lim,7Ju-Seog Lee,7Vikas Kundra,2
Michael J. Birrer,19Mien-Chie Hung,4,17Gabriel Lopez-Berestein,3,5,6and Anil K. Sood1,3,5,*
1Department of Gynecologic Oncology
2Department of Experimental Diagnostic Imaging
3Department of Cancer Biology
4Department of Molecular and Cellular Oncology
5Center for RNA and Non-Coding RNA
6Department of Experimental Therapeutics
7Department of Systems Biology
U.T. M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Unit 950, Houston, TX 77030, USA
8Department of Pathology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, MI 48201, USA
9Department of Cell and Cancer Biology, National Cancer Institute, Bethesda, MD 20892, USA
10Women’s Health Services, Henry Ford Health System, Detroit, MI 48202, USA
11Baylor College of Medicine, Department of Obstetrics and Gynecology, Houston, TX 77030, USA
12Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine,
Seoul 135-710, Korea
13Department of Surgery, University of Puerto Rico, San Juan, 00935, Puerto Rico
14Center for Reproductive Sciences, 505 Parnassus, University of California, San Francisco, CA 94143, USA
15Department of Pathology, Cheil General Hospital and Women’s Healthcare Center, Kwandong University College of Medicine, Seoul
16Hacettepe University, Nanotechnology and Nanomedicine Division, Ankara 06532, Turkey
17Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University and Hospital,
Taichung 40447, Taiwan
18Department of Biotechnology, Asia University, Taichung 41354, Taiwan
19Department of Medicine, Harvard Medical School, Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA
20These authors contributed equally to this work
tional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in
tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result
of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohi-
bin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by
reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with
ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important
In this work, we identify EZH2 as a key regulator of tumor angiogenesis. The increase in endothelial EZH2 is a direct result of
VEGF stimulation and indicates the presence of a paracrine circuit that promotes angiogenesis. Ezh2 silencing in the tumor-
associated endothelial cells using siRNA, packaged in the chitosan delivery system, resulted in significant growth inhibition
mediated by increased levels of the angiogenesis inhibitor, VASH1. Combined, these data provide a significant conceptual
advance in our understanding of the regulation of angiogenesis in ovarian carcinoma and support the potential for targeting
ezh2 as a therapeutic approach.
Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc. 185
Targeting the tumor vasculature is a particularly attractive
strategy because ofthe presumed genetic stability of endothelial
cells (Folkman, 1990). Anti-angiogenic therapeutic strategies are
predicted to be meritorious in ovarian cancer patients based on
tumor and endothelial VEGF overexpression, and response
characteristics noted in phase II clinical trials (Burger et al.,
2007; Jain et al., 2006; Spannuth et al., 2008). As such, seven
phase III clinical trials in primary and recurrent disease have
completed enrollment or are accruing. However, despite initial
progression. The mechanism for this acquired resistance is not
well described, but appears to be due in part to expansion or
expression of redundant (Relf et al., 1997) alterations in maturing
vasculature (Huang et al., 2004) and epigenetic mechanisms
(Kerbel, 2001a; Kerbel et al., 2001b); therefore, new anti-angio-
genesis targets are needed.
In search of such targets, we carried out genomic profiling
studies of endothelial cells from epithelial ovarian cancer and
from their normal counterparts (Lu et al., 2007a). Among the
regulators of gene expression and are involved in the stable
transmission of the repressive state of their target gene
throughout the cell cycle (Cavalli and Paro, 1998; Kingston
comb repressive complex 2 (PRC2), has intrinsic histone methyl
transferase (HMTase) activity and has been implicated in the
progression and metastasis of several cancers (Cha et al.,
2005; Raman et al., 2005). For instance, normalized ezh2
mRNA transcripts were significantly associated with invasive-
ness of bladder tumors and were significantly higher in grade
tionships havebeendescribed inbreast(Kleeretal.,2003),pros-
tate (Varambally et al., 2002), gastric (Matsukawa et al., 2006),
and squamous cell cancers of the oral pharynx (Kidani et al.,
2009). The association of EZH2 with the malignant phenotype
of many solid tumors and its function as a repressor of gene
targets led to the hypothesis that EZH2 could impact specific
angiogenic mechanisms of endothelial cell biology. Herein, we
focused on EZH2 mediated regulation of vasohibin1 (VASH1),
which is an endothelial cell specific and intrinsic negative regu-
EZH2 Expression in Human Ovarian Carcinoma
We first examined the clinical significance of EZH2 in 180
epithelial ovarian cancers. Increased tumoral EZH2 (EZH2-T)
expression, based on the histochemical score, was noted in
66% of samples and increased expression in the vasculature
(EZH2-Endo) was noted in 67% of the samples (Figures 1A
and 1C). Increased expression of EZH2-T and EZH2-Endo was
significantly associated with high-stage (p < 0.001) and high-
grade (p < 0.05; Table 1) disease. Increased EZH2-T was signif-
icantly associated with decreased overall survival (median
2.5 years versus 7.33 years, p < 0.001; Figure 1B). Similarly,
EZH2-Endo was predictive of poor overall survival (2.33 versus
8.33 years, p < 0.001; Figure 1D). On the basis of pathway-anal-
ysis predictions from our genomic profiling data comparing
endothelial cells from epithelial ovarian cancer with those from
normal ovarian tissues (Lu et al., 2007a), we next examined
potential associations between EZH2 and VEGF expression
and microvessel density (MVD). Tumors with increased VEGF
expression had significantly greater prevalence of increased
EZH2-Endo expression (p < 0.001; Figures 1E and 1F). More-
over, tumors with increased EZH2-Endo expression had signifi-
cantly greater MVD (p < 0.001 by Wilcoxon ranked sums test;
Figures 1G and 1H).
