sox2 expression associates with stem cell state in...

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SOX2 expression associates with stem cell state in human ovarian carcinoma

Running title: SOX2 in ovarian carcinoma stem cells

Petra M. Bareiss*1, Anna Paczulla*1, Hui Wang1, Rebekka Schairer1, Stefan Wiehr2, Ursula

Kohlhofer3, Oliver C. Rothfuss4, Anna Fischer3, Sven Perner5, Annette Staebler3; Diethelm

Wallwiener6, Falko Fend3, Tanja Fehm6, Bernd Pichler2, Lothar Kanz1, Leticia Quintanilla-Martinez3,

Klaus Schulze-Osthoff4,7, Frank Essmann4, Claudia Lengerke1

1Department of Internal Medicine II, University of Tuebingen, Tuebingen, Germany; 2Department of Preclinical

Imaging and Radiopharmacy, Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-

Foundation, University of Tuebingen, Tuebingen, Germany; 3Institute of Pathology, University of Tuebingen,

Tuebingen, Germany; 4Interfaculty Institute for Biochemistry, University of Tuebingen, Tuebingen, Germany; 5Institute of Pathology, University of Bonn, Bonn, Germany; 6Women´s Hospital, University of Tuebingen,

Tuebingen, Germany, 7German Cancer Consortium (DKTK) and German Cancer Research Center, Heidelberg,

Germany * equal contribution (Petra Bareiss and Anna Paczulla have contributed equally and share first authorship)

Author contributions:

1. Conception and design: P.M.B. and C.L.

2. Development of methodology: P.M.B, A.P., H.W., S.W., U.K., O.C.R., F.E., M.K:

3. Acquisition of data: P.M.B, A.P., R.S., H.W., S.W., U.K., A.F., F.E., L.Q.M.

4. Analysis and interpretation of data: P.M.B., A.P., R.S., S.W., O.C.R., S.P., A.S., B.P., L.Q.M.,

K.S.O., F.E., C.L.

5. Writing, review and/or revision of the manuscript: P.M.B., A.P., S.W., S.P., L.Q.M., K.S.O., F.E.,

C.L.

6. Administrative, technical, or material support: D.W., F.F., T.F., B.P., L.Q.M., L.K., K.S.O., F.E., C.L.

7. Study supervision: C.L. Correspondence

Claudia Lengerke, MD

University of Tuebingen

Department of Internal Medicine II

Otfried-Mueller-Strasse 10

72076 Tuebingen

Email: [email protected]

Tel.: +49 7071 29 82899; Fax: +49 7071 29 4524

Keywords: SOX2, ovarian, cancer stem cells, apoptosis

Conflicts of interest: none

on May 26, 2018. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 18, 2013; DOI: 10.1158/0008-5472.CAN-12-4177

http://cancerres.aacrjournals.org/

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Word count: 4853

Number of figures and tables: 7

Number of references: 46

Supplemental Material: 7 Supplemental Figures, Supplemental Material & Methods




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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. Intriguingly, however, studies

exploring the predictive value of SOX2 expression with respect to histopathological 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 cancer stem cell 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 demonstrate that SOX2 associates with stem cell state in ovarian

carcinoma and induction of SOX2 imposes cancer stem cell properties on SOC cells. We propose the

existence of SOX2-expressing ovarian cancer stem cells as a mechanism of tumor aggressiveness

and therapy resistance in human SOC.




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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 self-renewal properties (4), and targeted ablation lethally

disrupts epithelial tissue homeostasis (4). SOX2 expression is also found in neural stem cells, where it

promotes stemness by preventing default differentiation into neurons (5).

More recently, SOX2 expression has been demonstrated 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

histopathological 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 appears to be an early event in tumor development (13), indicating

potential roles in cancer stem cells (CSCs) 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). Due to the lack of

early specific symptoms, ovarian carcinoma is mostly diagnosed at advanced metastatic stages that

cannot be cured by surgical resection alone. In spite of initially good response rates to platin-based

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,




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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 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

SOX2-positive samples displayed SOX2 expression in less than 10% of tumor cells. Moreover, SOX2

expression was enhanced by culture conditions enriching for tumor stem cells. Detailed analyses

performed on SOX2-modified human serous ovarian carcinoma (SOC) cell lines and primary cells

demonstrate that indeed SOX2 expression induces CSC properties, such as expression of stemness

markers, tumor sphere formation and in vivo tumor initiating capacity as well as apoptosis resistance,

thereby strongly promoting in vivo tumorigenicity and enabling selective resistance to conventional

anti-cancer therapies.




