Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by Promoting Mesenchymal Differentiation

Abstract

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients' survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells' migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target. Copyright: ß 2012 Morone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Overexpression of CD157 Contributes to Epithelial
Ovarian Cancer Progression by Promoting Mesenchymal
Differentiation
Simona Morone1, Nicola Lo-Buono1, Rossella Parrotta1, Alice Giacomino1, Giulia Nacci1, Alfredo Brusco1,
Alexey Larionov1, Paola Ostano2, Maurizia Mello-Grand2, Giovanna Chiorino2, Erika Ortolan1,3*,
Ada Funaro1,3*
1Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy, 2Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia Valenta, Biella,
Italy, 3Research Center on Experimental Medicine (CeRMS), University of Torino, Torino, Italy
Abstract
Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no
prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient
outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for
improving patients’ survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed
in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we
demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes
morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial
markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to
anoikis,increasedanchorage-independentgrowth,cellmotilityandmesothelialinvasion.Conversely,knockdownofCD157in
OV-90andOC314cellsrevertsthemesenchymalphenotypeandreducesthecells’migratorypotential.Transcriptomeprofiling
analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing
OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells
a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high
expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and
cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and
responsetostress).Together,thesefindingsimplicateCD157intheprogressionofEOCtometastaticdiseaseandsuggestthat
CD157 may represent a valuable therapeutic target.
Citation: Morone S, Lo-Buono N, Parrotta R, Giacomino A, Nacci G, et al. (2012) Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by
Promoting Mesenchymal Differentiation. PLoS ONE 7(8): e43649. doi:10.1371/journal.pone.0043649
Editor: Sandra Orsulic, Cedars-Sinai Medical Center, United States of America
Received April 3, 2012; Accepted July 23, 2012; Published August 20, 2012
Copyright: ? 2012 Morone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by grants from the Italian Association for Cancer Research (AIRC, MFAG6312 and IG 11602 to EO), from the Italian Ministry for University and
Scientific Research (PRIN and 60% Projects to AF) and from the International Foundation for Research in Experimental Medicine. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: erika.ortolan@unito.it (EO); ada.funaro@unito.it (AF)
Introduction
Epithelial ovarian cancer (EOC) is an aggressive and lethal
gynecological malignancy. Over 70% of patients present with
advanced disease and, despite aggressive treatment, the 5-years
survival rate of patients with EOC is below 50%. This poor
prognosis results from the difficulty of diagnosis in the early clinical
stages and the lack of an effective therapy for advanced-stage
tumors. Understanding the biological mechanisms regulating the
progression of EOC is therefore critical for devising new treatment
options and improving patients’ survival.
EOC is thought to arise from the ovarian surface epithelium
that lines the ovary. EOC cells can shed from the primary tumor
and, because no anatomical barrier is present, spread directly
throughout the peritoneal cavity and then disseminate mainly via
the lymphatic system, developing the necessary defense mechan-
isms for survival under anchorage-independent conditions [1]. In
the tumor environment, localized proteolytic degradation of the
extracellular matrix (ECM) facilitates the migration of floating
cells, allowing them to anchor to the mesothelium and sub-
sequently invade it, establishing tumors at secondary sites. Tumor
dissemination implies a phenotypic conversion of epithelial cells,
which are not motile, into mesenchymal cells. This process has
remarkable similarities with the epithelial-mesenchymal transition
(EMT) occurring during embryonic development [2]. Indeed, type
3 or oncogenic EMT is increasingly recognized as a dynamic and
transient mechanism whereby cells in primary non-invasive
tumors acquire properties essential for migration, invasion,
metastatic dissemination and resistance to apoptosis [3]. The
EMT program can be induced by a variety of contextual signals
that cells might experience in the tumor microenvironment;
regardless of the trigger signals, activation of the EMT is
associated with poor clinical outcome in different types of tumors,
including ovarian cancer [4]. Cell surface molecules involved in
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the control of processes such as cell-cell, cell-ECM adhesion,
localized intraperitoneal migration and invasion of the peritoneum
by floating cells or cell aggregates (spheroids) are believed to play
a leading role in EOC progression and, ultimately, in patients’
outcome.
CD157/BST-1, a GPI-anchored member of a family of
NADase/ADP-ribosyl cyclase, is an ectoenzyme that cleaves
extracellular nicotinamide adenine dinucleotide (NAD+), generat-
ing cyclic ADP ribose (cADPR) and ADPR [5,6]. In addition,
CD157 establishes functional and structural interactions with
other transmembrane molecules thus acquiring the ability to
transduce intracellular signals [7–9]. Although CD157 was initially
characterized as a stromal [10] and myeloid surface glycoprotein
[11] involved in the control of cell migration and diapedesis [12],
we recently demonstrated that CD157 is also expressed by .90%
of primary EOC and that high levels of CD157 are associated with
rapid tumor relapse in patients with EOC. Consistently with these
findings, inhibition of CD157 activity, by a specific monoclonal
antibody (mAb) in vitro or by its weak expression in patients, is
associated with reduced tumor cell invasion and migration. The
association of CD157 with EOC aggressiveness has been further
substantiated by the observation that exogenous expression of
CD157 in scarcely motile, CD157-negative EOC cells sub-
stantially increases cell motility, a prerequisite for tumor cells
invasion into surrounding tissues [13].
The implication of CD157 in tumor cell motility and in-
vasiveness, and its association with poor outcome in ovarian
cancer patients, prompted us to further investigate its biological
role in EOC progression using engineered ovarian cancer cell lines
as an experimental model. The ultimate goal was to understand
how the function of CD157 might contribute to a more aggressive
ovarian cancer and whether CD157 might be helpful in assisting
the management of these patients.
Materials and Methods
Cell Lines and Reagents
The human EOC cell lines OVCAR-3 and OV-90 and the
non-malignant pleural mesothelial cell line Met-5A were pur-
chased from American Type Culture Collection (ATCC, Mana-
ssas, VA). The EOC cell lines TOV-21G and A2780 were
provided by M.F. Di Renzo (University of Turin, Italy) and
OC314 was provided by S. Ferrini (Institute for Cancer Research
and Treatment, Genoa, Italy). The anti-CD157 mAb (SY/11B5,
kindly provided by F. Malavasi, University of Turin, Italy) was
produced in the authors’ laboratories and affinity purified on
protein G (Sigma-Aldrich). The Alexa-488 labeled F(ab9)2fraction
of goat antibodies to mouse IgG or to rabbit IgG were from
Molecular Probes (Milan, Italy). Anti-E-cadherin mAb was from
BD Biosciences (Milan, Italy), anti-Snail, anti-Zeb1, anti-EpCAM,
anti-VCAN, anti-BMP7, anti-tubulin, anti-b-catenin, anti-N-
cadherin and anti-b-actin-horseradish peroxidase (HRP) mAb
were from Santa Cruz Biotechnologies (Santa Cruz, CA), anti-
lamin B1 was from Abcam (Cambridge, UK). TRITC-labelled
phalloidin used to detect F-actin was from Sigma-Aldrich.
GM6001 (matrix metalloproteases inhibitor) was from Enzo Life
Science (Vinci Biochem, Vinci, Italy).
CD157 Gene Transfection and ShRNA Lentiviral Particle
Transduction
Cells were transfected with the eukaryotic expression vector
pcDNA3.1 containing the cDNA for full-length CD157 or no
insert (mock), as described [13]. Cells were grown in RPMI-1640
or MCDB131/M199 (vol/vol) culture medium (Sigma-Aldrich,
Milan, Italy) supplemented with 10% fetal calf serum (FCS,
Biochrom Seromed, Milan, Italy). Cells were maintained at 37uC
and 5% CO2 and tested for Mycoplasma contamination.
