Download SOX2 Expression Associates with Stem Cell State in Human

Published OnlineFirst July 18, 2013; DOI: 10.1158/0008-5472.CAN-12-4177
Cancer
Research
Tumor and Stem Cell Biology
SOX2 Expression Associates with Stem Cell State in Human
Ovarian Carcinoma
Petra M. Bareiss1, Anna Paczulla1, Hui Wang1, Rebekka Schairer1, Stefan Wiehr2, Ursula Kohlhofer3,
Oliver C. Rothfuss4, Anna Fischer3, Sven Perner6, Annette Staebler3, Diethelm Wallwiener5, Falko Fend3,
Tanja Fehm5, Bernd Pichler2, Lothar Kanz1, Leticia Quintanilla-Martinez3, Klaus Schulze-Osthoff4,7,
Frank Essmann4, and Claudia Lengerke1
Abstract
The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency
in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression
was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in
regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein
expression with respect to histopathologic and clinical parameters report contradictory results in individual
tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of
SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells,
SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo
tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display
enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show
that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on
SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness
and therapy resistance in human SOC. Cancer Res; 73(17); 5544–55. 2013 AACR.
Introduction
Pluripotency-associated stem cell factors such as OCT4 and
SOX2 regulate cellular identity in embryonic stem cells and
facilitate the reprogramming of terminally differentiated
somatic cells back to a pluripotent stem cell state (1). SOX
proteins are also important regulators of early development in
different tissues, such as the foregut and lung, where for
example SOX2 expression controls bronchogenesis by inhibiting airway branching (2, 3). In adult mice, SOX2 is expressed in
different epithelial compartments marking cells with selfrenewal properties (4), and targeted ablation lethally disrupts
epithelial tissue homeostasis (4). SOX2 expression is also found
Authors' Affiliations: Departments of 1Internal Medicine II and 2Preclinical
Imaging and Radiopharmacy, Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation; 3Institute of Pathology;
4
Interfaculty Institute for Biochemistry; 5Women's Hospital, University of
Tuebingen, Tuebingen; 6Institute of Pathology, University of Bonn, Bonn;
and 7German Cancer Consortium (DKTK) and German Cancer Research
Center, Heidelberg, Germany
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
P.M. Bareiss and A. Paczulla contributed equally to this work and share first
authorship.
Corresponding Author: Claudia Lengerke, Department of Internal Medicine II, University of Tuebingen, Otfried-Mueller-Strasse 10, 72076 Tuebingen, Germany. Phone: 49-7071-29-82899; Fax: 49-7071-29-4524;
E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-12-4177
2013 American Association for Cancer Research.
5544
in neural stem cells, where it promotes stemness by preventing
default differentiation into neurons (5).
More recently, SOX2 expression has been shown in several
tumor types such as lung (6–10), breast (11–14), skin (15, 16),
prostate (17), ovarian (18, 19), sinonasal (20) as well as different
types of squamous carcinomas (21). However, the SOX2 expression pattern and the correlation with histopathologic status
and clinical outcome are highly variable among tumors, suggesting distinct roles of SOX2 in individual tumors. In breast
carcinoma, SOX2 expression is mostly detected in a minor
subset of tumor cells and seems to be an early event in tumor
development (13), indicating potential roles in cancer stem
cells (CSC) biology and involvement in reprogramming processes generating CSCs. In support of this notion, induction of
SOX2 expression in breast carcinoma cell lines was shown to
enhance CSC properties such as tumor sphere potential and
in vivo tumorigenicity (12). Moreover, SOX2 expression was
associated with positive lymph-nodal status in early-stage
breast carcinoma (13). In contrast, in human squamous cell
lung cancer, SOX2 protein overexpression was associated
with smaller tumor size, lower probability of metastasis, and
improved clinical outcome. Other than breast carcinoma,
squamous cell lung cancer samples displayed homogenous
SOX2 expression, arguing against specific roles of SOX2 in lung
CSCs.
Ovarian carcinoma has the seventh highest morbidity
rate of cancer in women (22). Because of the lack of early
specific symptoms, ovarian carcinoma is mostly diagnosed at
advanced metastatic stages that cannot be cured by surgical
Cancer Res; 73(17) September 1, 2013
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SOX2 in Ovarian Carcinoma Stem Cells
resection alone. Despite initially good response rates to Platinbased chemotherapies, relapse is a common event during
the clinical course of the disease (22). An explanation for
ovarian carcinoma relapse is provided by the tumor stem cells
hypothesis, proposing that conventional chemotherapeutic
approaches target the fast proliferating bulk of the ovarian
cancer cells, while sparing the tumor-initiating CSCs (23). The
isolation and molecular characterization of ovarian CSCs are
thus subjects of intense research. Previous studies have suggested ALDH1, CD133, CD44, and CD117 as ovarian CSC
markers, but plasticity and transition between stem and
non–stem cell states complicates their efficient isolation
(24–32).
In this study, we hypothesized that SOX2 expression associates with stem cell state in ovarian carcinoma. SOX2 protein
expression can be detected in 15% to 60.5% of ovarian carcinomas (18, 19), depending on the staining methodology. Supporting our hypothesis, we observed that the majority of SOX2positive (SOX2þ) samples displayed SOX2 protein expression in
less than 10% of tumor cells. Moreover, SOX2 expression was
enhanced by culture conditions enriching for tumor stem cells.
Detailed analyses conducted on SOX2-modified human serous
ovarian carcinoma (SOC) cell lines and primary cells show that
indeed SOX2 expression induces CSC properties, such as
expression of stemness markers, tumor sphere formation,
in vivo tumor-initiating capacity as well as apoptosis resistance, thereby strongly promoting in vivo tumorigenicity and
enabling selective resistance to conventional anticancer
therapies.
Materials and Methods
Cell culture, tumor spheres, and cell growth assays
Ovarian cancer cell lines (Caov-3, OVCAR-3, OVCAR-5;
DSMZ; last authentication on January 31, 2013 at DSMZ) and
primary cells obtained through dissociation of tissue samples
derived from 4 patients with SOC were cultured under standard conditions. For the tumor sphere culture assay, cells were
grown in ultralow attachment plates (Corning) with sphere
medium and daily-added growth factors [20 ng/mL fibroblast
growth factor (FGF), 20 ng/mL EGF; Sigma-Aldrich] as
described previously (33). Spheres were counted between day
5 and 9. To investigate serial sphere formation, spheres were
washed with PBS and dissociated to single cells by trypsinization. To assess cell growth, 50,000 cells were plated under
adherent conditions and counted on day 3, 6, and 9. For sphere
cultures conducted to enrich for CSC activity, OVCAR-3 and
Caov-3 cells were maintained under sphere culture conditions
for 21 days and primary SOC cells for 10 days before undergoing
assessment.