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Materials and Methods

Cell culture, tumor spheres and cell growth assays

Ovarian cancer cell lines (Caov-3, OVCAR-3, OVCAR-5; DSMZ, Braunschweig, Germany; last

authentification on January 31st 2013 at DSMZ) and primary cells obtained through dissociation of

tissue samples derived from four patients with SOC were cultured under standard conditions. For the

tumor sphere culture assay, cells were grown in ultra-low attachment plates (Corning, Tewksbury, MA)

with sphere medium and daily added growth factors (20 ng/ml FGF, 20 ng/ml EGF; Sigma-Aldrich,

Steinheim, Germany) as described (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 performed 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 (TMA) of primary human ovarian carcinomas

SOX2 and Ki67 positivity was investigated by immunohistochemistry using polyclonal goat anti-human

SOX2 (AF2018, R&D Systems, Wiesbaden, Germany) and monoclonal mouse anti-human Ki67 (clone

MiB-1, M7240, DakoCytomation, Glostrup, Denmark) on a TMA including 215 human primary ovarian

carcinomas from patients treated at the Women’s Hospital of the University of Tübingen. Detailed

information about the TMA construction and analysis are provided in the Supplemental Material. The

study was approved by the Ethics Committee of the University of Tuebingen.

Lentiviral transduction

Lentiviruses carrying SOX2 shRNA, SOX2 overexpression, corresponding empty GFP- and SOX2-

enhancer reporter constructs (12) were designed, produced and used for transduction as previously

reported (34-36). Details on lentiviral constructs and protocols are provided in the Supplemental

Material.

Flow cytometry analysis of stem cell markers, cell cycle and BrdU assays

To detect ALDH activity, the Aldefluor® assay was used according to the manufacturer’s guidelines

(Stem Cell Technologies, Grenoble, France). Cells were incubated in Aldefluor® assay buffer for 30




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min. Cells from each sample additionally treated with the ALDH inhibitor diethylaminobenzaldehyde

(DEAB) served as negative controls. For flow cytometric analyses anti-CD133 (Miltenyi Biotec,

Bergisch-Gladbach, Germany), anti-TRAIL receptor 1 and 2 antibodies (BioLegend, San Diego, CA)

were used. Dead cells were detected by DAPI staining (100 ng/ml). Cell cycle analysis using

propidium iodide (PI; Sigma) staining and BrdU incorporation assays using mouse anti-BrdU V450

antibody (Roche, Mannheim, Germany) were performed as previously described (35). Flow cytometric

analyses were performed using a FACS Canto II and data analysed using the FACSDiva™ software

(BD Biosciences, Heidelberg, Germany).

Apoptosis assays

Cells seeded at 50.000 cells/cm² were incubated overnight and then treated for 24 hours with

staurosporine (2.5 µM; Sigma-Aldrich) or SuperKiller TRAIL™ (25 ng/ml; Enzo Life Sciences,

Farmingdale, NY), or for 96 hours with cisplatin (5 µM, Medac, Wedel, Germany), carboplatin (100 µM,

Medac) or paclitaxel (5 nM, Bristol-Myers Squibb, New York, USA). Cells were harvested by

trypsinization, fixed in 70% ice-cold ethanol and incubated in PBS containing 50 µg/ml PI and 100

µg/ml RNase A. Cells with subdiploid DNA content (subG1) were assessed by flow cytometry.

Caspase-3/7 activity was assessed by the Caspase-Glo® 3/7 assay (Promega, Madison, WI) and

normalized to protein content following treatment with staurosporine (4 hours) or TRAIL (6 hours).

Immunoblot and immunocytochemistry analyses

Immunoblot and immunocytochemistry analyses were performed using mouse anti-actin (Li-COR

Biosciences, Lincoln, NE), rabbit anti-caspase-3 (Cell Signaling, Danvers, MA), rabbit anti-cleaved

caspase-3 (Asp175) (Cell Signaling), and rabbit anti-SOX2 (D6D9) XP (Cell Signaling) antibodies.

Detection was performed using IRDye® 800CW-conjugated goat anti-rabbit IgG or IRDye® 680 anti-

mouse IgG antibodies (Li-COR Biosciences) and an Odyssey Imager (Li-COR Biosciences). For

detection of the SOX2 knockdown HRP-linked anti-rabbit IgG antibody (Cell Signaling) and ECLTM

Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) were used.