VEGF Increases EZH2 Levels in Endothelial Cells
On the basis of our observations from clinical samples, we next
asked whether VEGF could directly regulate EZH2 levels in
endothelial cells. For these experiments, mouse ovarian endo-
thelial cells (MOEC) were cotransfected with the Renilla lucif-
erase plasmid and firefly luciferase plasmid either with or without
the ezh2 promoter construct. Cells were then treated with VEGF,
or conditioned media from SKOV3 ovarian cancer cells (SKOV3-
CM).There was a significant increase in ezh2promoter activity in
endothelial cells in response to VEGF, and conditioned media
(Figure 1I). To examine changes in EZH2 message, MOECs
were treated as indicated above and ezh2 mRNA was quantified
using real-time reverse transcription-polymerase chain reaction
(RT-PCR). Ezh2 mRNA expression levels were significantly
increased in endothelial cells in response to VEGF or SKOV3-
CM (Figure 1I). The increases in ezh2 promoter activity and
mRNA levels in response to SKOV3-CM or VEGF were blocked
with the VEGFR2 specific antibody DC101. Similarly, increased
EZH2 protein levels in response to VEGF were blocked by the
anti-VEGFR2 antibody (see Figure S1A available online).
Given that EZH2 levels were noted to be increased in tumor
and tumor-associated endothelial cells, we next asked whether
ovarian carcinoma. Orthogonal regression modeling between
these factors described a high coefficient of correlation
(r = 0.83, p < 0.001) (Figure S1B). To address whether tumor
derived VEGF affected endothelial EZH2, we utilized an
orthotopicmodel ofovariancancermetastasis.SKOV3ip1 tumor
bearing animals were treated with either control antibody or bev-
acizumab (selective for human VEGF). After treatment for
2 weeks, the tumors were harvested and tumor and endothelial
cells were isolated. Compared to normal endothelial cells, ezh2
levels were significantly higher in the tumor endothelial cells,
and this increase was substantially reduced in the bevacizumab
treated samples (Figure S1C). Conversely, bevacizumab had no
effect on tumor cell ezh2 levels (Figure S1D). To examine for
direct effects of EZH2 on endothelial cells, we transfected
EZH2 into MOEC, and examined for effects on tube formation
using in vitro assays. Compared to empty vector controls,
EZH2 promoted tube formation (p < 0.01), which was only mini-
mally blocked by a VEGFR2 inhibitor (Figure S1E). Similar results
were noted with HUVECs (data not shown).
Ezh2 Silencing Increases VASH1 in Endothelial Cells
To determine the mechanism by which EZH2 could promote
angiogenesis, we searched a database from a whole genome
EZH2 Effects on Angiogenesis
186 Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc.
Figure 1. EZH2 Expression in Human Ovarian Carcinoma
(A) Representative images of human tumors with low and high EZH2 expression based on immunohistochemical staining. The scale bar represents 50 mm.
(B) Kaplan-Meier curves of disease-specific mortality for patients whose ovarian tumors expressed high or low levels of EZH2 (EZH2-T). The log-rank test
(two-sided) wasusedtocomparedifferencesbetweenthetwogroups.IncreasedEZH2-Twassignificantly associatedwithdecreasedoverallsurvival(p<0.001).
(C) Representative images of human ovarian cancer vasculature (arrowheads point to endothelial cells) with low or high immunohistochemical staining for EZH2.
The scale bar represents 25 mm. Insets show blood vessels at higher magnification.
(D) Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EZH2 (EZH2-Endo). High EZH2-Endo
expression was predictive of poor overall survival.
(E) Representative images of human epithelial ovarian cancers with low or high immunohistochemical staining for VEGF. The scale bar represents 50 mm.
(F) VEGF expression was strongly associated with high EZH2-Endo expression levels.
(H) Differences in mean MVD based on EZH2-Endo expression levels in human epithelial ovarian cancers.
(I) VEGF increases ezh2 in endothelial cells. Results are in response to 6 hr treatment with VEGF (50 ng/mL), or conditioned medium (CM) from the noncancerous
ovarian epithelial cell line IOSE120, or the SKOV3 ovarian cancer cells. Fold changes represent the mean of triplicate experiments compared to untreated control
cells. *p < 0.01. Ezh2 promoter activity and mRNA levels are increased in mouse ovarian endothelial cells (MOEC) in response to VEGF, or conditioned media
from ovarian cancer cells. Error bars indicate SD. See also Figure S1.
EZH2 Effects on Angiogenesis
Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc. 187
ChIP-on-ChIP analysis that was performed in a separate study.
We found that an anti-angiogenic gene, vash1, directly binds to
ezh2. To validate this finding, we performed a ChIP assay of
or absence of VEGF (Figure 2A), which confirmed direct EZH2
binding to the vash1 promoter. Quantitative ChIP analysis
confirmed the enhanced binding of EZH2 to the vash1 promoter
in response to VEGF (Figure 2B). Next, we silenced the ezh2
gene inMOECs usingtwo
(Figure 2C), which resulted in a 3.6- to 3.8-fold increase in
vash1 (Figure 2D). Moreover, there was a significant increase in
vash1 promoter activity after ezh2 gene silencing (Figure S2).
To determine the mechanism by which EZH2 regulates vash1,
we performed methylation-specific PCR to detect vash1 methyl-
ation in endothelial cells in the presence of VEGF after silencing
ezh2. We found that VEGF treatment resulted in a 1.7-fold
increase in vash1 methylation compared to the controls
(Figure 2E). However, ezh2 silencing resulted in a 3.3-fold
decrease in VASH1 methylation in the VEGF-treated MOECs.