Analysis of a tissue microarray of primary human
ovarian carcinomas
SOX2 protein expression and Ki67 positivity were investigated by immunohistochemistry using polyclonal goat antihuman SOX2 (AF2018; R&D Systems) and monoclonal mouse
anti-human Ki67 (clone MiB-1, M7240; DakoCytomation) on a
tissue microarray (TMA) including 215 human primary ovarian
carcinomas from patients treated at the Women's Hospital of
www.aacrjournals.org
the University of Tuebingen (Tuebingen, Germany). Detailed
information about the TMA construction and analysis are
provided in the Supplementary Data. The study was approved
by the Ethics Committee of the University of Tuebingen.
Lentiviral transduction
Lentiviruses carrying SOX2 short hairpin RNA (shRNA),
SOX2 overexpression, corresponding empty GFP-, and SOX2enhancer reporter constructs (34) were designed, produced,
and used for transduction as previously reported (35–37).
Details on lentiviral constructs and protocols are provided in
the Supplementary Data.
Flow cytometry analysis of stem cell markers, cell cycle,
and BrdU assays
To detect aldehyde dehydrogenase (ALDH) activity, the
ALDEFLUOR assay was used according to the manufacturer's
guidelines (STEMCELL Technologies). Cells were incubated in
ALDEFLUOR assay buffer for 30 minutes. Cells from each
sample additionally treated with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) served as negative controls. For
flow-cytometric analyses, anti-CD133 (Miltenyi Biotec) and
anti-TRAIL receptor 1 and 2 antibodies (BioLegend) were used.
Dead cells were detected by 40 ,6-diamidino-2-phenylindole
(DAPI) staining (100 ng/mL). Cell-cycle analysis using propidium iodide (PI; Sigma) staining and bromodeoxyuridine
(BrdU, Roche) incorporation assays using mouse anti-BrdU
V450 antibody (BD Biosciences) were conducted as previously
described (36). Flow-cytometric analyses were conducted
using a FACSCanto II and data analysed using the FACSDiva
software (BD Biosciences).
Apoptosis assays
Cells seeded at 50,000 cells/cm2 were incubated overnight
and then treated for 24 hours with staurosporine (2.5 mmol/L;
Sigma-Aldrich) or SuperKiller TRAIL (25 ng/mL; Enzo Life
Sciences), or for 96 hours with cisplatin (5 mmol/L; Medac),
carboplatin (100 mmol/L; Medac), or paclitaxel (5 nmol/L;
Bristol-Myers Squibb). Cells were harvested by trypsinization,
fixed in 70% ice-cold ethanol, and incubated in PBS containing
50 mg/mL PI and 100 mg/mL RNase A. Cells with subdiploid
DNA content (sub-G1) were assessed by flow cytometry. Caspase-3/7 activity was assessed by the Caspase-Glo 3/7 assay
(Promega) and normalized to protein content following treatment with staurosporine (4 hours) or TRAIL (6 hours).
Immunoblot and immunocytochemistry analyses
Immunoblot and immunocytochemistry analyses were conducted using mouse anti-actin (LI-COR Biosciences), rabbit
anti-caspase-3 (Cell Signaling Technology), rabbit anti-cleaved
caspase-3 (Asp175; Cell Signaling Technology), and rabbit
anti-SOX2 (D6D9) XP (Cell Signaling Technology) antibodies.
Detection was carried out using IRDye 800CW-conjugated goat
anti-rabbit immunoglobulin G (IgG) or IRDye 680 anti-mouse
IgG antibodies (LI-COR Biosciences) and an Odyssey Imager
(LI-COR Biosciences). For detection of the SOX2 knockdown
horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody
(Cell Signaling Technology) and ECL Prime Western Blotting
Detection Reagent (GE Healthcare) were used.
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Published OnlineFirst July 18, 2013; DOI: 10.1158/0008-5472.CAN-12-4177
Bareiss et al.
Gene expression analyses
RNA isolation, cDNA preparation, and real-time gene
expression analyses were conducted as described previously
(13, 38) using a LightCycler 480 instrument and LightCycler
probes master mix (for SOX2, ALDH1, LIN28, NANOG, OCT4,
and GAPDH; Roche) or SYBR Green assay (for BBC3, PMAIP1,
and BCL2; Eurogentec). Primers and probes are listed in the
Supplementary Data. Relative expression levels were calculated after normalization to the reference gene glyceraldehyde-3phosphate dehydrogenase (GAPDH; Probe) or PBGD (SYBR
Green) by using the DDCT method.
Xenotransplantation model
NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (also termed NOD/SCID/
IL2Rg null, abbreviated as NSG) mice (39) were purchased
from The Jackson Laboratory and maintained under pathogen-free conditions. Control and SOX2-overexpressing
Caov-3 cells mixed with Matrigel (1:2; BD Biosciences)
were implanted subcutaneously in individual flanks of the
same mouse. Tumor growth was monitored by palpation of
the injection site and positron emission tomography (PET)/
magnetic resonance imaging (MRI) analysis conducted using
intravenously administered 11 to 15 MBq of 2[18F]fluoro-2deoxy-D-glucose (FDG) as described previously (40, 41) and
summarized in the Supplementary Data. Mice were euthanized 7 to 15 weeks after implantation. For histologic
analysis, mouse tissues were fixed in 4% formaldehyde,
paraffin-embedded, cut in 3 to 5 mm sections, and stained
with hematoxylin and eosin (H&E). Immunohistochemical
analysis was conducted as described previously (42) on an
automated immunostainer (Ventana Medical Systems)
according to the company's protocol for open procedures
with slight modifications. The antibody panel used included
SOX2 (SP76; Cell Marque), cleaved caspase-3 (Asp 175; Cell
Signaling Technology), Ki67 (SP6; DCS Innovative Diagnostik
Systeme), and EpCAM (BerEp4; Dako).
Statistical analyses
For all experiments, mean values are presented and error
bars represent the SE if not otherwise indicated. P values are
derived via the application of a two-tailed, unpaired Student
t test.
Results
SOX2 expression is enhanced in CSC-enriched SOC cell
cultures
SOX2 mRNA expression was investigated by real-time PCR
in SOC patient samples and cell lines including the lines
Caov-3 and OVCAR-3 harboring amplifications on the chromosome 3q (Fig. 1A). Heterogeneous expression of SOX2 was
noted (Fig. 1A and Supplementary Fig. S1) mirroring the
results documented in TMAs of human SOC samples (18, 19).