Gene expression analyses

RNA isolation, cDNA preparation and real-time gene expression analyses were performed as

described (13, 37) using a LightCycler® 480 instrument and LightCycler® probes master mix (for




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SOX2, ALDH1, LIN28, NANOG, OCT4, GAPDH; Roche) or SYBR Green assay (for BBC3, PMAIP1,

BCL2; Eurogentec, Cologne, Germany). Primers and probes are listed in the Supplemental Material.

Relative expression levels were calculated after normalization to the reference gene GAPDH (Probe)

or PBGD (SYBR Green) by using the ΔΔCT method.

Xenotransplantation model

NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (also termed NOD/SCID/IL2Rγnull, abbreviated as NSG) mice (38) were

purchased from Jackson Laboratory (Bar Harbor, ME, USA) 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 PET/MR analysis performed using i.v. administered 11-15 MBq of

[18F]FDG as described (39, 40) and summarized in the Supplemental Material. Mice were euthanized

7 to 15 weeks after implantation. For histological analysis, mouse tissues were fixed in 4%

formaldehyde, paraffin-embedded cut in 3-5 µm-sections and stained with H&E. Immunohistochemical

analysis was performed as described (41) on an automated immunostainer (Ventana Medical

Systems, Frankfurt, Germany) according to the company’s protocol for open procedures with slight

modifications. The antibody panel used included SOX2 (SP76; Cell Marque, Rocklin, CA), cleaved

caspase-3 (Asp 175, Cell Signaling), Ki67 (SP6, DCS Innovative Diagnostik Systeme, Hamburg,

Germany), and EpCAM (BerEp4, Dako, Hamburg, Germany).

Statistical analyses

For all experiments, mean values are presented and error bars represent the standard error if not

otherwise indicated. P-values are derived via the application of a 2-tailed, unpaired Student t-test.




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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 Suppl. Fig. 1) mirroring the results

documented in a TMA of human SOC samples (18, 19). Previous data on ovarian and breast cancer

cells reported that sphere cultures increase CSC frequency as compared to two-dimensional (2D)

adherent cultures (27, 33). Indeed, ovarian cancer cell lines grown as spheres for 21 days showed a

higher frequency of ALDHhighCD133+ putative CSCs (OVCAR-3 cells, Fig. 1B) and enhanced

expression of putative stem cell markers in comparison to 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 Suppl. Fig. 2A). 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. 2A and Suppl. Fig. 3A). 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,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, while 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 (SOX2+) in breast carcinoma and neural stem cells (12, 42) (Suppl. Fig. 4A). Puromycin




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selection was applied to select for efficiently transduced cells and SOX2+ cells visualized by

fluorescence. Supporting our previous data, SOX2+ cells were enriched in OVCAR-3 cells cultured as

spheres, as compared to 2D cultures (Suppl. Fig. 4B). In primary sphere assays, nearly every SOX2+

cell isolated by FACS gave rise to an individual tumor sphere (SOX2+ sphere), and SOX2+ spheres

were larger than those derived from reporter-negative cells (SOX2- spheres) (Fig. 4D and Suppl. Fig.

4C). Importantly, flow cytometry of SOX2+ primary spheres revealed a mixture of SOX2+ and SOX2-

cells, suggesting that SOX2+ cells undergo both self-renewal and differentiation processes giving rise

to both populations of cells. In contrast, primary SOX2- spheres remained SOX2-negative (Suppl. Fig.

4C). Consistent with these data, cells derived from SOX2- spheres exhausted their sphere generation

potential upon serial replating, while cells derived from SOX2+ spheres maintained sphere formation

(Fig. 4D).

Together, these data indicate that cells with self-renewal capacity segregate to the SOX2+

compartment and suggest that SOX2 induction can activate CSC molecular pathways and functional

properties in human SOC cells.

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 performed xenotransplantation

experiments of SOX2-overexpressing and control Caov-3 cells in immunopermissive NOD.Cg-

Prkdcscid IL2rgtmWjl/Sz (NSG) mice (38). The same numbers of SOX2-overexpressing and control Caov-

3 cells were implanted subcutaneously in the flanks of 8 weeks 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 post-injection in 1 out of 4 animals (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 performed in vivo PET/MRI analyses and ex vivo

immunohistological analyses at the end of the experiment. At week 15 post-inoculation (p.i.) 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




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induced much larger tumors than control cells (118.2±19.0 mm3 versus 40.4 mm3 at 15 weeks

following injection of 100.000 cells, and respectively 618.6±244.6 mm3 versus 50.98±21.8 mm3 when

500.000 cells were injected, Fig. 5B), which was also confirmed by immunohistological analysis (Fig.