Ezh2 gene silencing also decreased histone 3 methylation at
lysine 27 by 2.5-fold in endothelial cells (Figure 2F).
different siRNA sequences
E2F Mediated Regulation of Ezh2 in Endothelial Cells
It is known that VEGF can activate E2F transcription factors
(Zhu et al., 2003). Due to their suspected role in regulating
EZH2 levels, we first tested the effect of VEGF on E2F1-5
in MOECs. There was a significant increase in e2f1 and e2f3,
Table 1. Association of Clinical and Demographic Features with
EZH2 in Epithelial Ovarian Carcinoma
EZH2-T OverexpressionEZH2-Endo Overexpression
No Yesp ValueNo Yesp Value
Mean Age 59.8 years (range 37–89 years)
Low (I/II)20920 9
High (III-IV) 41 108<0.001 37112<0.001
High52 1120.04847 117 0.005
Other22180.002 2218 <0.001
Figure 2. Ezh2 Gene Silencing Increases
vash1 mRNA Expression in Endothelial
(A) ChIP assay of EZH2 binding to vash1 promoter
in response to VEGF in mouse ovarian endothelial
cells (MOEC). Cross-linked chromatin from MOEC
was treated with (+) or without (?) VEGF and
immunoprecipitated (IP) using EZH2 or mouse
IgGantibodies. The input and immunoprecipitated
DNA were subjected to PCR using primers corre-
sponding to the 3800–3584 base pairs upstream
of vash1 transcription start site. PCR products
were examined on ethidium bromide-stained
(B) Quantitative ChIP assay of EZH2 binding to
VASH1 promoter in response to VEGF in endothe-
lial cells. Treatment conditions are similar to those
described in (A). PCR products were examined by
Roche SYBR Green System for quantitative PCR.
(C) MOECs were transfected with control or
mouse ezh2 siRNA (two different sequences) and
harvested after 72 hr. Untransfected (UT) cells
were used as controls. RNA was isolated and sub-
jected to real-time quantitative RT-PCR. The fold
difference represents the mean of triplicate exper-
iments compared to control siRNA treated cells.
*p < 0.01.
(D) Fold change in vash1 mRNA levels in MOEC
after transfection with either control or ezh2 siRNA
(two different sequences). *p < 0.01.
(E)Theeffect of ezh2 gene silencingon ezh2 meth-
ylation in VEGF-treated MOECs was detected by
methylation specific PCR. The inhibitory units of
methylated vash1 were normalized by that of the
unmethylated vash1 and represent the mean of
triplicate experiments. *p < 0.05.
(F) Western blot of lysate collected 48 hr after
transfection of MOEC with control, VEGF-treated,
and mouse EZH2 siRNA-treated cells. Error bars
indicate SEM. See also Figure S2.
EZH2 Effects on Angiogenesis
188 Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc.
but not others, after treatment with VEGF (Figure 3A). To
determine which e2f transcription factors might be responsible
for increasing ezh2 levels, we next examined the effects of
VEGF after silencing either e2f1 or e2f3. Ezh2 levels were sig-
nificantly decreased in e2f1 and e2f3 silenced cells (Figure 3B).
To validate the binding of ezh2 promoter to E2F1 and/or E2F3
transcription factors,weperformed ChIP assaysofezh2tothese
transcription factors. As shown in Figure 3C, E2F1 and E2F3
bind directly to the ezh2 promoter, demonstrating that ezh2 is
a direct target of the E2F transcription factors (Wu et al., 2010).
Moreover, the VEGF mediated binding of EZH2 to the vash1
promoter was abrogated with e2f1 or e2f3 gene silencing
To determine the functional role of VASH1 in angiogenesis, we
performed migration and tube formation assays using MOECs
after vash1 gene silencing using two different siRNA sequences.
Both cell migration and tube formation were significantly
increased after vash1 gene silencing (Figures S3A and S3B).
Similar results were obtained with HUVEC (data not shown).
In Vivo ezh2 Gene Silencing
On the basis of our in vitro findings, we next asked whether ezh2
gene silencing in vivo would affect tumor growth and angiogen-
esis. Before conducting the ezh2 targeted in vivo experiments,
we developed and characterized chitosan (CH) nanoparticles
for systemic delivery of siRNA into both tumor cells and tumor-
associated vasculature. Several formulations of CH with siRNA
(siRNA/CH) were tested (Figure S4A) and optimized (Figures
S4B–S4I) and the 3:1 ratio (CH/TPP) nanoparticles showed the
greatest (75%) incorporation efficiency (Figure S4D). Therefore,
for all subsequent experiments, we used the siRNA/CH3 nano-
particles due to their small size, slight positive charge, and
high incorporation efficiency of siRNA.
Prior to performing proof-of-concept in vivo efficacy studies,
we tested the efficiency of siRNA delivery into orthotopic ovarian
tumors. Nonsilencing siRNA labeled with Alexa-555 was incor-
porated into CH nanoparticles and injected intravenously (i.v.)
into mice bearing HeyA8 orthotopic tumors (17 days after intra-
peritoneal [i.p.] inoculation of tumor cells). Tumors were har-
vested at 15 hr and 3, 5, or 7 days (3 mice per time point) after
injection and examined for extent of siRNA delivery. At all time
points, punctated emissions of the siRNA were noted in the peri-
nuclear regions of individual cells. SiRNA was noted in >80% of
fields examined after a single intravenous injection. To confirm
delivery of siRNA in the vasculature, we also stained slides for
CD31. Indeed, siRNA was delivered into the tumor-associated
endothelial cells, suggesting potential applications for targeting
the tumor vasculature (Figure 4A). To confirm intracellular
delivery of siRNA, we created 3D reconstructions of the tumors
using confocal microscopy. Lateral views of the optical sections
clearly demonstrated the presence of siRNA within the tumor
cells (Figure 4B). However, very little siRNA was taken up by
macrophages as determined by labeling tissues with f4/80
(Figure S4J). To examine the biodistribution of siRNA into other
organs, we also examined sections of liver, lung, kidney, heart,
spleen, and brain, and detected siRNA delivery in most of these
organs (Figure S4K). We also utilized optical imaging to assess
biodistribution. Fluorescence corresponding to siRNA uptake
was seen in tumor and various organs, such as kidney, liver,
lung, and spleen (Figure 4C). Semiquantitative assessment of
fluorescence confirmed increased uptake of siRNA in HeyA8
tumors and various organs in mice injected i.v. with Cy
5.5 labeled siRNA/CH compared to those injected with unla-
beled siRNA/CH (Figure 4D).