Previous data on ovarian and breast cancer cells reported
that sphere cultures increase CSC frequency as compared
with two-dimensional (2D) adherent cultures (27, 33).
Indeed, ovarian cancer cell lines grown as spheres for 21
days showed a higher frequency of ALDHhighCD133þ putative CSCs (OVCAR-3 cells; Fig. 1B) and enhanced expression
5546
Cancer Res; 73(17) September 1, 2013
of putative stem cell markers in comparison with 2D cultures (Caov-3 cells; Fig. 1C). Consistent with a role of SOX2 as
a stem cell marker in SOC, SOX2 expression was also
enhanced in sphere cultures of Caov-3 as well as primary
ovarian carcinoma cells (Fig. 1C).
SOX2 modulates CSC properties in human SOC cells
To explore the functional role of SOX2 in ovarian carcinoma,
we stably suppressed SOX2 expression in OVCAR-3, the SOC
line with the highest basal SOX2 expression, using two different
lentiviruses containing SOX2-inhibitory shRNAs (Fig. 2A and
Supplementary Fig. S2A). Furthermore, Caov-3 cells displaying
low-basal SOX2 expression as well as primary SOC cells derived
from patients were treated with SOX2 lentiviruses to study
the effects of SOX2 overexpression (Fig. 2B and Supplementary Fig. S3A). Cells transduced with empty GFP-lentiviruses
were used as controls.
Induction of SOX2 expression in both Caov-3 and primary
SOC cells was able to enhance the expression of other
putative stem cell markers (LIN28, NANOG, OCT4, and
ALDH1; Fig. 3A and B), suggesting that activation of SOX2
expression is sufficient to facilitate the transition to a stem
cell–like state. Consistent with this notion, enhanced tumor
spheres formation was observed in SOX2-overexpressing
cells, whereas SOX2 knockdown induced the opposite effect
(Fig. 4A). Notably, the effect on tumor sphere formation was
also documented in primary SOC cells and was even more
pronounced upon serial replating (Fig. 4B). Single cell tumor
sphere assays further confirmed the higher frequency of
sphere-initiating cells in SOX2-expressing versus control
cells (Fig. 4C).
To further explore the role of SOX2 as a CSC marker in human
SOC, we treated OVCAR-3 cells with a lentiviral SOX2-reporter
construct, previously described to recognize cells with high
SOX2 promoter activity (SOX2þ) in breast carcinoma and neural
stem cells (Supplementary Fig. S4A; refs. 12, 43). Puromycin
selection was applied to select for efficiently transduced cells
and SOX2þ cells were visualized by fluorescence. Supporting
our previous data, SOX2þ cells were enriched in OVCAR-3 cells
cultured as spheres, as compared with 2D cultures (Supplementary Fig. S4B). In primary sphere assays, nearly every SOX2þ
cell isolated by fluorescence-activated cell sorting (FACS) gave
rise to an individual tumor sphere (SOX2þ sphere), and SOX2þ
spheres were larger than those derived from reporter-negative
cells [SOX2-negative (SOX2) spheres; Fig. 4D; Supplementary
Fig. S4C]. Importantly, flow cytometry of SOX2þ primary
spheres revealed a mixture of SOX2þ and SOX2 cells, suggesting that SOX2þ cells undergo both self-renewal and differentiation processes giving rise to both populations of cells. In
contrast, primary SOX2 spheres remained SOX2 (Supplementary Fig. S4C). Consistent with these data, cells derived from
SOX2 spheres exhausted their sphere generation potential
upon serial replating, whereas cells derived from SOX2þ spheres
maintained sphere formation (Fig. 4D).
Together, these data indicate that cells with self-renewal
capacity segregate to the SOX2þ compartment and suggest
that SOX2 induction can activate CSC molecular pathways
and functional properties in human SOC cells.
Cancer Research
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SOX2 in Ovarian Carcinoma Stem Cells
A
2.5
250
1
0.1
0
0
-O
V3
A
O
VC
A
O
VC
B
#1
#2
#3
#4
SK
R
-5
R
R
A
O
VC
ao
C
-8
0.5
0.2
-3
150
2
v3
Relative SOX2 gene expression
4.5
350
C
OVCAR-3
Caov-3
P < 0.0001
% Positive cells
60
50
40
P = 0.0481
30
20
10
0
ALDH+
CD133+
Relative gene expression
Spheres
2D culture
25
15
5
3
2
1
0
SOX2
ALDH+/CD133+
***
11
9
9
7
7
***
3
1
0
LIN28
OCT4
***
11
5
ALDH1
Patient #2
Patient #1
***
***
SOX2 ALDH1 LIN28 NANOG OCT4
Induction of SOX2 strongly enhances in vivo
tumorigenicity in a NSG mouse model
To explore the relevance of SOX2 expression in SOC cells
in vivo, we carried out xenotransplantation experiments of
SOX2-overexpressing and control Caov-3 cells in immunopermissive NSG mice (39). The same numbers of SOX2-overexpressing and control Caov-3 cells were implanted subcutaneously in the flanks of 8-week-old female mice as indicated, and
tumor induction was monitored every second week by palpation of the injection sites. To avoid bias through different
animal hosts, SOX2-overexpressing and control cells were
injected in the right and left flank of the same mouse. When
500,000 cells were injected per flank, control Caov-3 cells
generated tumors at 4 weeks postinjection in 1 of 4 animals
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Patient samples
e–03
450
Relative gene expression in
spheres vs. 2D
Figure 1. Enhancement of SOX2
expression in CSC-enriched
spheres cultures. A, SOX2 shows
heterogeneous gene expression in
ovarian cancer cell lines and
patient samples. B, OVCAR-3 cells
cultured as spheres for 21 days are
high
þ
enriched for ALDH CD133
putative ovarian CSCs as
measured by flow cytometry.
C, enhanced gene expression of
SOX2 and other putative stem cell
genes in Caov-3 cells cultured for
21 days and patient samples
cultured for 10 days under sphere
conditions, as compared with
corresponding 2D-cultured cells.
Relative gene expression was
analyzed by real-time PCR after
normalization to GAPDH. Shown
are the fold changes in relative
gene expression of cells cultured in
spheres (gray box) relative to cells
cultured in 2D (dashed line). Data
represent the mean values SEM
from 3 or more independent
biologic experiments carried out in
triplicates for the Caov-3 cell line,
and respectively from technical
triplicates in the patient samples
( , P < 0.05; , P < 0.01;
, P < 0.005; n.s., not significant).
Cell lines
e–04
***
5
3
1
0
n.s.
**
*
n.s.