5C). Interestingly, PET-quantification of [18F]FDG uptake (39) revealed similar metabolic activity in

SOX2-overexpressing and control tumors at both measured time-points (8 and 15 weeks post

injection, p.i., Fig. 5B), indicating that the inductive effects 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

[18F]FDG at the measured time points.

Interestingly, the pronounced difference of in vivo tumorigenicity between SOX2-overexpressing and

control cells was not due to enhanced cellular proliferation, as revealed by the similar results of the

Ki67 staining performed 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/MR imaging results where 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 performed a limiting dilution in vivo transplantation assay: 10.000, 1.000, 100 and 10 SOX2-

overexpressing and control Caov-3 cells were transplanted as described above in the contralateral

flanks of n=5 mice per group. In contrast to the results observed with higher numbers of cells (Fig. 5 A

and C), no palpable tumors were documented at seven weeks post-transplantation. However,

immunohistological 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. 5E). Staining

with antibodies against human EpCAM (Suppl. Fig. 5) and CA125 (not shown) confirmed correct

detection of human SOC cells. Notably, microscopical tumors were detected more frequently from

SOX2-overexpressing as compared to control cells, which was especially evident with the lowest

number of injected cells (Fig. 5E and F).

SOX2 expression does not affect cell proliferation but enhances the apoptosis resistance of

SOC cells




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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 expression and Ki67 staining on a tissue

microarray (TMA) of 215 human primary ovarian carcinomas comprising 143 high-grade SOC (see

Supplemental Material for details on TMA construction). In 136 out of 143 high-grade SOC samples,

both SOX2 and Ki67 staining was available. Although SOX2 expression was found in 64.6% of high-

grade SOC samples (Fig. 6C, Suppl. Fig 1 and 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 above, the histologic analysis of the murine tumors revealed higher levels of active

caspase-3 in SOX2-overexpressing 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),

since in vitro 2D-culture experiments showed reduced levels of spontaneous apoptosis in SOX2-

overexpressing Caov-3 as well as OVCAR-3 cells (Fig. 6D). To test whether SOX2 expression

modulates apoptosis, 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-B, Suppl.

Fig. 2 and 6). We also assayed activity of caspase-3/7 in substrate cleavage assays (Fig. 7A-B) and

processing of caspase-3 by immunoblot analysis for cleaved caspase-3 (Suppl. Fig. 7), 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, Suppl. Fig. 7), indicating SOX2

expression as a molecular driver of chemotherapy resistance in ovarian carcinoma. Notably, the

enhanced sensitivity of OVCAR-3 cells due to SOX2 knockdown was reverted by lentiviral re-

expression of ectopic SOX2 (Fig. 7D). These data demonstrate that the observed phenotype

specifically depends on SOX2 expression levels. Analogue experiments performed in a third cell line

(OVCAR-5) and using an alternative SOX2 shRNA sequence furthermore confirmed these results

(Suppl. Fig. 2 and data not shown).

In an attempt to elucidate the molecular basis for SOX2-induced resistance to TRAIL-mediated

apoptosis, we initially 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




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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 controlled by the BCL2 protein

family, we analyzed the expression of pro-apoptotic (PUMA/BBC3 and NOXA/PMAIP1) and anti-

apoptotic genes (BCL2) by real-time PCR. In line with the observed apoptosis resistance,

overexpression of SOX2 induced enhanced expression of anti-apoptotic BCL2 while reducing

expression of the pro-apoptotic proteins PUMA/BBC3 and NOXA/PMAIP1 (Fig. 7F).




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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. Based on its prominent role in pluripotent stem

cell stemness, SOX2 expression has been proposed as a general feature of CSCs (12, 27, 29, 43).

Emerging data, however, demonstrate 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 non-adherent sphere conditions that enrich for

CSCs, breast cancer cells up-regulated 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 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 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 in line with the observed homogenous SOX2 expression in all tumor cells,

since 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). Since 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 (44).