CD157 expression in OV-90 and OC314 cells was silenced by
lentiviral delivery of pLV-puro (Biosettia, San Diego, CA)
encoding a short-hairpin RNA (shRNA) targeting BST-1 mRNA
(targetsequences59-GAGTCAGACTGCTTGTATA-39
(shCD157)and59-CCTGAGCGATGTTCTGTAT-39
(shCD157#2); scrambled sequence 59-TTCTCCGAACGTGT-
CACGTT-39). Particles were generated as previously described
[14]. Cells were incubated with appropriate lentiviral supernatants
and Polybrene (8 mg/ml, Sigma-Aldrich). Transduced cells un-
derwent selection in 2 mg/ml puromycine (Santa Cruz Biotechnol-
ogies) for 3 days.
Western Blot Analysis
Total cell lysates were obtained by incubation in RIPA lysis
buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP-40, 0.5%
Sodium Deoxycholate, 1 mM EDTA, 0.1% SDS supplemented
with 1 mM Na3VO4, 5 mM NaF, 50 mg/ml aprotinin and
leupeptin). Cytosolic extracts were obtained by incubating cells
for 10 minutes at 4uC with hypotonic buffer solution containing
20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 and
50 mg/ml Protease Inhibitor Cocktail (Sigma-Aldrich). The
suspension was treated with a 0.5% NP40 solution and the
obtained homogenate was centrifuged (3.000 rpm, 10 minutes at
4uC). Nuclear extracts were prepared by incubating the pellet
obtained as described above in RIPA Lysis buffer for 30 minutes.
After 30 minutes centrifugation at 14.000 rpm at 4uC, protein
concentration was determined using the Bradford assay (Bio-Rad
Laboratories). Western blotting was performed as previously
described [13]. Briefly, equal amounts (30 mg) of protein extracts
from cells were separated by 10% SDS-polyacrylamide gel
electrophoresis (PAGE) under non-reducing conditions and
electro-transferred on a polyvinylidene difluoride (PVDF) mem-
branes, then blocked and probed with the indicated mAb. After
incubation with the appropriate HRP-conjugated antibodies
(Santa Cruz Biotechnologies), the immunoreactive bands were
detected by enhanced chemiluminescence (Perkin Elmer, Monza,
Italy). Images were captured with a ChemiDocTMXRS+ System
and densitometry analysis was performed with Image LabTM
Software (Bio Rad, Milan, Italy).
Cell Colony Scattering Assay and Soft Agar Colony
Formation
Cells (500/well) were seeded in 6-well plates and maintained in
culture medium supplemented with 5% FCS for 14 days, then
washed, fixed in methanol for 30 minutes, stained with crystal
violet (Sigma-Aldrich) and visualized using an IX70 inverted
microscope equipped with a UC30 camera and the CellF analysis
software (Olympus Biosystems).
In soft agar assays 66102cells were mixed with 0.45% agar
solution in RPMI containing 10% FCS and layered on top of
0.9% base agar layer in 24-well plates. Assays were performed in
triplicate. After 2–3 weeks at 37uC in a 5% CO2 incubator,
colonies were visualized with an inverted microscope and counted.
Cell-cell Adhesion and Cell Aggregation Assays
Cell-cell adhesion experiments were performed as described
[15]. Briefly, single cell suspensions were seeded on 0.5% agarose-
coated culture dishes (1 ml/well, 30 minutes at 37uC) to prevent
cell adhesion, and slowly shaken for 2 h at 37uC.
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Figure 1. Ectopic expression of CD157 alters cell morphology, cell-cell interactions and anoikis in OVCAR-3 cells. (A) sqRT-PCR of
ectopic CD157 expression in OVCAR-3 cells (top). GAPDH was used as an internal control. Western blot analysis of CD157 in OVCAR-3/CD157 and
OVCAR-3/mock cells (bottom). The anti-b-actin mAb was used as a loading control. (B–C) Morphology of OVCAR-3/mock and OVCAR-3/CD157 cells.
Representative colonies visualized after crystal violet staining using an IX70 inverted microscope equipped with a UC30 camera and the CellF analysis
software (Olympus Biosystems) are shown. (B, scale bar: 200 mM; C, scale bar: 20 mM). (D) Confocal microscopy analysis of F-actin. Cells were grown
on gelatin-coated coverslips, fixed with 2% PFA, permeabilized with 0.2% Triton-X 100 and stained with phalloidin-TRITC. Samples were analyzed
using an Olympus FV300 laser scanning confocal microscope. Cells were imaged using a 606oil immersion objective (1.4 NA). (Scale bar: 20 mM).
Microphotographs in B, C, and D were then reproduced in black and white. (E) OVCAR-3 cells were subjected to a cell-cell adhesion assay and clusters
(.5 cells) were counted in 20 different fields/dish. Results represent the mean 6 SEM of four independent experiments. **P,0.01, two-tailed t test.
(F) Aggregates generated using the hanging drop method and overnight incubation at 37uC were mechanically dispersed and then photographed
under phase contrast microscope (scale bar: 50 mM). (G) Induction of anoikis in OVCAR-3/CD157 and OVCAR-3/mock cells after 72 h of culture on
poly-HEMA-coated plates. Phase-contrast microscopy images show the formation of large floating aggregates in OVCAR3/mock cells and small
aggregates or single isolated cells in OVCAR3/CD157 cells (scale bar: 200 mM). (H) After 24, 48 and 72 h of anchorage-independent growth, cells were
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Cell aggregation assays were performed using the hanging drop
method, as described [16]. Briefly, cells (56103/25 ml of RPMI
1640 medium with 5% FCS) were seeded onto the inner surface of
the lid of a Petri dish. To prevent evaporation, 10 ml phosphate
buffered saline (PBS) were placed into the dish. After overnight
incubation at 37uC, cell aggregates were mechanically dispersed
and photographed using a phase-contrast microscope.
Anoikis Assay
Cells (56105/well) were cultured on 20 mg/ml poly-HEMA
(polyhydroxyethylmethacrylate,
plates for 24 to 72 h. Then, cell aggregates were dispersed and
the cells were fixed with ice-cold 75% ethanol (vol/vol) overnight
at 4uC. Cells were treated with 100 mg/ml RNase A (Sigma-
Aldrich, 30 minutes at 37uC), stained with 10 mg/ml propidium
iodide (10 minutes at 4uC) and samples were analyzed with
a FACSCanto (Becton Dickinson, Mountain View, CA, USA).
Data analysis was performed using ModFit LTTMcell cycle
analysis software (Verity Software House, Topsham, ME). Anoikis
was determined by measuring the percent of sub-G1 cells.
Sigma-Aldrich)-treated 6-well
Immunofluorescence Staining and Confocal Microscopy
In confocal microscopy experiments, cells were grown to
subconfluence on gelatin-coated coverslips, fixed with 4% para-
formaldehyde (PFA) for 30 minutes at 20uC, washed and
permeabilized with 0.1% Triton X-100 in phosphate-buffered
saline with 1% normal goat serum and 1% BSA for 15 minutes at
20uC. Cells were incubated with the indicated primary antibodies
followed by Alexa Fluor-488-conjugated secondary antibodies.
The samples were analyzed with an Olympus FV300 laser
scanning confocal microscope equipped with a Blue Argon
(488 nm) laser, a Green Helium Neon (543 nm) laser, and
FluoView 300 software (Olympus Biosystems, Hamburg, Ger-
many). Cells were imaged using a 606oil immersion objective (1.4
NA) and 106 ocular lens. Nomarski images were obtained by
differential interference contrast (DIC) optical components in-
stalled on an IX71 inverted microscope. Semiquantitative analysis
of E-cadherin junctional staining was performed by counting
a minimum of 10 fields per sample (at least 200 cells overall) and
scoring as positive the number of cells with two remaining
fluorescent cell-cell borders.