To examine the in vivo effects of ezh2 gene silencing on tumor
growth, we used ezh2 siRNA directed to either the human (tumor
cells; ezh2 Hs siRNA/CH) or mouse (endothelial cells; ezh2 Mm
siRNA/CH) sequence. The expression levels of EZH2 in ovarian
cancer cells are shown in Figure S5A; the specificity of siRNA
was confirmed by testing each siRNA in both MOEC and
human tumor (HeyA8) cells (Figure S5B). After intravenous injec-
tion of either control siRNA/CH, ezh2 Hs siRNA/CH, ezh2 Mm
Figure 3. E2F-Mediated Regulation of ezh2 and vash1
(A) Expression levels of e2f transcription factors in mouse ovarian endothelial
cells (MOEC) after treatment with VEGF. *p < 0.01.
(B) Effect of VEGF and either control, e2f1, or e2f3 siRNA (two different
sequences) on ezh2 mRNA levels. The fold change in levels of mRNA expres-
sion represents the mean of triplicate experiments. *p < 0.01.
(C) Quantitative ChIP assay of E2F1 and E2F3 binding to ezh2 promoter in
response to VEGF in MOEC. Crosslinked chromatin from MOECs treated
with (+) or without (?) VEGF 50 ng/ml was immunoprecipitated using E2F1,
E2F3, or mouse IgG antibodies. The input and immunoprecipitated DNA
upstream of ezh2 transcription site. PCR products were examined by Roche
SYBR Green System for quantitative PCR.
siRNA and with (+) or without (?) VEGF 50 ng/ml was immunoprecipitated
using EZH2 or mouse IgG antibodies. The input and immunoprecipitated
DNA were subjected to PCR using primers corresponding to the 3800–3584
base pairs upstream of vash1 transcription site. PCR products were examined
by Roche SYBR Green System for quantitative PCR. Error bars indicate SEM.
See also Figure S3.
EZH2 Effects on Angiogenesis
Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc. 189
siRNA/CH, or the combination of ezh2 targeted siRNAs into
HeyA8 tumor-bearing mice (n = 3 mice per group at each time
point), tumors were harvested at different time points and exam-
ined for EZH2 protein levels. EZH2 levels were decreased by
24 hr after single injection of ezh2 Hs siRNA/CH with return of
expression to baseline expression levels after 96 hr (Figure 5A).
Ezh2 gene silencing was also confirmed with real-time RT-PCR
analysis of tumor and endothelial cells (Figures S5D and S5E).
To determine the localization of EZH2 silencing after siRNA/CH
for EZH2 and CD31. This experiment further demonstrated that
ezh2 Hs siRNA/CH resulted in ezh2 silencing in the tumor cells
whereas ezh2 Mm siRNA/CH silenced ezh2 only in the tumor
endothelial cells (Figure 5B).
To determine the therapeutic efficacy of ezh2 gene silencing,
we used a well-characterized orthotopic model of ovarian carci-
noma. Seven days after injection of tumor cells into the perito-
neal cavity, mice were randomly allocated to one of the following
groups (n=10 mice per group): (1) control siRNA/CH; (2) ezh2 Hs
siRNA/CH; (3) ezh2 Mm siRNA/CH; and (4) combination of ezh2
Hs siRNA/CH plus ezh2 Mm siRNA/CH. Mice were sacrificed
when animals appeared moribund due to significant tumor
burden (4–5 weeks after cell injection depending on the cell
line). As shown in Figure 5C and Figure S5C, in both models,
treatment with ezh2 Mm siRNA/CH resulted in a significant
decrease in tumor burden compared to control siRNA/CH
(62% reduction in HeyA8; p < 0.02 and 40% reduction in SKO-
V3ip1, p < 0.03). Ezh2 Hs siRNA/CH as a single-agent had
modest effects on tumor growth (p < 0.04 for HeyA8; and
p = 0.18 for SKOV3ip1) compared with control siRNA/CH.
However, the greatest reduction was observed with the combi-
nation of ezh2 Hs siRNA/CH plus ezh2 Mm siRNA/CH (83%
reduction in HeyA8, p < 0.001 and 65% reduction in SKOV3ip1,
p < 0.001). To test for potential off-target effects, we tested the
efficacy of three additional mouse ezh2 siRNA sequences with
similar effects on tumor growth (data not shown).
To evaluate the effects of EZH2 on other parameters of tumor
growth, we examined tumor incidence and number of nodules
(Table S1). The combination of ezh2 Hs siRNA/CH plus ezh2
Mm siRNA/CH resulted in a significant reduction in tumor
nodules in both HeyA8 (p = 0.002 versus control siRNA/CH
treated group) and SKOV3ip1 tumors (p = 0.004 versus control
siRNA/CH treated group). The decrease in tumor burden
occurred despite having comparable tumor incidence. The
mean mouse body weight was similar among the different
groups (data not shown), suggesting that feeding and drinking
habits were not affected.
Effect of ezh2 Targeting on Tumor Vasculature
To identify potential mechanisms underlying the efficacy of ezh2
silencing on ovarian tumors, we examined its effects on several
biological end points including MVD, pericyte coverage (DES-
MIN) and cell proliferation (PCNA). Ezh2 Mm siRNA/CH and the
combination therapy groups had significantly lower microvessel
density (Figure 6A) compared to the ezh2 Hs siRNA/CH and
control siRNA/CH treated SKOV3ip1 tumors. Pericyte coverage
(assessed with DESMIN and alpha smooth muscle actin [ASMA]
staining) was increased in ezh2 Mm siRNA/CH and the combina-
tion groups compared to the other two groups, suggesting
greater vascular maturation (Figure 6A and Figure S6B). Combi-
nation treatment with ezh2 Hs siRNA/CH and ezh2 Mm siRNA/
CH also resulted in a significant reduction in cell proliferation
(Figure S6C) and increased apoptosis (Figure S6C). Similar
Figure 4. In Vivo siRNA Delivery with Chitosan Nanoparticles
Distribution of siRNA after single intravenous injection of Alexa-555 siRNA/CH
nanoparticles in orthotopic HeyA8 tumor bearing nude mice.
(A) Fluorescent siRNA distribution in tumor tissue. Hematoxylin and eosin,
original magnification 2003 (left); tumor tissues were stained with anti-CD31
(green) antibody to detect endothelial cells (right). The scale bar represents
(B) Fluorescent siRNA distribution in tumor tissue. Sections (8 mm thick) were
stained with Sytox green and examined with confocal microscopy (scale bar
represents 20 mm) (left); lateral view (right). Photographs taken every 1 mm
were stackedand examined from the lateral view. Nuclei were labeled withSy-
tox green and fluorescent siRNA (red) was seen throughout the section. At all
time points, punctated emissions of the siRNA were noted in the perinuclear
regions of individual cells, and siRNA was seen in >80% of fields examined.