SOX2 ALDH1 LIN28 NANOG OCT4
(Fig. 5A). Lowering the number of transplanted cells to 100,000
or 50,000 cells per animal delayed tumor formation from
control Caov-3 cells (Fig. 5A). In contrast, SOX2-overexpressing
cells robustly induced tumors in all transplanted animals and
accelerated the appearance of palpable tumor masses (Fig. 5A).
To further consolidate these observations, we conducted
in vivo PET/MRI analyses and ex vivo immunohistologic
analyses at the end of the experiment. At week 15 postinoculation of 100,000 cells, tumors were detected on both
sites in all mice by the sensitive PET/MRI method (Fig. 5D).
However, quantitative image analysis of tumor volumes
revealed that SOX2-overexpressing cells induced much
larger tumors than control cells (118.2 19.0 vs. 40.4
mm3 at 8 weeks following injection of 500,000 cells, and
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Bareiss et al.
A
B
SOX2 knockdown
SOX2 overexpression
Caov-3
OVCAR-3
P < 0.0001
P = 0.0127
100
Relative SOX2
gene expression
Relative SOX2
gene expression
1.00
0.75
0.50
0.25
75
50
25
0
0.00
Control
SOX2
Control
shSOX2
Protein expression
Control SOX2
Control shSOX2
SOX2
35 kDa
SOX2
35 kDa
Actin
40 kDa
Actin
40 kDa
respectively 618.6 244.6 vs. 50.98 21.8 mm3 at 15 weeks
when 100,000 cells were injected; Fig. 5B), which was also
confirmed by immunohistologic analysis (Fig. 5C). Interestingly, PET-quantification of FDG uptake (40) revealed similar
metabolic activity in SOX2-overexpressing and control tumors
at both measured time points (8 and 15 weeks postinjection,
postinoculation; Fig. 5B), indicating that the inductive effects
A
SOX2
Control
600
220
205
Fold relative gene expression
in SOX2-overexpressing vs. control cells
B
Caov-3
***
**
7
***
***
***
5.0
1.0
0
SOX2
Control
***
5
***
***
SOX2 ALDH1 LIN28 NANOG OCT4
0
***
SOX2 ALDH1 LIN28 NANOG OCT4
Patient #4
Patient #3
4
3
***
1
SOX2
Control
***
***
*
2
*
n.s.
1
25
***
SOX2
Control
***
15
2
n.s.
***
n.s.
Figure 3. SOX2 expression
enhances expression of putative
stem cell genes. Caov-3 (A) and
primary SOC patient-derived cells
(B) were analyzed by real-time PCR
and normalized for GAPDH. Shown
are the fold changes in relative
gene expression in cells cultured in
spheres (gray box) versus cells
cultured in 2D (dashed line). Shown
are data from one representative
out of 3 independent biologic
experiments carried out in
triplicates for Caov-3 cells and
from technical triplicates in primary
cells ( , P < 0.05; , P < 0.01;
, P < 0.005; n.s., not significant).
1
0
0
SOX2 ALDH1 LIN28 NANOG OCT4
5548
of SOX2 on tumor formation were not mediated by modulation of metabolic activity. However, due to their larger mass,
tumors derived from SOX2-overexpressing cells partially displayed necrotic areas revealing a heterogeneous uptake of
FDG at the measured time points.
Interestingly, the pronounced difference of in vivo tumorigenicity between SOX2-overexpressing and control cells was
Patient #1
3
0.5
Figure 2. Modulation of SOX2
expression. Modulation of SOX2
mRNA and protein expression in
ovarian cell lines after lentiviral
SOX2 knockdown (A) or SOX2
overexpression (B) in comparison
with control lentiviruses. Top,
relative gene expression levels
normalized to GAPDH, as
measured by real-time PCR
analysis of 3 representative
independent biologic experiments,
each carried out in triplicates.
Bottom, immunoblot analyses
representative for 3 independent
biologic replicates.
Cancer Res; 73(17) September 1, 2013
SOX2 ALDH1 LIN28 NANOG OCT4
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SOX2 in Ovarian Carcinoma Stem Cells
SOX2 overexpression
OVCAR-3
15
P = 0.0161
10.0
7.5
5.0
2.5
0.0
Control
SOX2
Number of spheres
per 100 cells
Number of spheres
per 100 cells
12.5
SOX2 overexpression
B
SOX2 knockdown
Caov-3
Patient #1
P = 0.0185
10
5
0
Patient #2
P < 0.001 (GFP+)
P < 0.001 (all)
P = 0.0016 (GFP+)
35
P = 0.0165 (all) 15
Number of spheres
per 100 cells
A
35
P = 0.0166 (GFP+)
P = 0.0292 (all)
15
P < 0.001 (GFP+)
P < 0.001 (all)
30
30
25
25
10
10
20
20
15
15
5
10
5
10
5
5
0
Control SOX2
Control shSOX2
0
0
0
Control SOX2
Control
SOX2
Control
SOX2
GFP–
GFP+
Primary spheres Secondary spheres Primary spheres Secondary sphere
SOX2 overexpression - single spheres
Caov-3
20
P = 0.0372
15
10
5
0
Control
SOX2
10
P = 0.0021
5
5
P = 0.0085
10
5
0
0
SOX2
Control
Control SOX2
Secondary spheres
Tertiary spheres
SOX2
Quaternary spheres
SOX2 reporter cells
D
OVCAR-3
P < 0.001
60
40
20
0
SOX2 – SOX2+
Primary spheres
25
20
15
10
5
0
30
30
P < 0.001
SOX2 –
Number of spheres
per 100 cells
Number of spheres
per 100 cells
30
80
25
20
P = 0.0202
15
10
SOX2+
Secondary spheres
5
0
SOX2 – SOX2+
Tertiary spheres
Number of spheres
per 100 cells
100
Number of spheres
per 100 cells
10
0
Control
Primary spheres
15
15
Number of spheres per
20 plated cells
25
P < 0.001
15
Number of spheres per
20 plated cells
30
Number of spheres from one
dissociated single sphere
Number of spheres
per 100 plated single cells
C
25
20
P = 0.0167
15
10
5
0
SOX2 – SOX2+
Quaternary spheres
Figure 4. SOX2 increases the sphere-forming potential of ovarian cell lines and primary tumor cells. A, SOX2-modified and control Caov-3 and OVCAR-3 cells
(plated 1,250 cells/well) were scored for primary sphere formation after 9 and 5 days, respectively. Shown are data from 3 independent biologic
experiments carried out in triplicates. B, primary SOC cells were transduced with lentiviruses for GFP-tagged SOX2 or a GFP control. Cells were then directly
þ
plated at a density of 625 cells per well without prior FACS. After 5 days, sphere formation of transduced (GFP ) and nontransduced (GFP ) cells was
microscopically scored. Primary spheres were subsequently dissociated to single cells and used for the replating experiments to investigate secondary
sphere formation. Shown are data from technical triplicates. C, single SOX2-overexpressing and control Caov-3 cells were assessed for their sphere-forming
potential in 96-well plates. Shown are data from 3 independent biologic experiments. Secondary spheres were generated by replating of cells
dissociated from one individual primary sphere in each well. For tertiary and quaternary spheres, pooled spheres dissociated to single cells were replated at a
density of 20 cells per well. D, SOX2þ and SOX2 OVCAR-3 cells isolated by FACS were plated in sphere conditions 100 cells per well and spheres
counted after 7 days. For all replating assays, spheres were pooled, dissociated to single cells, and replated as indicated or at a density of 100 cells
per well. Primary and secondary sphere formation was analyzed in 3 or more, tertiary and quaternary sphere formation in 2 independent biologic experiments
carried out in triplicates (C and D).