RNA Extraction and Reverse Transcriptase-PCR
Total RNA (2 mg) extracted from 70–80% confluent cultures
using TRIZOLH reagent (Invitrogen, S. Giuliano Milanese, Italy)
was reverse-transcribed with the M-MLV Reverse Transcriptase
(Invitrogen) and Oligo-dT primers. cDNA was amplified using
KAPA2G Fast HotStart DNA Polymerase (Kapa Biosystems,
Cambridge, MA). Each cycle consisted of denaturation at 94uC for
10 seconds, annealing for 10 seconds and extension at 72uC for
1 second. In semi-quantitative analysis (sqRT-PCR), the appro-
priate number of cycles for remaining within the exponential
phase was determined for each substrate. The primers used are
reported in Table S1. PCR products were then analyzed by
agarose gel electrophoresis.
SYBR Green Real-time RT-PCR (qRT-PCR)
Total RNA was extracted using the RNeasy mini kit (Qiagen,
Milan, Italy) according to the manufacturer’s directions. qRT-
PCR was performed using SYBR Green JumpStart Taq Ready-
Mix (Sigma-Aldrich) and an ABI 7500 Fast Sequence Detection
System (Applied-Biosystems, Foster City, CA, USA). PCR cycling
conditions were performed for all samples as follows: 95uC for
10 minutes, followed by 40 cycles at 95uC for 15 seconds and
60uC for 1 minute. The primers used are listed in Table S2. PCR
reactions for each template were done in triplicate in 96-well
plates. The comparative CT method (Applied Biosystems) was
used to determine gene expression in CD157-transfected relative
to the value observed in the corresponding control cells, using TBP
as normalization control.
Wound-healing Assay
Cells were seeded in six-well plates to confluence and a scratch
was then made across the monolayer, as previously described [13].
Images of the wounded area were recorded at the times indicated.
Each experiment was performed in quadruplicate and repeated at
least three times.
Transmesothelial Migration Assays and Confocal
Microscopy Analysis
Met-5A cells (26105) were labeled using a CellBriteTMRed
Cytoplasmic Membrane Staining kit according to the manufac-
turer’s directions (Biotium Inc. Hayward, CA), then seeded on
fibronectin-coated (10 mg/ml) coverslips and allowed to grow to
confluence. Ovarian cancer cells (1.56105) were stained with
5 mM CFSE (Carboxyfluorescein succinimidyl ester, Molecular
Probes) and seeded on the Met-5A monolayer for 6 h (OVCAR-3
cells) or 2.5 h (OV-90 cells) at 37uC. Non-adherent tumor cells
were carefully removed, the sample fixed with 2% PFA and then
analyzed using an Olympus FV300 laser scanning confocal
microscope. Cells were imaged using a 606 oil immersion
objective (1.4 NA) by sequential scanning of the XY planes
recorded along the Z-axis (step size: 1.25 mm). Cells were counted
on the top, median and bottom stage and the ratio of cells on the
bottom stage to total cells represents the percentage of cells that
migrated through the monolayer [17]. Where indicated, cells were
treated for 1 h with GM6001 (25 mg/ml) before seeding onto the
Met-5A mesothelial cell monolayer.
Gelatin and Casein Zymography Assays
OVCAR-3 and OV-90-transfected cells were seeded in 48-well
plates and grown to 80% confluence, then incubated for an
additional 48 h in FCS-free medium. Matrix metalloproteinases
(MMPs) MMP2 and MMP9 activity in the conditioned medium
was analyzed using 10% SDS-PAGE containing 1 mg/ml gelatin
(gelatin zymography) [18], and MMP7 activity was visualized
using 12% SDS-PAGE containing 1 mg/ml casein (casein
zymography) [19]. Gels were stained with Coomassie brilliant
Blue G-250 to visualize protease activity. Images were captured
with a ChemiDocTMXRS+ System and densitometry analysis was
performed using Image LabTMSoftware.
fixed, permeabilized, stained with propidium iodide and analyzed with a FACSCanto. Data analysis was performed with ModFit LTTMcell cycle analysis
software. Anoikis in OVCAR-3/mock and OVCAR-3/CD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean
6 SEM of three independent experiments. *P,0.05; **P,0.01, two-tailed t test. (I) Representative histograms of cell cycle status of mock and CD157-
positive OVCAR-3 cells after 48 h of anchorage-independent growth. (J) Anchorage-independent growth of OVCAR-3/CD157 and mock cells was
analyzed by soft agar colony formation assay. Graph represents average number of colonies formed from three independent experiments 6 SEM
after 3 weeks incubation of cells in soft agar. *P,0.05, two-tailed t test.
doi:10.1371/journal.pone.0043649.g001
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Spheroid Disaggregation Assay
Spheroids were generated with the hanging drop method and
their disaggregation on fibronectin-coated plates (10 mg/ml) was
measured after 12 h incubation at 37uC, as described elsewhere
[13]. Briefly, 96-well plates were coated with 10 mg/ml fibronectin
and blocked with BSA (1 mg/ml) for 1 h at 37uC. Spheroids (8–10
spheroids/well) were seeded in serum-free RPMI-1640 medium.
To track individual spheroids over time, each well was photo-
graphed 30 minutes after seeding (time 0) and after 12 h. The
pixel area of the spheroids was measured at time 0 and 12 h, and
the fold change in area was calculated as the ratio between the
pixel area of the spheroids at 12 h and at time 0.
Microarray Hybridization, Data Collection and Analysis
Total RNA was extracted from duplicate 70–80% confluent
control cell lines (OVCAR-3/mock and OV-90/mock) and testing
cell lines (OVCAR-3/CD157 and OV-90/CD157), microarray
probe preparation, hybridization, scanning and image analysis
were performed as previously described [20]. Two replicates, with
dye swap, were performed for each sample. Raw data elaboration
was carried out with Bioconductor [21], using R statistical
language [22] applying a cut-off on the P-value, adjusted for
multiple testing by the Benjamini-Hochberg approach (,0.01),
followed by filtering on expression level (logFC .1 or ,21 in at
least one cell line, excluding transcripts with opposite sign). This
criterion allowed the identification of transcripts with concordant
modulation and took into account intrinsic biological differences of
distinct cell lines that might be reflected in the amplitude of fold
changes.
Gene ontology, canonical pathway, and functional network
analyses were performed using both the DAVID Knowledgebase
[23] and MetaCore software from GeneGo Inc., applying a cut-off
on enrichment P-values (,0.05). Gene expression data sets have
been deposited into the Gene Expression Omnibus (GEO)
database, ID: GSE36364.
(http://www.ncbi.nlm.nih.gov/geo/query/acc.
cgi?token=flktjkcsuyygcrm&acc=GSE36364).
Statistical Methods and Data Analysis
Unless otherwise indicated, values are expressed as means 6
SEM. Comparisons between two groups were carried out using an
unpaired two-sided Student’s t test for normal distributed
variables. Statistical analyses were performed using SPSS Statistics
17 software (Chicago, IL) and GraphPad Prism 5 software (San
Diego, CA). All statistical tests were two-sided. For all analyses,
differences were considered significant at P,0.05 (*P,0.05,
**P,0.01, ***P,0.001 versus control) and ns for not significant.
Results
CD157 Expression Modulates Ovarian Cancer Cells
Morphology and Cell-cell Interaction
We chose OVCAR-3 ovarian cancer cells as the most suitable
model for studying the effects induced by CD157 expression since
they have an epithelial phenotype [24], are poorly invasive,
scarcely motile on plastic and barely anchorage-independent [25].
To explore the biological significance of CD157 in ovarian cancer
progression, we stably transfected full length CD157 in CD157-
negative OVCAR-3 cells (Figure 1A). Light microscopy images
revealed that mock cells grew as tightly connected clusters
composed of cells with typical cobblestone-like epithelial mor-
phology. In contrast, OVCAR-3/CD157 cells exhibited a more
scattered distribution and elongated shape, a distinctive feature of
fibroblast-like cells, and formed poorly organized junctions
between adjacent cells (Figure 1B,C). Consistently with this
observation, phalloidin staining in OVCAR-3/CD157 cells
revealed remodeling in actin cytoskeleton architecture which is
a prerequisite for cancer cell motility and invasion [26], and is
considered a characteristic of mesenchymal differentiation. In-
deed, in OVCAR-3/CD157 cells, cortical actin, which is prevalent
in mock cells, was largely replaced by the formation of F-actin
stress fibers throughout the cells and accumulation at the adhesion
sites, typical features of a cell spreading response (Figure 1D). The
morphological features along with the reorganization of F-actin
suggest that exogenous expression of CD157 in OVCAR-3 cells
drives tumor cells toward morphological changes reminiscent of
mesenchymal-like differentiation.