(C and D) Optical imaging of organs and tumors from HeyA8 tumor-bearing
mice treated with either Cy5.5 siRNA/CH or unlabeled siRNA/CH. (C) shows
fluorescence intensity overlaid on white light images of different mouseorgans
and tumor.(D) shows asemiquantitative evaluation of fluorescence intensity in
different mouse organs. Error bars indicate SD. *p < 0.05;**p < 0.01. See also
EZH2 Effects on Angiogenesis
190 Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc.
effects of ezh2 silencing on MVD, pericyte coverage, and prolif-
eration were noted in the HeyA8 model (Figure S6D). To test the
effect of ezh2 gene silencing on intratumor hypoxia, we
measured viable hypoxic areas by staining tumor sections with
Figure 5. Effects of ezh2 Gene Silencing on
In Vivo Ovarian Cancer Growth
(A) Western blot of lysates from orthotopic tumors
collected 24, 48, 72, and 96 hr after a single injec-
tion of control siRNA/CH or human (ezh2 Hs
(B) Ezh2 gene silencing in HeyA8 tumor as well as
tumor endothelial cells. Tumors collected after
48 hr of single injection of control siRNA/CH,
ezh2 Hs siRNA/CH, or ezh2 Mm siRNA/CH and
bar represents 50 mm.
CH on tumor weight inorthotopic mouse modelsof
ovarian cancer. Error bars indicate SEM. *p < 0.05;
**p < 0.001. See also Table S1 and Figure S5.
siRNA/CH treated tumors, ezh2 Mm
siRNA/CH treated tumors had modest
increase in hypoxia (Figures S6E and
S6F), which is consistent with effects of
increased after ezh2 gene silencing in
the tumor endothelial cells (Figure S6A).
VASH1 in mediating the antitumor effects
of ezh2 silencing, we examined the
effects of vash1 silencing in combination
with ezh2 Mm siRNA/CH. The antitumor
effect of ezh2 silencing in the tumor
vasculature was completely reversed by
vash1 silencing (Figure 6B; Table S2),
indicating that VASH1 is required for
mediating the antitumor effects of ezh2
EZH2 expression is related to VASH1
expression in human epithelial ovarian
samples for VASH1. The best-fit linear
regression model demonstrated a signifi-
cant inverse relationship (R2= ?0.59;
p < 0.001) between endothelial EZH2
and VASH1 scores (Figure 6C). Specifi-
cally, presence of high EZH2 expression
was associated with low VASH1 expres-
sion, which was otherwise elevated in
the absence of or in the presence of low
EZH2 expression. We also examined
mRNA levels of ezh2 and vash1 in endo-
thelial cells isolated from three normal
ovarian and ten epithelial ovarian cancer
Compared to normal ovarian endothelial
cells, vash1 levels were significantly lower in samples with high
versus low ezh2 levels (Figure 6D). To assess vessel maturation
(pericyte coverage), the 37 human ovarian cancer samples were
immunostained for ASMA. Tumors with low endothelial EZH2
EZH2 Effects on Angiogenesis
Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc. 191
Figure 6. Effect of ezh2 Targeting on Tumor Vasculature
(A) Effect of tumor (ezh2 Hs siRNA/CH) or endothelial (ezh2 Mm siRNA/CH) targeted ezh2 siRNA on microvessel density (MVD) and pericyte coverage. Tumors
harvested after 4–5 weeks of therapy were stained for CD31 (MVD; red) and DESMIN (pericyte coverage; green). The scale bar represents 50 mm. The bars in the
graphs correspond sequentially to the labeled columns of images at left. *p < 0.01; **p < 0.001.
(B) Effects of vash1 gene silencing on tumor growth in vivo. Nude mice injected with SKOV3ip1 ovarian cancer cells into the peritoneal cavity were randomly
divided into six groups (10 mice per group): (1) control siRNA/CH (control si); (2) ezh2 Mm siRNA1/CH (EZH2-1 si); (3) ezh2 Mm siRNA2/CH (EZH2-2 si); (4)
ezh2 Mm siRNA3/CH (ezh2-3 si); (5) vash1 Mm siRNA1/CH (vash1 si); and (6) combination of ezh2 Mm siRNA1/CH plus vash1 Mm siRNA/CH. *p < 0.05. MVD
is shown graphically in the adjacent graph.
(C) Endothelial VASH1 protein expression is plotted against endothelial EZH2 expression in 37 epithelial ovarian cancer specimens. The best-fit linear regression
model isdepictedwith95%confidence limits (R2=?0.59,p<0.001).Thelinearlinesintersecting with100oneachaxisrepresent predeterminedcut-off valuesof
‘‘high’’ versus ‘‘low’’ expression. The presence of EZH2 expression was associated with low expression of VASH1, which was otherwise elevated in the absence
of or in the presence of low EZH2 expression.
(D) Vash1 mRNA levels were measured in endothelial cells isolated from normal ovarian (n = 3), and epithelial ovarian cancer (n = 10) samples using quantitative
RT-PCR. The final mRNA levels in the tumor endothelial cells were converted to ratios of decreased (%1) or increased (>1) relative to levels of mRNA in normal
ovarian endothelial cells (*p < 0.01).
(E) Vessel maturation was examined by determining the extent of pericyte coverage in human epithelial ovarian cancer samples using an anti-ASMA antibody.
*p < 0.01. Error bars indicate SEM. See also Table S2 and Figure S6.
EZH2 Effects on Angiogenesis
192 Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc.
had a significantly higher percentage of blood vessels with peri-
cyte coverage (p < 0.01; Figure 6E).