not due to enhanced cellular proliferation, as revealed by the
similar results of the Ki67 staining conducted on explanted
tumors (Fig. 5C). SOX2-overexpressing tumors displayed
more necrotic areas and a higher apoptotic activity, as
shown by the active caspase-3 staining (Fig. 5C). These
findings are in line with the in vivo PET/MRI results where
www.aacrjournals.org
necrotic areas were detected in all SOX2-overexpressing
tumors (Fig. 5B).
Overall, these data strongly suggest that SOX2 mediates
tumorigenicity in SOC cells by facilitating transition to a CSC
state with enhanced tumor-initiating properties. To further
explore this hypothesis, we conducted a limiting dilution
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Bareiss et al.
500,000 cells
100,000 cells
5
Control
SOX2
4
Number of
palpable tumors
Number of
palpable tumors
5
3
2
1
0
50,000 cells
5
Control
SOX2
4
Number of
palpable tumors
A
3
2
1
2
4
Weeks
4
3
2
1
0
0
0
Control
SOX2
01
0 2 4 6 8 10 12 14
Weeks
6
1,250
Volumetry (mm3)
B
3
5 7 9 11
Weeks
500,000
cells
1,000
100,000
cells
750
500
250
0
n=1 n=4
Co
l
ro
nt
SO
%ID/cc FDG
4
X2
500,000
cells
3
n=4 n=4
15 wks p.i.
8 wks p.i.
100,000
cells
2
1
0
n=1 n=4
8 wks p.i.
n=4 n=4
15 wks p.i.
SOX2
Control
H&E 12.5x
SOX2 12.5x
Ki 67 12.5x
Caspase-3 12.5x
Control
C
SOX2
SOX2
Control
H&E 50x
in vivo transplantation assay: 10,000, 1,000, 100, and 10 SOX2overexpressing and control Caov-3 cells were transplanted as
described earlier in the contralateral flanks of n ¼ 5 mice per
group. In contrast to the results observed with higher numbers
of cells (Fig. 5A), no palpable tumors were documented at 7
weeks posttransplantation. However, immunohistologic analysis of the injection sites revealed microscopic human tumor
cell clusters in animals injected with 10,000 or 1,000 cells, but
not 100 or 10 cells (H&E staining; Fig. 5D and E). Staining
with antibodies against human EpCAM (Supplementary Fig.
S5) and CA125 (not shown) confirmed correct detection of
human SOC cells. Notably, microscopic tumors were detected
5550
Immunohistologic tumor
detection after 7 wks
Cells injected
SOX2
E
D
Cancer Res; 73(17) September 1, 2013
SOX2 Control
10,000
4/5
3/5
1,000
4/5
1/5
100
0/5
0/5
10
0/5
0/5
Control
Figure 5. SOX2 expression confers
tumorigenic potential in vivo.
SOX2-overexpressing and control
Caov3 cells were injected as
indicated at same numbers
contralaterally in the flanks of NSG
mice (n ¼ 5 mice/group). A, as
compared with controls, SOX2overexpressing tumors were
detected faster and at higher
frequency by palpation. B, SOX2
expression results in increased
tumor size but unaltered metabolic
activity, as revealed by FDG PET/
MRI (left, representative image of a
tumor-bearing mouse scanned
15 weeks after cell inoculation;
right, tumor volumetry and
quantification of tracer
accumulation). C, SOX2overexpressing tumors show
similar Ki67 and enhanced active
caspase-3 staining by
immunohistochemistry. D, limiting
dilution assays reveal increased
tumor cell cluster formation of
SOX2-overexpressing versus
control cells, as shown by
immunohistochemical analysis of
the injection site 7 weeks after
inoculation. Tumor clusters were
detected in animals injected with
10,000 or 1,000 cells, but not with
100 or 10 cells. E, H&E staining of
representative samples derived
from mice inoculated with 1,000
cells. Pictures were taken at the
indicated magnification. p.i.,
postinoculation.
H&E 630x
more frequently from SOX2-overexpressing as compared with
control cells, which was especially evident with the lowest
number of injected cells (Fig. 5D and E).
SOX2 expression does not affect cell proliferation but
enhances the apoptosis resistance of SOC cells
Modulation of SOX2 expression did not alter cell-cycle
progression, BrdU incorporation (Fig. 6A), or cell growth of
in vitro 2D-cultured OVCAR-3 and Caov-3 cells (Fig. 6B). To
further explore whether SOX2 regulates SOC cell proliferation,
we analyzed SOX2 protein expression and Ki67 staining on a
TMA of 215 human primary ovarian carcinomas comprising
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SOX2 in Ovarian Carcinoma Stem Cells
SOX2 overexpression
SOX2 knockdown
Control
SOX2
60
50
40
30
20
10
40
30
20
10
0
S–M–G2-phase
G0-phase
40
7.5
Control
SOX2
5.0
2.5
0
0 1 2 3 4 5 6 7 8 9 10
Days
S–M–G2-phase
SOX2 knockdown
35
% BrdU-positive cells
% BrdU-positive cells
Control
shSOX2
50
0
G0-phase
SOX2 overexpression
Caov-3
60
% PI-positive cells
% PI-positive cells
70
B
OVCAR-3
Number of viable cells
(x105)
Caov-3
30
20
10
0
Control
30
25
20
15
10
5
0
SOX2
Control
shSOX2
Number of viable cells
(x105)
A
12.5
10.0
OVCAR-3
Control
shSOX2
7.5
5.0
P = 0.0053
2.5
0
0 1 2 3 4 5 6 7 8 9 10
Days
C
Primary SOC samples
D
SOX2 overexpression
SOX2 knockdown
OVCAR-3
Caov-3
90
80
70
60
50
40
30
20
10
0
12
14
12
Sub-G1 fraction %
Sub-G1 fraction %
% Ki67 positivity
P = 0.004
10
8
6
4
2
0
Negative Low Medium High
10
8
6
4
2
0
Control
SOX2
Control
shSOX2
SOX2 protein positivity
Figure 6. SOX2 effects on cell proliferation and basal apoptosis. SOX2 expression does not influence cell-cycle distribution, BrdU uptake (A) or cell
growth (B) of 2D-cultured cells. C, SOX2 protein expression and Ki67 staining in primary high-grade SOC samples. Analysis was conducted on
a TMA comprising 143 high-grade SOCs. D, SOX2 expression modulates basal apoptosis as measured by percentages of sub-G1 cells. Shown are
data from 3 or more independent biologic experiments carried out in triplicates in Caov-3 and OVCAR-3 cells (A, B, and D).