The formation of organized intercellular contacts critically
affects the growth pattern of EOC cells and, in particular, may
counteract their dissociation from the tumor mass, thus limiting
the peritoneal dissemination of the tumor. To determine the effects
of CD157 on the propensity of tumor cells to form aggregates
under anchorage-independent conditions, an in vitro situation
mimicking the early stages of the metastatic process occurring
in vivo, we measured the formation of cell aggregates under slow
agitation. The results showed that OVCAR-3/CD157 cells
formed significantly fewer clusters than mock cells (P,0.01)
(Figure 1E). The impaired ability of CD157-positive cells to form
large, organized clusters in the absence of adhesion with a substrate
was confirmed by a cell aggregation assay. In these experimental
conditions, OVCAR-3/mock cells generated a huge number of
Figure 2. CD157 overexpression alters the expression of epithelial and mesenchymal markers. (A) Confocal microscopy analysis of E-
cadherin and (B) b-catenin expression in OVCAR-3/CD157 and mock cells. Cells were grown on a gelatin-coated coverslip, fixed, permeabilized and
stained with anti-E-cadherin, and anti-b-catenin antibodies followed by secondary Alexa Fluor-488-labelled antibody. Samples were analyzed with an
Olympus FV300 laser scanning confocal microscope and by Nomarski differential interference contrast (DIC) optics. For E-cadherin, a fluorescence
image merged with DIC image is shown (scale bar: 50 mM). Semiquantitative analysis of E-cadherin junctional staining was determined by counting
a minimum of 10 fields/sample (at least 200 cells overall) and scoring as positive cells with two remaining fluorescent intercellular borders. For b-
catenin, a fluorescent image is shown. Inset: amplified view of an individual OVCAR-3/CD157 cell exhibiting diffuse b-catenin staining in the plan of
focus cutting through the nucleus. Asterisks correspond to nucleoli. (C) Western blotting for E-cadherin and b-catenin in OVCAR-3/CD157 and mock
cells. Densitometry quantifies the expression level of E-cadherin and b-catenin relative to b-actin. (D) b-catenin levels in nuclear and cytoplasmic
fractions of OVCAR-3/mock and OVCAR-3/CD157 cells were determined by western blot analysis. a-tubulin and lamin B1 (LamB1) were used as
cytoplasmic and nuclear loading controls, respectively. Densitometry quantifies the expression level of b-catenin relative to the proper control. (E)
sqRT-PCR for E-cadherin, N-cadherin, b-catenin and for E-cadherin transcriptional repressors in OVCAR-3/CD157 and mock cells. Densitometry
quantifies the levels of expression of E-cadherin repressors relative to GADPH. (F) qRT-PCR for Zeb1, Snail and Twist1. The comparative CT method
was used to determine gene expression in CD157-transfected cells relative to the value observed in the mock cells, using TBP as normalization
control. Histograms report the means 6 SEM of three qRT–PCR independent experiments, each conducted in triplicate. *P,0.05, ***P,0.001; ns, not
significant; two-tailed t test. (G) Western blot analysis showing the level for Snail and Zeb1 in OVCAR-3/mock and OVCAR-3/CD157 cells. Densitometry
quantifies the expression level of both proteins relative to b-actin. Results shown in each panel are representative of three independent experiments
with similar results.
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compact and large clusters able to resist mechanical disruption,
while OVCAR-3/CD157 cells formed small and scarcely cohesive
clusters, apt to be destroyed (Figure 1F).
In patients, cell detachment from the primary tumor and loss of
contact with the ECM cause anoikis (detachment-induced
apoptosis) in a large proportion of cells; only a reduced number
of tumor cells acquire the ability to avoid anoikis and subsequently
form invasive foci. Consistently, the expression of anti-apoptotic
molecules, which confer resistance to anoikis, has been shown to
promote metastasis in selected experimental models [27]. The
observed association between high CD157 expression and tumor
relapse in patients fostered the hypothesis that CD157 might
provide protection against anoikis. As shown in Figure 1G, after
72 h in suspension, OVCAR-3/CD157 cells appeared as small
aggregates or single cells, whereas control cells were organized in
large, floating clusters. Comparison of anoikis sensitivity of
OVCAR-3/CD157 and mock cells highlighted that the former
had a significantly lower number of apoptotic cells than OVCAR-
3/mock cells, at any of the times considered (Figure 1H). Cell cycle
analysis confirmed that, after 48 h under anchorage-independent
conditions, OVCAR-3/CD157 cells included a larger fraction of
cycling cells compared to the control (Figure 1I). Next, we
investigated whether the increased resistance to anoikis might
influence the ability of OVCAR-3/CD157 cells to form colonies in
soft agar, an in vitro conventional measure of tumorigenicity.
Results in Figure 1J show that OVCAR-3/CD157 cells formed
a greater number of colonies than OVCAR-3/mock cells. These
data indicate that the ectopic expression of CD157 in OVCAR-3
cells is critical for cell cohesion, protects floating cells from anoikis
and enhances in vitro tumorigenicity.
CD157 Regulates Epithelial and Mesenchymal Protein
Markers
The accepted paradigm of oncogenic mesenchymal differenti-
ation is that tumor cells reduce or lose their markers of epithelial
cells and express de novo mesenchymal markers. Numerous reports
have shown that this change in phenotype impairs cell-cell
adhesion and communication and assists the dissemination of
the tumor [28–30]. We therefore explored the relationship
between the observed CD157-induced morphological alterations
and mesenchymal differentiation of OVCAR-3 cells. Confocal
microscopy analysis revealed that E-cadherin staining (a pro-
totypic epithelial marker) was significantly lower in CD157-
positive cells than in OVCAR-3/mock cells (Figure 2A). The
cytoplasmic domain of E-cadherin has been shown to bind to the
cytosolic protein b-catenin, which in turn provides anchorage to
the actin cytoskeleton [31]. When E-cadherin is repressed, b-
catenin is released and re-localizes in the cytoplasm before being
targeted for degradation or translocating to the nucleus. As
expected, b-catenin expression was lower in OVCAR-3/CD157
cells than in the control, and the weak residual staining was
confined to the cytoplasm (Figure 2B, right panel) and, at least in
part, it was localized into the nucleus (Figure 2B, inset). In
OVCAR-3/mock cells E-cadherin and b-catenin staining was
predominantlyassociated with
(Figure 2A,B left panels). Western blotting of whole cell lysates
confirmed the decreased expression of both E-cadherin and b-
catenin in OVCAR-3/CD157 cells (Figure 2C). Cell fractionation
and subsequent analysis of the cytoplasmic and nuclear fractions
showed nuclear accumulation of b-catenin in OVCAR-3/CD157
cells (Figure 2D). SqRT-PCR revealed that the reduction of E-
cadherin observed in OVCAR-3/CD157 cells was accompanied
by increased expression of N-cadherin transcript (Figure 2E).
However, the induction of N-cadherin was not appreciable at
protein level (data not shown). The observed CD157-induced
effects were not limited to a single ovarian cancer cell line, indeed,
transient transfection of CD157 in CD157-negative TOV-21G
epithelial cells (Figure S1A) was accompanied by decreased
expression of E-cadherin and slightly increased expression of N-
cadherin proteins (Figure S1B), and caused accumulation of b-
catenin in the nuclear extracts (Figure S1C). Moreover, in A2780
cells (which lack expression of E-cadherin and present a mixed
epithelial/mesenchymal phenotype [32]), exogenous expression of
CD157 (Figure S1A) enhanced the basal level of N-cadherin,
driving cells toward a mesenchymal differentiation (Figure S1B).