To identify potential direct effects of ezh2 gene silencing on
tumor cells, we performed a series of in vitro assays. Ezh2 siRNA
reduced migration by 64% to 71% (p < 0.01), and invasion by
63% to 72% (p < 0.01) in the SKOV3ip1 cells (Figures S5F and
S5G). There were no significant effects on cell viability
(Figure S5H). Similar results were noted with the HeyA8 cells
(data not shown). To address the potential effects of tumor cell
ezh2 gene silencing on invasion in vivo, we injected SKOV3ip1
tumorcellsdirectlyinto theovaryandtheanimalswere randomly
allocated to the following groups (n = 10 mice per group): (1)
control siRNA/CH; and (2) ezh2 Hs siRNA/CH. After 4 weeks of
treatment, the mice were dissected and the aggregate tumor
weight was 30% lower (p = 0.03) in the ezh2 Hs siRNA/CH
treated animals. Although 50% of the mice in the control group
developed para-aortic lymph node metastasis, none had nodal
metastasis in the ezh2 Hs siRNA/CH group (p < 0.001). More-
over, the ovarian tumors in the control Hs siRNA/CH group had
a more infiltrative growth pattern compared to the ezh2 Hs
siRNA/CH group (Figure S5I). To identify genes potentially
affectedbyEZH2 inovarian cancer cells,weperformed genomic
analyses of SKOV3ip1 cells after treatment with either control or
decreased expression (Figure S5J). Among these, gene
networks of connective tissue growth factor (CTGF) were signif-
icantly reduced, which are known to regulate tumor cell migra-
tion and invasion (Figure S5K) (Chu et al., 2008; Cicha and
Our results describe a mechanism by which VEGF increases
EZH2 levels in the tumor vasculature. EZH2, in turn, contributes
to tumor angiogenesis by inactivating the antiangiogenic factor,
ysis predictions of genomic profiling data of tumor endothelial
cells (Lu et al., 2007a) expand our understanding of tumor angio-
genesis (Figure 7). Moreover, we have developed and character-
ized a highly effective method of gene silencing in tumor cells as
well as in the blood vessels that support their growth.
PcG proteins play a critical role in determining cell fate during
both normal and pathologic processes. Two separate subsets of
PcG complexes (PRC1 and PRC2) have been described in
humans (Cao and Zhang, 2004). PRC1 is thought to be involved
in maintenance of repression, whereas PRC2 plays a role in initi-
ating repression. The PRC2 complex consists of the EZH2, EED,
and SUZ proteins (Cao and Zhang, 2004). Altered expression of
these proteins has been implicated in cancer pathogenesis
(Raman et al., 2005; Cao and Zhang, 2004). However, prior to
our work, the role of EZH2 in angiogenesis was not known.
Angiogenesis is regulated by the balance of various proangio-
genic stimulators, such as VEGF, and several angiogenesis
inhibitors such as angiostatin, and endostatin (Folkman, 1990).
On the basis of findings from genomic profiling of endothelial
cells from ovarian cancer versus those from normal ovaries, we
discovered that EZH2 expression is significantly increased in
tumor-associated endothelial cells (Lu et al., 2007a). VEGF is
well recognized as a proangiogenic factor in ovarian and other
cancers (Spannuth et al., 2008), and its receptors are expressed
by endothelial and other cell types including tumor and perivas-
cular cells (Spannuth et al., 2009). In the current study, we
showed that VEGF can directly increase EZH2 levels in endothe-
lial cells, which in turn inactivates a potent antiangiogenic factor,
VASH1. Silencing ezh2 gene resulted in demethylation of vash1
in endothelial cells. This finding is consistent with the role of
EZH2 in controlling DNA methylation of EZH2-targeted genes
concomitant with reducing H3K27 (McGarvey et al., 2007).
VASH1 is known to inhibit endothelial-cell migration, prolifera-
tion, and tube formation (Shen et al., 2006). VASH1 expression
can be induced by VEGF as part of a negative feedback mecha-
nism in endothelial cells (Hosaka etal., 2009; Tamaki et al., 2009;
Watanabe et al., 2004; Yoshinaga et al., 2008). VASH1 limits
tumor-associated angiogenesis and increases vessel maturity
(Kern et al., 2009). Given that VASH1 levels are low in the setting
of high EZH2, vash1 siRNA alone had no significant effect on
tumor growth. However, vash1 levels in tumor endothelial cells
are increased in response to ezh2 gene silencing, and vash1
siRNA blocked the inhibitory effects of ezh2 gene silencing on
A number of therapeutic antiangiogenic targets have recently
emerged supporting the clinical rationale of exploiting tumor and
endothelial cell interactions. The most mature and promising
among ovarian cancer patients involves VEGF and its receptors.
For instance, bevacizumab, a chimeric monoclonal antibody
targeting VEGF-A has demonstrated impressive single agent
activity (objective response and nonprogression at 6 months)
in recurrent ovarian cancer patients and more recently, signifi-
cant efficacy (PFS) in combination with chemotherapy adminis-
tered in the frontline adjuvant setting (Burger et al., 2007; Burger
et al., 2010). However, this compound and others in its class in-
discriminately target VEGF, leading to a number of clinically
significant and sometimes lethal side effects (Stone et al.,
2010). In addition, new strategies targeting angiogenesis are
needed as the above therapies are rarely curative and a growing
population of patients are demonstrating resistance to anti-
VEGF agents. EZH2-targeting represents an innovative strategy,
which has activity on both tumor cells and tumor-associated
vasculature. The differential expression between normal and
tumor vasculature may be hypothesized to have less off-target
associated toxicity. Nevertheless, combinatorial approaches
with VEGF targeting may augment efficacy and are attractive
for further preclinical exploration.
Although a number of important targets in tumor and endothe-
lial cells have been identified, many of these are difficult to target
with small molecule inhibitors and monoclonal antibodies. This
limitation prompted us to use RNA interference as a means to
target ezh2. We have recently demonstrated that a neutral nano-
liposomal carrier allows efficient systemic delivery of siRNA into
orthotopic tumors (Landen et al., 2005; Thaker et al., 2006).
However, because of limited delivery of siRNA into the tumor-
associated endothelial cells with this approach, we sought to
into both tumor and tumor-associated endothelial cells. Chito-
san is desirable for biological applications due to properties
such as low immunogenicity and low toxicity (Kumar, 2000).