143 high-grade SOC (see Supplementary Data for details on
TMA construction). In 136 of 143 high-grade SOC samples,
both SOX2 and Ki67 stainings were available. Although SOX2
expression was found in 64.6% of high-grade SOC samples (Fig.
6C; Supplementary Fig. S1; data not shown), Ki67 positivity was
not dependent on the SOX2 expression level, which is in
contrast to findings in other tumor entities (11, 17).
As mentioned earlier, the histologic analysis of the murine
tumors revealed higher levels of active caspase-3 in SOX2overexpressing tumors (Fig. 5C). However, enhanced caspase-3
activation is most likely a secondary effect in response to
restrictive in vivo environmental factors (e.g., insufficient blood
supply due to disproportionate tumor growth overriding
tumor's capacity of vessel recruitment), as in vitro 2D culture
experiments showed reduced levels of spontaneous apoptosis
in SOX2-overexpressing Caov-3 as well as OVCAR-3 cells (Fig.
www.aacrjournals.org
6D). To test whether SOX2 expression modulates apoptosis
sensitivity, we incubated the cells with staurosporine and the
death ligand TRAIL to activate the intrinsic and extrinsic
pathways of apoptosis. Flow-cytometric quantitation of subG1 cell populations revealed enhanced apoptosis in response to
staurosporine and TRAIL in the SOX2 knockdown cells, whereas SOX2 overexpression conferred enhanced resistance (Fig. 6A
and B and Supplementary Figs. S2 and S6). We also assayed
activity of caspase-3/7 in substrate cleavage assays (Fig. 7A and
B) and processing of caspase-3 by immunoblot analysis for
cleaved caspase-3 (Supplementary Fig. S7), confirming the
resistance-mediating potential of SOX2 expression. Importantly, SOX2 expression in Caov-3 cells also mediated resistance to
carboplatin, cisplatin, and paclitaxel (Fig. 7C and Supplementary Fig. S7), indicating SOX2 expression as a molecular driver
of chemotherapy resistance in ovarian carcinoma. Notably, the
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Bareiss et al.
SOX2 overexpression
30
20
10
SOX2
Control
10
0
Control
P = 0.0001
50
40
30
20
10
0
Relative RLU (%)
75
50
25
400
300
200
100
0
SOX2
Chemotherapy
treatment
Control
D
50
25
shSOX2
Control
30
20
10
Control
shSOX2
175
75
0
40
0
P < 0.0001
100
500
50
SOX2
Control
shSOX2
600
100
C
20
Relative RLU (%)
0
30
P = 0.0036
60
Sub-G1 fraction %
(specific apoptosis)
40
OVCAR-3
P < 0.0001
60
40
Sub-G1 fraction %
(specific apoptosis)
50
SOX2 knockdown
Caov-3
P = 0.0060
Sub-G1 fraction %
(specific apoptosis)
Sub-G1 fraction %
(specific apoptosis)
P = 0.0073
Control
SOX2 overexpression
OVCAR-3
60
0
TRAIL treatment
SOX2 knockdown
Caov-3
Relative RLU (%)
B
Staurosporine treatment
Relative RLU (%)
A
150
125
100
75
50
25
0
SOX2
Control
shSOX2
Rescue of SOX2 expression in SOX2 knockdown OVCAR-3 cells
SOX2 overexpression
TRAIL treatment
Staurosporine treatment
0.50
0.25
0.00
SOX2 knockdown
Caov-3
1,000
Control
SOX2
800
600
400
200
0
TRAILR1
OVCAR-3
Difference of median
fluorescence intensity
Difference of median
fluorescence intensity
SOX2 overexpression
1,200
40
30
20
10
0
TRAILR2
3,000
Control
shSOX2
2,000
1,000
0
TRAILR1
TRAILR2
50
40
30
20
10
0
Control shSOX2 shSOX2
Control shSOX2 shSOX2
+ SOX2
Cisplatin Carboplatin Paclitaxel
E
50
P = 0.0120
60
Control
shSOX2
shSOX2
+ SOX2
F
+ SOX2
SOX2 overexpression
Caov-3
120
P = 0.0001
Control
SOX2
100
80
60
40
20
0
BBC3
PMAIP1
Proapoptotic
Relative gene expression
of BCL2
0.75
60
Sub-G1 fraction %
(specific apoptosis)
Sub-G1 fraction %
(specific apoptosis)
1.00
P = 0.0073
P = 0.0014 P < 0.0001
P < 0.0001 P = 0.0078
P = 0.0232
P = 0.0040
Relative gene expression
Control
SOX2
70
60
50
40
30
20
10
0
Relative SOX2
gene expression
Sub-G1 fraction %
(specific apoptosis)
Caov-3
P = 0.0042
250
200
150
100
50
0
Control
SOX2
Antiapoptotic
Figure 7. SOX2 expression induces apoptosis resistance. Elevated SOX2 expression reduces apoptotic responses after treatment with staurosporine (A),
TRAIL (B), and chemotherapeutic drugs (cisplatin, carboplatin, and paclitaxel; C). D, SOX2 overexpression restores apoptosis resistance in SOX2-knockdown
cells. E and F, SOX2 modulation does not change TRAIL-R1 and TRAIL-R2 surface expression, but influences mRNA expression of BCL2, BBC3, and
PMAIP1. Shown are percentages of sub-G1 cells and caspase-3/7 activities carried out in Caov-3 and OVCAR-3 cells (A–D) and differences of median
fluorescence intensities (E). Relative gene expression levels were determined by real-time PCR and normalized to PBGD (D and F). The results of each panel
represent mean values SEM from 3 independent biologic experiments carried out in triplicates. RLU, relative light units.
enhanced sensitivity of OVCAR-3 cells due to SOX2 knockdown
was reverted by lentiviral reexpression of ectopic SOX2 (Fig.