These results indicate that morphological and phenotypic changes
induced by ectopic expression of CD157 in ovarian cancer cells
are consistent with their mesenchymal differentiation.
inter-epithelialjunctions
CD157 Modulates the Expression of Transcription
Repressors of E-cadherin
Reduced expression of E-cadherin can be achieved in multiple
ways, among which, transcriptional repression has recently
emerged as a fundamental mechanism for the dynamic silencing
of the E-cadherin gene (CDH1) during tumor progression [3,33].
Using sqRT-PCR and qRT-PCR, we determined whether CD157
expression was associated with increased transcription of some
known repressors (such as Snail, Slug, Zeb1, Zeb2 and Twist1).
Higher mRNA expression levels for Snail, Zeb1 and Twist1 (but
not for Zeb2 and Slug) were observed in OVCAR-3/CD157 as
compared to OVCAR-3/mock cells (Figure 2E). qRT-PCR
confirmed that Snail and Zeb1 mRNA expression was increased
by .50% in CD157-positive cells compared to control cells, while
Twist1 mRNA expression did not significantly differ in CD157-
positive versus negative cells (Figure 2F). Western blot analysis
confirmed the increased expression of Snail and Zeb1 proteins in
OVCAR-3/CD157 (Figure 2G), in TOV-21G/CD157 and in
A2780/CD157 cells (Figure S1B) compared to the corresponding
mock controls. These results suggest that ectopic expression of
Figure 3. CD157 overexpression protects OV-90 cells from anoikis and enhances motility. (A) sqRT-PCR (left) and western blot analysis
(right) of CD157 in OV-90/CD157 and OV-90/mock cells. GAPDH and b-actin were used as internal controls, respectively. (B) Morphology of colonies
formed by OV-90/mock and OV-90/CD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 mM. (C) sqRT-
PCR analysis of E-cadherin and N-cadherin in OV-90/mock and OV-90/CD157 cells. Densitometry quantifies the levels of mRNA expression of the
indicated molecules relative to GAPDH. (D) Effect of CD157 overexpression on anoikis. After 48, 72. 96 and 192 h of anchorage-independent growth,
cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Anoikis in OV-90/mock and OV-90/CD157 cells was determined by
measuring the percent of sub-G1 cells. Results represent the mean 6 SEM of three independent experiments. *P,0.05; **P,0.01, two-tailed t test. (E)
Anchorage-independent growth of OV-90/CD157 and mock cells was analyzed by soft agar colony formation assay. Graph represents average
number of colonies formed from three independent experiments 6 SEM after 3 weeks incubation of cells in soft agar. ***P,0.001, two-tailed t test.
(F) Effect of CD157 expression on cell migration in a scratch-wound assay in OV-90/CD157 and mock cells. Cells were grown as monolayers, wounded,
and photographed at time 0 and at 24 hr. Wound edges are indicated by black dashed lines (scale bar: 200 mM). (G) The ability of cells to close the
wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 hr. Results represent the percentage
reduction of the average wound width and are expressed as the mean 6 SEM of three independent experiments. *P,0.05; two-tailed t test.
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CD157 in ovarian cancer cells enhances the expression of Snail
and Zeb1 transcriptional repressors, driving EMT.
CD157 Expression Enhances Motility and Invasion of
Mesothelium by EOC Cells in vitro
Mesenchymal differentiation makes an essential contribution to
cancer progression because it endows tumor cells with motile and
invasive skills [29,34]. Recently, we demonstrated that exogenous
expression of CD157 in OVCAR-3 cells substantially increased
cell motility [13] a prerequisite for cancer progression and for
invasive migration of tumor cells into surrounding tissues. To
evaluate the impact of increasing levels of CD157 on the behavior
of tumor cells with an intermediate basal CD157 expression, we
transfected the full-length CD157 in OV-90 EOC cells which are
highly invasive and express low E-cadherin and CD157
(Figure 3A,C). OV-90/mock cells generated colonies with a typical
epithelial shape, while OV-90/CD157 cells formed scattered
colonies (Figure 3B), mirroring those formed by OVCAR-3/
CD157 cells. Moreover, OV-90/CD157 displayed increased
resistance to anoikis. The effect was appreciable after 48 h of
anchorage-independent culture
(Figure 3D). The increased viability of OV-90/CD157 cells under
anchorage independent culture was paralleled by increased ability
to form colonies in soft agar (Figure 3E). Moreover, OV-90/
CD157 (similarly to OVCAR-3/CD157 cells [13]) exhibited
a migratory potential two fold higher than the corresponding
mock cells, as assessed using a wound healing assay (Figure 3F,G).
Despite the correlation between expression levels of CD157 and
tumor cell motility and tumorigenicity, OV-90 cells expressing
high CD157 or basal CD157 showed no substantial differences in
the E-cadherin and N-cadherin mRNA expression (Figure 3C).
Next, to determine the contribution of endogenous CD157
expressed by OV-90 cells in tumor cell behavior, OV-90 cells
were retrovirally transduced with a shRNA that targets the CD157
mRNA (OV-90/shCD157), resulting in efficient knockdown of
CD157 expression (Figure 4A). OV-90/shCD157 cells appeared
even more compact and organized in tight colonies than the OV-
90/scramble cells (Figure 4B). This morphological change was
accompanied by upmodulation of E-cadherin and downmodula-
tion of Snail, Twist and Slug gene transcription (Figure 4C).
Moreover, OV-90/shCD157 cells displayed increased sensitivity
to anoikis (Figure 4D), reduced ability to form colonies in soft agar
(Figure 4E) and a striking impaired migratory ability compared to
cells transduced with a control shRNA (Figure 4F,G), suggesting
that endogenous CD157, although it is expressed at low level,
influences OV-90 cell motility and tumorigenicity. These findings
were confirmed in a second cell model. We transduced OC314
cells (expressing CD157 levels comparable to that of OV-90 cells)
with two independent shRNA resulting in efficient (OC314/
shCD157) and partial (OC314/shCD157#2) decrease in CD157
andpersistedovertime
Figure 4. Morphological and functional modifications induced
by CD157 knockdown in OV-90 cells. (A) sqRT-PCR (left) and
western blot analysis (right) showing OV-90 cells retrovirally transduced
with a shRNA that targets the human CD157 mRNA, resulting in
efficient knockdown of CD157 expression. GAPDH and b-actin were
used as internal controls, respectively. (B) Morphology of colonies
formed by OV-90/scramble and OV-90/shCD157 cells. Representative
colonies visualized after crystal violet staining are shown. Scale bar:
200 mM. (C) sqRT-PCR for E-cadherin, N-cadherin and Snail, Twist1 and
Slug transcriptional repressors in OV-90/scramble and OV-90/shCD157
cells. Densitometry quantifies the levels of mRNA expression of the
indicated molecules relative to GAPDH. (D) Anoikis assay. After 48, 72,
96 and 192 h under anchorage-independent growth, cells were fixed,
stained with propidium iodide and analyzed with a FACSCanto. Data
analysis was performed with ModFit LTTMcell cycle analysis software.
Anoikis in OV-90/scramble and OV-90/shCD157 cells was determined by
measuring the percent of sub-G1 cells. Results represent the mean 6
SEM of three independent experiments. *P,0.05; **P,0.01, two-tailed t
test. (E) Anchorage-independent growth of OV-90/scramble and OV-90/
shCD157 cells was analyzed by soft agar colony formation assay. Graph
represents the average number of colonies/field formed from three
independent experiments 6 SEM after 3 weeks incubation of cells in
soft agar. *P,0.05, two-tailed t test. (F) Effect of CD157 knockdown on
OV-90 cell migration in a scratch-wound assay. Cells were grown as
monolayers, wounded, and photographed at time 0 and at 24 h (scale
bar: 200 mM). Wound edges are indicated by black dashed lines. (G) The
ability of OV-90/scramble and OV-90/shCD157 cells to close the wound
was calculated by measuring 20 randomly chosen distances along the
wound edge at time 0 and at 24 h. Results represent the percentage
reduction of the average wound width and are expressed as the mean
6 SEM of three independent experiments. **P,0.01, two-tailed t test.
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mRNA and protein expression, respectively (Figure S2A).