These properties make use of chitosan for systemic in vivo
siRNA delivery highly attractive (Howard et al., 2006). Indeed,
EZH2 Effects on Angiogenesis
Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc. 193
our data demonstrate highly effective delivery of siRNA incorpo-
rated into chitosan nanoparticles into both tumor and tumor-
associated endothelial cells. Such an approach may add
a powerful tool to the armamentarium for targeting angiogenesis
In summary, molecular and genetic manipulations have
identified EZH2 as a key regulator of tumor angiogenesis here,
but these effects do not rule out the possibility that EZH2 has
oncogenic functions in tumor cells (Cao and Zhang, 2004;
Raman et al., 2005). For example, EZH2 has been implicated in
cellulartransformation,proliferation, andavoidance ofapoptosis
(Tonini et al., 2008). However, to the extent that targeting tumor
endothelial cells provides therapeutic benefit (Burger et al.,
2007; Jain et al., 2006), interfering with EZH2 in the tumor and
endothelial cells might represent an important strategy for treat-
ment of ovarian and other cancers.
Cell Lines and Culture
The HeyA8 and SKOV3ip1 human epithelial ovarian cancer cells were main-
tained as described previously (Kamat et al., 2007; Lu et al., 2007b). The deri-
vation and characterization of the mouse ovarian endothelial cells (MOEC) has
been described previously (Langley et al., 2003). HUVECs were purchased
from Cambrex (Walkersville, MD) and maintained with heparin and genta-
micin/amphotericin-B, as previously described (Ptasinska et al., 2007).
Ezh2 Promoter Construct
The ezh2 promoter was amplified by PCR from the Roswell Park Cancer Insti-
tute human BAC library 11, Clone-ID RP11-992C19 purchased from the Chil-
dren’s Hospital Oakland Research Institute (Oakland, CA), and then cloned
into the pGL3-Basic Vector (Promega Corp., Madison, WI). The ezh2 promoter
dures) with XhoI and HindIII restriction endonuclease sites added to the ends.
Purified PCR product was then cloned upstream of the luc+ gene in the pGL3-
Basic Vector (Promega Corp.).
Luciferase Reporter Assay
Relative activity of the ezh2 promoter in MOEC was determined by luciferase
reporter assays. Cells were transfected in low-serum medium (0.5% serum)
with the firefly luciferase plasmid, either empty vector (pGL3-Basic) or the
ezh2 promoter construct vector (EZH2prom-pGL3-Basic), in 12-well plates
using Effectene Transfection Reagent from QIAGEN (Valencia, CA). Cells
were then maintained in low-serum medium for 18 hr, washed in PBS, and
treated in triplicate at 37?C for 6 hr. Treatments included recombinant human
rhVEGF165(VEGF; 50 ng/mL; Peprotech, Rocky Hill, NJ), in fresh medium plus
0.5% serum, and conditioned media from immortalized ovarian surface
epithelium (IOSE120) or SKOV3 ovarian cancer cells. After treatment, cells
were washed and processed using the Dual-Luciferase Reporter Assay
Chromatin Immunoprecipitation Assay
Cells were cultured in low serum medium (0.5% serum) for 18 hr after being
transfected with siRNA for 48 hr and then treated with or without VEGF
(50 ng/mL) for 6 hr. After treatment, Chromatin immunoprecipitation (ChIP)
assays were performed using EZ ChIP kit (Millipore, Temecula, CA) as
described by the manufacturer. In brief, crosslinked cells were collected,
lysed, sonicated, and subjected to immunoprecipitation with EZH2 antibody
(Cell signaling) or mouse IgG (mIgG) control. Immunocomplexes were
collected with protein A/G agarose beads and eluted. Cross-links were
reversed by incubating at 65?C. DNA was extractedand purified for PCR using
primers (see Supplemental Experimental Procedures) corresponding to the
3800–3697 base pairs upstream of the vash1 transcription start site.
Real-Time Quantitative PCR
Cells were seeded at 1.0 3 104cells per well in 96-well plates in complete
medium and incubated at 37?C for 24 hr, and then in low-serum medium
(0.5% serum) for 18 hr, minus EGF and VEGF supplements where appropriate.
Real-time quantitative RT-PCR was performed using 50 ng total RNA isolated
from treated cells using the RNeasy Mini Kit (QIAGEN). Relative expression
values were obtained using the average of three reference genes and the
2?DDCTmethod as described previously, and normalized to control for percent
fold changes (Donninger et al., 2004).
SiRNA Constructs and Delivery
SiRNAs were purchased from QIAGEN, Dharmacon (Chicago, IL), or Sigma-
Aldrich (Woodlands, TX). A nonsilencing siRNA that did not share sequence
homology with any known human mRNA from a BLAST search was used as
control for target siRNA, and the same sequence with Alexa-555 tag was
used for determining uptake and distribution in tumor and other organs
in vivo. In vitro transient transfection was performed as described previously
(Landen et al., 2005).
DNA Extraction and Methylation Analysis
After DNA extraction, methylation analysis was done using a methylation kit
(EZ-96 gold; Zymo Research, Orange, CA). MethPrimer software was used
for the prediction of CpG island of vash1 and design of methylation specific
primers (methylated vash1 promoter: 50-TTAGGGATTTACGTATCGACGT-30
(forward); 50-AAACGACAAACTCCAACCG-30(reverse); and unmethlyated
vash1 promoter: 50-TTTTTTTTAGGGATTTATGTATTGATGT-30(forward); 50-C
TAAACAACAAACTCCAACCACA-30(reverse). PCR conditions were 94?C for
5 min with hot start, then 94?C for 45 s, 56?C for 45 s, and 72?C for 45 s
titative measurement of methylated and unmethylated VASH1. Methylated
VASH1 was normalized to unmethylated VASH1. The experiments were
repeated three times.