7D). These data show that the observed phenotype specifically
depends on SOX2 expression levels. Analog experiments carried out in a third cell line (OVCAR-5) and using an alternative
SOX2 shRNA sequence furthermore confirmed these results
(Supplementary Fig. S2; data not shown).
In an attempt to elucidate the molecular basis for SOX2induced resistance to TRAIL-mediated apoptosis, we initially
5552
Cancer Res; 73(17) September 1, 2013
analyzed surface expression of the TRAIL receptors 1 and 2 by
flow cytometry. However, no significant difference in the
expression level of TRAIL-R1 or TRAIL-R2 was detected in
OVCAR-3 and Caov-3 cells in response to SOX2 knockdown and
overexpression, respectively (Fig. 5E). Therefore, SOX2 expression modulates apoptosis sensitivity downstream of these
death receptors, as usually seen in so-called type II cells that
depend on amplification of death receptor signaling via the
intrinsic apoptotic pathway. As the intrinsic pathway is
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SOX2 in Ovarian Carcinoma Stem Cells
controlled by the BCL2 protein family, we analyzed the expression of proapoptotic (PUMA/BBC3 and NOXA/PMAIP1) and
antiapoptotic genes (BCL2) by real-time PCR. In line with the
observed apoptosis resistance, overexpression of SOX2
induced enhanced expression of antiapoptotic BCL2, whereas
reducing expression of the proapoptotic proteins PUMA/BBC3
and NOXA/PMAIP1 (Fig. 7F).
Discussion
SOX2 is a key regulator for maintaining the pluripotency
and self-renewal of embryonic stem cells and contributes to
the reprogramming of differentiated somatic cells back to a
pluripotent stem cell state. More recently, enhanced SOX2
expression has been detected in several epithelial tumors
(6, 7, 9–19) suggesting that SOX2 also regulates tumorigenesis.
On the basis of its prominent role in pluripotent stem cell
stemness, SOX2 expression has been proposed as a general
feature of CSCs (12, 27, 29, 44). Emerging data, however, show
divergent SOX2 expression patterns and functions across
tumors, suggesting that SOX2 adopts specific roles in individual tumor types. In breast cancer cells, for instance, SOX2 was
shown to promote CSC characteristics such as in vitro tumor
sphere formation and in vivo tumorigenicity (12). When cultured under nonadherent sphere conditions that enrich for
CSCs, breast cancer cells upregulated SOX2 expression, indicating a tight link between SOX2 expression and functional
stem cell state. Furthermore, immunohistochemical analysis of
primary breast carcinomas revealed a heterogeneous SOX2
protein expression in only a minority of tumor cells (13),
consistent with the putative role of SOX2 as a breast CSC
marker. In contrast, squamous cell lung cancers (9) mostly
display homogenous distribution of SOX2 protein among
tumor cells, suggesting that in this tumor entity SOX2 might
also influence non-CSCs. The difference in upstream regulatory mechanisms reported for SOX2 in individual tumor types
further support this hypothesis. In squamous cell lung cancers,
SOX2 overexpression is mostly linked to SOX2 gene amplification on the chromosome 3q26 (9). This is in line with the
observed homogenous SOX2 protein expression in all tumor
cells, as genetic amplification events likely persist upon CSC
differentiation. In contrast, in breast carcinomas elevated
SOX2 expression has been largely detected in the absence of
chromosomal amplifications and relies on yet unknown
upstream regulatory mechanisms (13). Because epigenetic
mechanisms essentially participate in stem cell reprogramming, it is possible that SOX2 expression in breast CSCs is
triggered by epigenetic events, such as altered SOX2 promoter
methylation as previously reported in glioblastoma (45).
In SOC, high SOX2 protein expression is associated with
histopathologically and clinically aggressive disease (18, 19, 46).
Similar as in breast carcinoma (13), we found that SOX2þ
expressing ovarian carcinomas display a heterogeneous
expression pattern with mostly less than 10% of tumor cells
expressing SOX2 protein, indicating that SOX2 might preferentially regulate the ovarian CSC compartment. Indeed, SOC
sphere cultures enriched for putative ovarian CSCs induced
increased SOX2 enhancer activity and SOX2 expression as
compared with 2D cultures. Consistently, SOX2þ cells enriched
www.aacrjournals.org
by detection of the SOX2 enhancer reporter generated tumor
spheres from nearly every cell and showed self-renewal and
differentiation properties in serial replating assays. SOX2
cells, in contrast, gave rise to significantly less primary spheres,
and most importantly, could not preserve sphere initiation
properties beyond tertiary spheres.
To explore whether SOX2 expression is sufficient to mediate
stemness in ovarian carcinoma cells, we modulated SOX2
expression in human SOC cell lines and primary cells by
lentiviral SOX2 knockdown and overexpression. Ectopic SOX2
expression enhanced the in vitro tumor sphere potential and
expression of stemness genes such as OCT4, LIN28, NANOG,
and ALDH1, whereas the SOX2 knockdown showed opposite
effects. However, although the frequency of sphere-initiating
cells was greatly enhanced by SOX2 overexpression in Caov3
and patient-derived SOC cells, primary tumor spheres were
initiated by only a fraction of SOX2-overexpressing cells. In
contrast, SOX2þ cells isolated via positivity for the SOX2
enhancer reporter generated spheres from nearly every cell.
A possible explanation for this finding is that, even though
SOX2 can facilitate transition to a stem cell state, this transition
occurs only in a subset of tumor cells. Alternatively, particularly
high SOX2 expression levels, as detected by the SOX2 enhancer
reporter, are needed to accomplish the transition to a CSC
state, which might not be uniformly induced in all cells by
lentiviral SOX2 expression.
Upon xenotransplantation in NSG mice, SOX2-overexpressing cells induced tumors earlier and more frequent than
control SOC cells. In vivo PET/MRI analyses as well as histologic
analyses of xenotransplanted mice confirmed larger tumor
volumes from SOX2-overexpressing than control SOC cells.
Because data in prostate as well as breast cancer suggested
that SOX2 promotes tumorigenesis by inducing cell proliferation
(11, 17), we tested whether cell growth and proliferation were
affected by SOX2. Surprisingly, mouse tumors derived from
SOX2-overexpressing and control cells showed similar Ki67
staining. In addition, no differences in cell-cycle distribution,
BrdU incorporation, or cell growth were observed in 2D cultures
of SOX2-modified and control cells. Tumors generated from
SOX2-expressing and control cells showed also similar metabolic activity in the PET assay. These results were further
corroborated by TMA analyses of primary ovarian carcinomas,
which revealed no correlation between the SOX2 protein
expression level and Ki67 positivity. Furthermore, limiting
dilution experiments suggest that SOX2 overexpression enhanced the frequency of tumor-initiating cells, as increased tumor
cell clusters could microscopically be detected in animals
transplanted with low numbers of SOX2-overexpressing as
compared with control cells. Thus, our data suggest that the
enhanced tumorigenicity of SOX2-overexpressing ovarian carcinoma cells does not rely on enhanced cell proliferation, but
is rather due to the induction of a CSC state.