OC314/shCD157 cells exhibited significantly reduced motility,
while OC314/shCD157#2 cells showed only slightly reduced
motility as compared to OC314/scramble cells. (Figure S2B,C).
Overall, these results strongly support a physiological role of
CD157 in migration of selected EOC cell lines and highlight
a direct correlation between CD157 expression levels and the
efficiency of tumor cell motility.
The adhesion of single cells (or cell aggregates) to and migration
throughout the mesothelium are key steps during EOC metasta-
tisation. The ability of CD157-positive and negative tumor cells to
invade the mesothelium was compared using a specially designed
3D assay (Figure 5A,B). The evaluation of transmigration
efficiency showed that forced expression of CD157 resulted in
a significant increase in transmesothelial migration of both
OVCAR-3 and OV-90 cells, as compared to the respective mock
cells (Figure 5C), highlighting a pro-invasive function of CD157 in
EOC cells. Collectively, these data indicate that CD157 expression
impacts on tumorigenicity and invasiveness of ovarian cancer cells
in vitro.
Figure 5. CD157 overexpression promotes invasion of mesothelium by OVCAR-3 and OV-90 cells and increases matrix
metalloproteinase expression and activity. (A) Ovarian cancer cell migration through a mesothelial monolayer. Met-5A mesothelial cells were
labeled with CellBriteTMRed and grown to confluence on fibronectin-coated coverslips. CFSE-stained tumor cells were plated onto the monolayer.
After 6 h (OVCAR-3 cells) or 2.5 h (OV-90 cells) at 37uC, sample were fixed and analyzed using an Olympus FV300 laser scanning confocal microscope
by sequential scanning of the XY planes recorded along the Z-axis (step size: 1.25 mM). Series of confocal optical XY images were processed using a 3D
reconstruction program (bioView3D software, Bio-Image Informatics, University of California, Santa Barbara, CA). Top, orthogonal and bottom views
are shown (scale bar: 50 mM). (B) Mesothelial layer integrity was verified by confocal microscopy analysis in CellBriteTMRed-labeled samples. Samples
were analyzed with an Olympus FV300 laser scanning confocal microscope and by Nomarski differential interference contrast (DIC) optics. Scale bars :
50 mM. (C) Transmesothelial migration of OVCAR-3 and OV-90 cells. Where indicated, OVCAR-3/CD157, OV-90/CD157 and the corresponding mock
cells were treated for 1 h with GM6001 (25 mg/ml) before seeding onto Met-5A mesothelial cell monolayer. Results are expressed in terms of percent
of transmigrated cells calculated as the ratio of cells that crossed the mesothelial layer to the total number of cells in the field. At least ten fields have
been counted for each sample. Results are expressed as the mean 6 SEM of three independent experiments. P values were derived from analysis of
variance (ANOVA) with Dunnett correction. **P,0.01, ***P,0.001, ns, not significant. (D) sqRT-PCR expression of MMP2, MMP7, MMP9 and TIMP3 in
engineered OVCAR-3 (left panel) and OV-90 cells (right panel). Densitometry quantifies the levels of expression of MMP compared with GADPH. (E)
Gelatin zymography and casein zymography were used to quantify MMP2, MMP9 and MMP7 activity, respectively, in cell-free conditioned media
from OVCAR-3/CD157, OV-90/CD157 and the corresponding mock cells (left panel). Zymographic bands from all samples were quantified by
densitometry (right panel). The enzyme activities are expressed as arbitrary units. Results are from one representative experiment performed in
triplicate and are expressed as the mean 6 SD.
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CD157 Expression Enhances Matrix Metalloproteinase
Expression and Activity
Tumor invasion is supported by active MMPs secreted by
tumor or stromal cells, which constitute the most prominent family
of proteinases associated with tumorigenesis [35]. Hence, follow-
ing the observation that the higher the expression of CD157, the
greater the ability of tumor cells to invade the mesothelium, we
hypothesized that CD157 expression could promote MMP activity
thus fuelling tumor cell transmigration. To address this issue,
MMP expression was determined by measuring MMP mRNA and
proteolytic activity. Forced expression of CD157 was character-
ized by increased expression of MMP2 and MMP7 in OVCAR-3
cells, and of MMP2 and MMP9 in OV-90 cells. Concomitantly,
a remarkable reduction of the tissue inhibitor of metalloproteinases
3 (TIMP3) was appreciable in both cell lines (Figure 5D). The
results of zymography assays showed an increased gelatino- or
caseino-lytic activity in conditioned media derived from CD157-
overexpressing cells (Figure 5E). The increased proteolytic activity
significantly contributes to the enhanced trans-mesothelial migra-
tion of ovarian cancer cells with high levels of CD157. Indeed, in
the presence of GM6001 (a broad-spectrum inhibitor of MMPs)
both OVCAR-3/CD157 and OV-90/CD157 cells transmigrated
with an efficiency comparable to that of the corresponding
untreated mock cells. GM6001 also interfered with trans-
mesothelial migration of the highly invasive OV-90/mock cells
but not of the barely invasive OVCAR-3/mock cells (Figure 5C).
These results reflect an intense proteolytic activity associated with
CD157 overexpression in EOC cells which significantly con-
tributes to increase the invasive potential characterizing cells
expressing high CD157.
CD157 Overexpression Influences Spheroid Formation
and Disaggregation
The formation of cellular spheroids capable of floating in the
abdominal cavity and overcoming the environmental stresses is an
important step during tumor dissemination in EOC patients [36].
We generated spheroids from OV-90 cells and determined
whether the CD157-induced phenotype can overthrow the
spherical architecture acquired by OV-90 cells grown in
suspension. Accordingly with their natural propensity to form
spheroids [37], OV-90-mock cells gave rise to compact spherical
clusters (Figure 6A, top panel); in contrast, OV-90/CD157 cells
formed irregular clusters composed of loosely associated cells
(Figure 6A, bottom panel). The disruption of the spherical
architecture was accompanied by a considerable increase in the
invasive properties of cells. Indeed, when these disorganized
spheroids were seeded onto fibronectin-coated dishes a number of
cells rapidly escaped from the cell aggregates and invaded a large
area of the surrounding matrix, whereas, under similar conditions,
OV-90/mock spheroids maintained their quite compact structure
and cells leaking from these spheroids invaded a significantly
smaller area than the OV-90/CD157 cells (Figure 6B, C). These
results further support the association between high CD157
expression and the enhanced invasive proclivity of EOC cells.
Gene Expression Profiling Identifies Molecular Changes
Induced by the Overexpression of CD157 in OVCAR3 and
OV-90 Cells
To dissect the transcriptional changes that may mediate the
tumor aggressiveness associated with high CD157 expression, we
performed microarray gene expression analysis of OVCAR-3 cells
with and without CD157 and OV-90 cells with increased or basal
Figure 6. High CD157 expression influences spheroid formation and disaggregation in OV-90 cells. (A) Representative morphology of
spheroids formed by OV-90/mock (top) and OV-90/CD157 (bottom) (scale bar: 100 mM). (B) Phase contrast microscopy images of spheroid
disaggregation on fibronectin. A representative spheroid of OV-90/mock and OV-90/CD157 cells at time 0 and at 12 h are shown (scale bar: 200 mM).
(C) Spheroids were photographed at time 0 and at 12 h, and the increase in the size of disaggregation area calculated as described in Materials and
Methods is reported. The means 6 SD of the fold change in area of 20 spheroids per condition of a representative experiment, repeated (n=3), are
shown. ***P,0.001, two-tailed t test.