Orthotopic In Vivo Model of Ovarian Cancer and Tissue Processing
Female athymic nude mice (NCr-nu) were purchased from the NCI-Frederick
Cancer Research and Development Center (Frederick, MD) and maintained
as previously described (Landen et al., 2005). All mouse studies were
approved by the Institutional Animal Care and Use Committee. Mice were
cared for in accordance with guidelines set forth by the American Association
for Accreditation of Laboratory Animal Care and the US Public Health Service
Figure 7. Analysis of Putative ezh2 Path-
Pathway diagrams were generated with the assis-
tance of Pathway Studio software (Ariadne, Rock-
ville,MD). Amodel isreported inwhichVEGF stim-
ulation leads to increased expression of e2f
transcription factors, which directly modulates
ezh2 levels. EZH2, a transcriptional repressor,
causes vash1 silencing by promoter methylation
and subsequently increases angiogenesis.
EZH2 Effects on Angiogenesis
194 Cancer Cell 18, 185–197, August 17, 2010 ª2010 Elsevier Inc.
Policy on Human Care and Use of Laboratory Animals. For intra-ovarian injec-
tion, SKOV3ip1 cells (1 3 106) were injected directly into the left ovarian paren-
chyma through a left flank incision, as previously described (Lu et al., 2008).
For therapy experiments, each siRNA was given twice weekly at a dose of
150 mg/kg body weight. At the time of sacrifice, mouse and tumor weight,
number, and distribution of tumors were recorded. Individuals who performed
imenswerefixed eitherwithformalin,OCT(Miles, Elkhart, IN),or snapfrozenin
Immunofluorescence and Confocal Microscopy
Staining for EZH2, CD31, DESMIN, and ASMA was performed using frozen
tissue as described previously (Lu et al., 2007b). Pericyte coverage was deter-
mined by the percent of vessels with R50% coverage of DESMIN- or ASMA-
positive cells in five random fields at 2003 magnification for each tumor.
Nude mice bearing HeyA8 tumors (i.p.) were given Cy 5.5 labeled siRNA/CH
(n = 5) or unlabeled siRNA/CH (n = 6) i.v., or nothing (n = 2). Forty-eight hours
later, fluorescence imaging of excised tumor and organs was performed using
the Xenogen IVIS 200 system. Cy5.5 fluorophore excitation (lexcitation =
678 nm) and emission (lemission= 703 nm) filter sets were used. Using Living
image 2.5 software, regions of interest (ROI) were drawn for each organ and
total flux (photons/second or p/s) was measured as photons/s/cm2/steradian
Western Blot Analysis
Western blot analysis was performed as previously reported (Halder et al.,
2006; Landen et al., 2005).
For quantification of MVD in the mouse tumor samples, the number of blood
vessels staining positive for CD31 (1:800 dilution, PharMingen, San Diego,
CA) was recorded in ten random 0.159 mm2fields at 2003 magnification
fashion. For human ovarian cancer samples, immunohistochemistry for EZH2
(1:400 dilution; Zymed, San Francisco, CA), CD34 (1:20 dilution; BioGenex
Laboratories, San Ramon, CA (Ali-Fehmi et al., 2005; Des Guetz et al., 2006;
Ino et al., 2006), VEGF (1:100 dilution; Santa Cruz Biotechnology, Santa
Cruz, CA), DESMIN (1:200 dilution, Dako, Carpinteria, CA), ASMA (1:500
dilution; Abcam, Cambridge, MA), VASH1 (1:200 dilution; Abcam, Cambridge,
MA), or NG2 (1:500 dilution, Santa Cruz Biotechnology) was performed, as
described previously (Ali-Fehmi et al., 2005). For EZH2 and VEGF, the stained
(H-score; >100 defined as high expression and % 100, low expression),
according to the method described by McCarty et al. (1985) (Ali-Fehmi et al.,
2005; Merritt et al., 2008), which considers both the intensity of staining and
thepercentageofcells stained. MVDand pericyte coverage inclinicalsamples
was quantified as described above.
Human Ovarian Cancer Specimens
After approval by the Institutional Review Board, 180 paraffin-embedded
epithelial ovarian cancer specimens (collected between 1985 and 2004) with
available clinical outcome data and confirmed diagnosis by a board-certified
gynecologic pathologist were obtained from the Karmanos Cancer Institute
tumor bank. The study was exempt from informed consent because it used
previously collected residual tissue samples.
Differences in continuous variables were analyzed using the Mann-Whitney
rank sum or t test. The relationship between EZH2 expression and MVD was
determined using the Wilcoxon ranked sums test. Statistical analyses were
performed using SPSS 12.0 for Windows (SPSS, Chicago, IL). A two-tailed
p < 0.05 was considered statistically significant. Kaplan-Meier survival plots
were generated and comparisons made using the log-rank statistic. Bivariate
orthogonal regression was used to describe the correlation between tumor
and endothelial EZH2 expression (H-score).
The microarray data have been deposited into NCBI GEO under accession
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and two tables and can be found with this article online at doi:10.
The authors thank Donna Reynolds, and Fang Wang for their technical exper-
tise and helpful discussion. We also thank Dr. Vickie Williams for reviewing the
manuscript. Portions of this work were supported by the NIH (CA 110793,
109298, P50 CA083639, P50 CA098258, CA128797, RC2GM092599), the
Ovarian Cancer Research Fund, Inc. (Program Project Development Grant),
the DOD (OC073399, W81XWH-10-1-0158, BC085265), NSC-96-3111-B,
the Zarrow Foundation, the Marcus Foundation, the Kim Medlin Fund,
and the Betty Anne Asche Murray Distinguished Professorship. W.A.S.,
A.M.N., A.R.C., and R.S. are supported by NCI-DHHS-NIH T32 Training Grant
(T32 CA101642). K.M. is supported by the GCF/OCRF Ann Schreiber Ovarian
Cancer Research grant and an award from the Meyer and Ida Gordon Founda-
tion2.M.M.K.S. issupportedbytheNIH/NICHDWRHR Grant (HD050128) and
the GCF-Molly Cade Ovarian Cancer Research Grant. M.C.H. and L.Y.L. are
supported by the NSC 97-3111-B-039.
Received: August 10, 2009
Revised: February 15, 2010
Accepted: June 24, 2010
Published: August 16, 2010
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