Another feature regulating tumor formation is the apoptosis sensitivity of tumor cells. CSCs are assumed to
possess enhanced apoptosis resistance, facilitating tumor
generation and escape from conventional chemotherapies.
Intriguingly, SOX2-expressing tumors from transplanted
Caov-3 cancer cells showed enhanced caspase-3 activity
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Bareiss et al.
as compared with tumors from control cells. Nevertheless,
analyses of SOX2-modified Caov-3, OVCAR-3, and OVCAR-5
cells consistently showed that higher SOX2 levels prevented
apoptosis in response to both intrinsic (e.g., staurosporine,
chemotherapies, etc.) and extrinsic (e.g., TRAIL, etc.) stimuli,
indicating that SOX2 confers apoptosis resistance, a property
classically attributed to CSCs. The obviously discrepant results
between caspase-3 activation observed in vitro and in vivo
might be explained by several reasons, including a potentially
increased hypoxia of the larger SOX2-expressing tumors,
which might result in secondary necrosis.
In ovarian cancer cells, SOX2 seems to regulate stemness,
tumor-initiating capacity, and apoptosis resistance, which are
main features characterizing CSCs, while not modulating proliferation. The molecular mechanisms of SOX2-mediated stemness remain largely unexplored. In this study, we observed
robust induction of OCT4, LIN28, NANOG, and partly ALDH1
upon SOX2 activation. This could be a direct SOX2-induced
transcriptional effect or mediated by the fact that SOX2 activation induces a stem cell state characterized by expression of
these markers. In embryonic stem cells, SOX2 interacts with the
pluripotency proteins OCT4, NANOG, and LIN28. In line, suppression of OCT4 and LIN28 by RNA interference was recently
shown to inhibit ovarian cancer cell growth and survival (29). To
elucidate the pathways underlying SOX2-mediated apoptosis
resistance, we first studied the surface expression of TRAIL
receptors, which was not affected by SOX2 expression and,
hence, indicated an involvement of receptor downstream
events. Indeed, expression analysis of apoptosis-regulatory
genes revealed that SOX2 modulated the expression of certain
BCL2 members. In SOX2-overexpressing Caov-3 cells, expression of the antiapoptotic gene BCL2 was enhanced, whereas the
expression of the proapoptotic genes PUMA/BBC3 and NOXA/
PMAIP1 was reduced. These data indicate that SOX2 modulates
the balance of central apoptosis regulators, thereby changing
apoptosis sensitivity. New therapeutic approaches that target
BCL2 proteins to enhance apoptosis may therefore be a valuable
tool for targeting SOX2þ putative ovarian CSCs. Further studies
are needed to explore in detail the mechanisms of apoptosis
protection governed by SOX2 and to investigate whether BCL2,
PUMA/BBC3, and NOXA/PMAIP1 or related genes are direct
transcriptional targets of SOX2 in ovarian carcinoma. In support
of this assumption is the recent identification of SOX2-binding
regions in the BCL2 and NOXA/PMAIP1 genes using a ChiP-Seq
approach of glioblastoma cells (47).
In summary, our data in ovarian carcinoma cell lines and
patient-derived tumor samples suggest that in this tumor entity
SOX2 expression is a CSC marker and can induce CSC properties
such as stemness, tumor-initiating capacity, and apoptosis
resistance. SOX2 expression in putative ovarian CSCs enables
their selective survival to conventional chemotherapies and
promotes their in vivo tumorigenicity. We propose that SOX2expressing CSCs contribute to therapy resistance and disease
relapse in patients with ovarian carcinoma and that targeting
SOX2 will improve clinical treatment of ovarian carcinoma
by enhancing apoptotic responses to conventional chemotherapies and exhausting the CSC fraction.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: P.M. Bareiss, K. Schulze-Osthoff, C. Lengerke
Development of methodology: P.M. Bareiss, A. Paczulla, H. Wang, R. Schairer,
S. Wiehr, L. Quintanilla-Martinez, F. Essmann
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): P.M. Bareiss, A. Paczulla, S. Wiehr, U. Kohlhofer,
A. Fischer, A. Staebler, D. Wallwiener, F. Fend, T. Fehm, L. Quintanilla-Martinez,
F. Essmann
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): P.M. Bareiss, A. Paczulla, R. Schairer, S. Wiehr,
U. Kohlhofer, O.C. Rothfuss, A. Staebler, F. Fend, B. Pichler, L. QuintanillaMartinez, F. Essmann, C. Lengerke
Writing, review, and/or revision of the manuscript: P.M. Bareiss, S. Wiehr,
O.C. Rothfuss, S. Perner, A. Staebler, D. Wallwiener, F. Fend, T. Fehm, K. SchulzeOsthoff, F. Essmann, C. Lengerke
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.M. Bareiss, B. Pichler, L. Kanz,
F. Essmann, C. Lengerke
Study supervision: C. Lengerke
Acknowledgments
The authors thank Jana Ihring, Caroline Herrmann, and Sabrina Grimm
(Department of Internal Medicine II, University of Tuebingen) for help with
apoptosis assays and FACS analysis; Maren Koenig (Department of Preclinical
Imaging and Radiopharmacy, Laboratory for Preclinical Imaging and Imaging
Technology of the Werner Siemens-Foundation, University of Tuebingen) for
excellent technical PET/MRI support; Claudia Kloss and Dennis Thiele (Institute
of Pathology, University of Tuebingen) for help with murine histopathological
analyses; Holm Zaehres (Max-Planck Institute, M€
unster, Germany) for providing
the human SOX2 cDNA; and Olga Kustikova, Axel Schmabach, and Christopher
Baum (Hannover Medical School, Hannover, Germany) for help with lentiviral
constructs.
Grant Support
The work of C. Lengerke was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB773, project C6) and the Baden-W€
urttembergStiftung ("Adult Stem Cells II" Program). P.M. Bareiss was supported by the
University of Tübingen Fortüne Program.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received November 9, 2012; revised April 29, 2013; accepted June 5, 2013;
published OnlineFirst July 18, 2013.
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Cancer Res; 73(17) September 1, 2013
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SOX2 Expression Associates with Stem Cell State in Human
Ovarian Carcinoma
Petra M. Bareiss, Anna Paczulla, Hui Wang, et al.
Cancer Res 2013;73:5544-5555. Published OnlineFirst July 18, 2013.
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