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expression of CD157. According to our selection criteria, 378
unique significantly modulated transcripts (163 upregulated and
215 downregulated) (Table S3 and S4), corresponding to 480
probes, were shared by OVCAR-3/CD157 and OV-90/CD157
cells (Figure 7A,B). To validate the microarray data, ten
upregulated genes (namely, EPCAM, CTTNBP2, BMP7, LAD1,
Figure 7. Gene expression profiling of OVCAR-3 and OV-90 cells overexpressing CD157. (A) Hierarchical clustering applied to the
expression matrix of genes similarly regulated in both OVCAR-3 and OV-90 cells overexpressing CD157, using Euclidean distance as similarity metrics
and complete linkage as the linkage method. A red-to-green gradient was used to indicate, for each gene, levels of up- or down-regulation. (B) Dot
plot shows 378 significantly modulated genes (163 up-regulated and 215 down-regulated) shared by OVCAR-3/CD157 and OV-90/CD157 cells. Single
genes are indicated by red (up) and green (down) data points. (C, D) A panel of modulated genes was selected and validated by qRT-PCR. (C) Fold
changes of the various indicated genes in OVCAR-3 and OV-90 cells following CD157 overexpression are shown. (D) qRT-PCR validation of the genes
shown in panel C. The comparative CT method was used to determine gene expression in CD157-transfected cells relative to the value observed in
the mock-transfected cells, using TBP as normalization control. Histograms report the means 6 SD of a qRT–PCR experiment conducted in triplicate.
(E) The expression of VCAN, EpCAM and BMP7 was examined in OVCAR-3/CD157, OV-90/CD157 and the corresponding control cells by western blot
analysis. Densitometry quantifies the expression level of the indicated proteins relative to b-actin. Results shown are representative of three
independent experiments with similar results.
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Figure 8. Gene ontology analysis of genes modulated by CD157 overexpression. Go analysis of differentially induced or repressed genes
shared by OVCAR-3/CD157 and OV-90/CD157 cells with respect to enrichment of genes with assignments to specific biological processes. The
number of genes in a specific biological process is indicated in brackets.
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VCAN, IGFBP2, NR2F1, HS6ST2, TPD52L1, E2F5) and three
down-regulated genes (namely ADM, MAP9, SYNE1) were chosen
for validation by qRT-PCR based on their levels of expression
(Figure 7B). The results of qRT-PCR confirmed those obtained by
our microarray analysis, with minor differences in amplitude in the
fold change expression (Figure 7C,D). Interestingly, among the
validated genes, EPCAM, VCAN, HS6ST2 and TPD52L1, whose
expression was increased in CD157-overexpressing cells, proved to
be decreased in OV-90/shCD157 cells (Figure S3). The modu-
lation of selected genes such as BMP7, EpCAM and VCAN
entailed an increase in the proteins encoded by them (Figure 7E),
indicating that high CD157 expression alters tumor cell pheno-
type.
Functional grouping and assessment of the Gene Ontology
(GO) designations of the 378 deregulated genes in CD157-
transfected cells indicated that upregulated genes included a sub-
stantial number of genes known to be implicated in development,
which also includes EMT, (such as S100A4, BMP7, WNT10A,
WNT6, FGF9, FZD4, FZD7, SFRP1, EPCAM and ten homeobox
HOXB3, DLX1, TSHZ1, HOXB8, HOXB5, SIX1, ONECUT2,
MNX1, HOXA10, HOXB9, among others), as well as genes related
to the control of cell motility and migration (for example, VAV3,
FUT8, PODXL, EFNB1, TPM1, SEMA6A, CTTNBP2, PVRL1,
SIX1, VCAN, NR2F2, NR2F1), and genes involved in the ECM-
receptor interactions (such as COL4A1, COL4A2, ITGAV, ITGB4,
LAMC2). The list of downregulated genes comprised genes playing
a role in the control of protein-DNA complex assembly (such as
H1F0, HIST1H2AC, HIST2H2AA4, HIST1H2AD, HIST1H2AE,
HIST1H3D, SIRT1, HIST1H4H) and of apoptosis and cell death
(such as CASP5, TP53I3, IL6, CASP4, CASP9, ERBB4, DUSP1,
CARD16, GULP1, BCL2A1, CASP1, PPP1R15A, EMP2) (Figure 8
and Table S5).
GeneGo pathways and process networks analysis of genes
representing the signature of CD157-overexpressing OVCAR-3
and OV-90 cells revealed a significant enrichment of genes
belonging to selected cell adhesion and ECM/cytoskeleton
remodeling signaling pathways, whose implication in tumor
progression is well documented. Similar process networks were
also on the top of the list. Moreover, networks with highest scores
included Notch signaling, connective tissue degradation (associat-
ed for example, with upregulation of the metalloproteinase
ADAM15 and down-regulation of TIMP3), apoptosis (associated
for example, with down-modulation of key elements of the caspase
cascade and the regulation of the neuregulin/erbB pathway) and
development, including regulation of EMT and blood vessel
morphogenesis (Table S5). Collectively, these data indicate that
forced expression of CD157 in EOC cells modulates the
transcription of a spectrum of genes encoding proteins involved
in crucial aspects of ovarian cancer dissemination.
Discussion
In this study we addressed the relevance of CD157 in the
induction of EOC aggressiveness and provided evidence that
CD157 overexpression is associated with dramatic variations in
tumor cell morphology, decreased cell-cell interactions, increased
anchorage independent growth, motility, and mesothelial invasion.
Indeed, ectopic expression of CD157 in OVCAR-3 cells, as well as
its increased expression in OV-90 cells, resulted in reduced cell-cell
contacts and adherens junction organization and enhanced cell
spreading, improving the ability of these tumor cells to move and
migrate as compared to the corresponding mock cells. The
exogenous expression of CD157 proved to be sufficient to convert
immobile OVCAR-3 epithelial cells into mesenchymal-like cells
characterized by weak contacts between neighboring cells, in-
creased motility, invasiveness, tumorigenicity, and improved
resistance to anoikis, all properties known to be fundamental
prerequisites for the progression of primary tumors to metastatic
disease [38]. These findings suggest that CD157 may function as
a potent driver of EOC progression. The observed association
between high levels of CD157 and the likelihood of disease
recurrence in patients with EOC implicitly supports a role of
CD157 in the control of tumor progression also in vivo [13].
There is a growing consensus that the events that convert
adherent and strictly connected tumor epithelial cells into
migratory cells capable of invading the ECM and establishing
distant metastases are reminiscent of the EMT occurring during
development [39]. In the ovary, EMT is a physiological process
during the postovulatory repair; in pathological contexts, such as
in tumors, however, it may have a detrimental effect, promoting
metastasis [29]. One of the leading events for EMT is the
downregulation of E-cadherin expression and function, which is
considered the hallmark of this process [30]. Despite the fact that
primary EOC express E-cadherin, advanced tumors have reduced
E-cadherin expression or none at all, suggesting that down-
regulation of E-cadherin is associated with the acquisition of the
invasive phenotype by EOC cells [40]. Exogenous expression of
CD157 in OVCAR-3 and TOV-21G cells that present typical
epithelial features, proved to suppress E-cadherin and to enhance
N-cadherin expression with consequent intracellular relocation
and partial nuclear translocation of b-catenin. In A2780 cells that
present a mixed epithelial/mesenchymal phenotype, forced
expression of CD157 increased the basal level of N-cadherin
and other mesenchymal traits. The CD157-driven differentiation
toward a mesenchymal phenotype is mainly choreographed by
Snail and Zeb1 transcriptional repressors. It is now evident that,
beside inducing the EMT program, these transcriptional factors
also confer resistance to apoptosis [33]. Furthermore, ZEB1 can
contribute to stemness maintenance thus enhancing the ability of
tumor cells to both disseminate and to fuel the growth of
metastases [41].
A crucial step in the progression of EOC is the release of tumor
cells into the peritoneal cavity. Once detached from the original
site, tumor cells disseminating throughout the peritoneum lose
their attachment to the neighboring cells and to the ECM,
resulting in anoikis [42]. We found that exogenous expression of
CD157 rescues tumor cells from anoikis despite it reduces the
ability of cells to form large aggregates, which are considered
a defense of tumor cells from anoikis. This apparent inconsistency
with what is thought to be the rule in ovarian cancer, suggests that
i) small aggregates of cells provide more favorable conditions for
anchorage-independent tumor cell survival than large aggregates,
and that ii) tumor cells expressing high CD157 likely develop
strategies relying on specific pro-survival signals, allowing both
individual cells and small aggregates that have detached from the
tumor to escape anoikis and to grow under anchorage-indepen-
dent conditions. The identification of the molecular basis of these
signals is expected to shed light on this issue. This observation
leads us to assume that in patients with advanced ovarian cancer,
high expression of CD157 may confer resistance to cell death
induced by the loss of adhesive supports, thus generating
a subpopulation of viable, highly malignant cells that might
account for a rapid tumor relapse.
Mesenchymal differentiation of EOC cells implies increased
secretion of MMPs which degrade and remodel the ECM, paving
the way to the establishment of metastases and the sprouting of
new vessels. Apart from this conventional activity, MMPs are
emerging as key modulators of the tumor microenvironment
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contributing to the formation of a metastatic niche [43]. Our data
show that CD157 regulates secretion of tumor-specific MMPs,
such as MMP2 and MMP7 (in OVCAR-3 cells) which play
a major role in early metastasis [44] and MMP9 (in OV-90 cells),
which is implicated in matrix invasion [45], is elevated in invasive
ovarian cancer specimens as well as ovarian carcinomatous ascites,
and correlates with lymph node metastasis [46]. The significant
reduction of TIMP3 (an endogenous inhibitor of MMPs) and
increase of ADAM15 transcripts, shared by both cell lines,
emphasize an imbalance of MMPs functions in tumor cells
overexpressing CD157.
In ovarian cancer, proteolytic degradation of ECM assists the
release of cells from the tumor mass and allows them to anchor to
and invade through the mesothelium establishing secondary
lesions and, at a later stage, to metastasize to distant organs.
Using a co-culture system to model aspects of the metastatic
process occurring in vivo [17], we demonstrated that the extent of
transmesothelial migration achieved by each EOC cell line
correlates with the level of CD157 and is influenced by the
activation of MMPs, further implicating CD157 in the control of
a crucial step of ovarian cancer metastasis, that is, mesothelial
invasion. We observed that the overexpression of CD157 in OV-
90 cells (showing a naturally low level of CD157) was able to
further increase their constitutive motility, reciprocally CD157
gene silencing reduced their basal motility, and that the extent of
CD157 silencing correlated with motility in OC314 cells.
Collectively, these data provide clear experimental evidence of
a relationship between CD157 levels and the progression of
metastatic ovarian cancer and support a direct role of CD157 in
regulating the aggressiveness of EOC cells reinforcing our previous
observation that high expression of CD157 is associated with
adverse clinical outcome in patients [13].
To gain further insight into CD157 function in EOC pro-
gression, we investigated gene expression changes following
CD157 transfection in OVCAR-3 and OV-90 cells. According
to our in vitro results, the analysis of genes deregulated in each line
confirmed the acquisition of clear mesenchymal traits in OVCAR-
3/CD157 cells (including downregulation of CDH1) but not in
OV-90/CD157 cells in which epithelial and mesenchymal traits
coexist (GEO database, ID: GSE36364). The analysis of genes
showing concordant modulation in both cell lines led to the
identification of 378 significantly altered genes, representing the
signature of both OVCAR-3 and OV-90 cells overexpressing
CD157. The overall picture inferred from the analysis of these
genes indicated that high CD157 expression results in strength-
ening of biological functions that favor tumor progression (for
example, cell differentiation, cell motility and migration), and
weakening of selected biological processes that hinder tumor
progression, such as apoptosis, cell death and response to stress.
Although the analysis of transcriptomic profiles alone is not
enough to permit definitive conclusions on the overall effects of
CD157-mediated EOC aggressiveness, however, its consistency
with the experimental data and clinical observations strongly
support the view that CD157 is directly implicated in the control
of ovarian cancer progression. The coexpression of molecules such
as BMP7, VCAN and EpCAM in EOC cells expressing high
CD157 further substantiates this view since these molecules are
considered negative prognostic markers involved in the control of
the progression of various types of tumor. For instance, BMP7
overexpression has been implicated in EMT in prostate cancer
[47] and with increased cell migration and invasion in breast
cancer [48]. VCAN is a mesenchymal marker whose increased
expression in ovarian cancer correspond to tumor progression,
metastatic dissemination and poorer survival outcome [49,50].
EpCAM is considered both an epithelial marker and ovarian
cancer stem-like cells marker whose expression is associated with
poor prognosis in ovarian cancer patients [51,52]. By virtue of this
double role attributed to EpCAM, the observed correlation
between CD157 and EpCAM expression in EOC cells does not
contradict the ability of CD157 to promote a mesenchymal-like
phenotype. Indeed, emerging evidence suggests that acquisition of
EMT and induction of cancer stem-like cell properties are
interrelated and contribute to tumor recurrence and metastatic
growth in several tumors [53]. The connection between EMT and
stemness could confer a crucial advantage to a single tumor cell by
increasing its ability to both disseminate and to begin a stemness-
associated growth and differentiation program in the metastasis.
The hypothesis that CD157 serves as a point of convergence in
conferring mesenchymal and stem cell-like differentiation to
ovarian cancer cells is very intriguing but it is beyond the scope
of this study.
Although the results of this study were derived from a limited
number of cell lines, the identification of a set of deregulated genes
in EOC with high CD157 strongly suggests a list of candidate
genes for further validation and functional analysis in patients with
EOC.
In summary, these data provide mechanistic support to our
previous studies [13], and demonstrate that the functional
contribution of CD157 to EOC progression relies on its ability
to switch on a differentiation program that allows cancer cells to
overlook the rules of epithelial tissue architecture and to advance
in their malignant progression. At the moment we don’t know
whether CD157 regulates EOC progression and aggressiveness
per se, or as part of a multimolecular complex governing cell
motility during metastatic progression. However, since CD157 is
devoid of transmembrane and cytoplasmic domain, and on the
basis of our previous results [9], we hypothesize that CD157 may
cooperate with other transmembrane receptors to fulfill its
functions. Lateral partners of the CD157 interactome and
mechanisms regulating CD157 interactions in ovarian cancer
are currently under investigation in our lab.
Additional studies are needed to further validate the tumori-
genic potential of OVCAR-3/CD157 and OV-90/CD157 cells in
animal models; however, the in vitro data demonstrate that high
expression of CD157 is sufficient to increase tumor aggressiveness
and tumorigenicity in several epithelial ovarian cancer cell lines. It
is tempting to speculate that CD157 might be a promising
therapeutic target for therapies aimed at controlling the invasion
and dissemination of the peritoneal cavity by ovarian cancer cells.
Supporting Information
Figure S1
of epithelial and mesenchymal markers in TOV-21G and
A2780 ovarian cancer cells. (A) sqRT-PCR and western blot
analysis of CD157 in mock- or CD157-transfected TOV-21G and
A2780 cells. The anti-b-actin mAb and GAPDH were used as
internal controls. (B) Western blot analysis of E-cadherin, b-
catenin, N-cadherin EMT markers, and Zeb-1 and Snail
transcription factors in total extracts from vector- or CD157-
transfected TOV-21G and A2780 cells. Densitometry quantifies
the expression level of the indicated proteins relative to b-actin. (C)
b-catenin protein level in cytoplasmic and nuclear fractions of
TOV-21G/mock and TOV-21G/CD157 cells were determined
by western blot analysis. a-tubulin and lamin B1 were used as
cytoplasmic and nuclear loading controls, respectively. Results
shown are from a representative experiment repeated at least twice
with similar results.
CD157 overexpression alters the expression
CD157 Promotes Ovarian Cancer Progression
PLOS ONE | www.plosone.org15August 2012 | Volume 7 | Issue 8 | e43649