In serous ovarian carcinoma, high SOX2 protein expression is associated with histopathologically and

clinically aggressive disease (18, 19). Similar as in breast carcinoma (13), we found that SOX2-

positive ovarian carcinomas display a heterogeneous expression pattern with mostly less than 10% of

SOX2-positive tumor cells, 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 to 2D-cultures. Consistently, SOX2+ cells




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enriched by detection of the SOX2 enhancer reporter generated tumor spheres from nearly every cell

and demonstrated 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, SOX2+ 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 histological analyses of

xenotransplanted mice confirmed larger tumor volumes from SOX2-overexpressing than control SOC

cells. Since 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 tissue microarray analyses of primary ovarian carcinomas, which

revealed no correlation between the SOX2 expression level and Ki67. Furthermore, limiting dilution

experiments suggest that SOX2 overexpression enhanced the frequency of tumor-initiating cells, since

increased tumor cell clusters could microscopically be detected in animals transplanted with low

numbers of SOX2-overexpressing as compared to control cells. Thus, our data suggest that the




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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 as compared to tumors from control cells. Nevertheless,

analyses of SOX2-modified Caov-3, OVCAR-3 and OVCAR-5 cells consistently demonstrated that

higher SOX2 levels prevented apoptosis in response to both intrinsic (e.g. staurosporine,

chemotherapies) and extrinsic (e.g. TRAIL) 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 appears 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 anti-apoptotic gene BCL2 was enhanced, while the

expression of the pro-apoptotic 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 SOX2-positive 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




17

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 (45).

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 cancer stem cell 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 SOX2-expressing CSCs contribute to therapy resistance and

disease relapse in ovarian carcinoma patients and that targeting SOX2 will improve clinical treatment

of ovarian carcinoma by enhancing apoptotic responses to conventional chemotherapies and

exhausting the cancer stem cell fraction.

Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (SFB773) and the Baden-

Württemberg-Stiftung (“Adult Stem Cells II” Program). We thank Jana Ihring, Caroline Herrmann and

Sabrina Grimm (University of Tuebingen, Department of Internal Medicine II) for help with apoptosis

assays and FACS analysis, Maren Koenig (University of Tübingen, Department of Preclinical Imaging

and Radiopharmacy, Laboratory for Preclinical Imaging and Imaging Technology of the Werner

Siemens-Foundation), for excellent technical PET/MR imaging support, Claudia Kloss and Dennis

Thiele (University of Tübingen, Institute of Pathology) for help with murine histopathological analyses,

Holm Zaehres (Max-Planck Institute, Münster, Germany) for providing the human SOX2 cDNA, Olga

Kustikova, Axel Schmabach and Christopher Baum from the Hannover Medical School (Hannover,

Germany) for help with lentiviral constructs.




18

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Figure legends Figure 1: Enhancement of SOX2 expression in CSC-enriched spheres cultures. (A) SOX2 shows

heterogeneous expression in ovarian cancer cell lines and patient samples. (B) OVCAR-3 cells

cultured as spheres for 21 days are enriched for ALDHhighCD133+ putative ovarian cancer stem cells

as measured by flow cytometry. (C) Enhanced expression of SOX2 and other putative stem cell genes

Caov-3 cells cultured for 21 days and patient samples cultured for 10 days under sphere conditions,

as compared to 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 (grey box) relative to cells cultured in 2D (dashed line). Data represent the mean

values ± SEM from three or more independent biological experiments performed 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)

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 to

control lentiviruses. The upper panels show relative gene expression levels normalized to GAPDH, as

measured by real-time PCR analysis of three representative independent biological experiments each

performed in triplicates. The lower panels show immunoblot analyses representative for three

independent biological replicates.

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 (grey box) versus

cells cultured in 2D (dashed line). Shown are data from one representative out of three independent

biological experiments performed 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).

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 1250 cells/well) were scored

for primary sphere formation after 9 and 5 days, respectively. Shown are data from three independent

biological experiments performed in triplicates. (B) Primary SOC cells were transduced with




21

lentiviruses for GFP-tagged SOX2 or a GFP control. Cells were then directly plated at a density of 625

cells/well without prior FACS. After 5 days sphere formation of transduced (GFP+) and non-

transduced (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 three

independent biological 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/well. (D) SOX2+ and

SOX2- OVCAR-3 cells isolated by FACS were plated in sphere conditions 100 cells/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/well. Primary and secondary sphere formation was

analyzed in three or more, tertiary and quaternary sphere formation in two independent biological

experiments performed in triplicates (C, D).

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=5 mice per group). (A) As compared to controls, SOX2-overexpressing 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 [18F]FDG PET/MRI imaging (left panel: representative

image of a tumor-bearing mouse scanned 15 weeks after cell inoculation; right panel: tumor volumetry

and quantification of tracer accumulation). (C) SOX2-overexpressing 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.

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




22

protein expression and Ki67 staining in primary high-grade SOC samples. Analysis was performed on

a tissue microarray comprising 143 high-grade SOCs. (D) SOX2 expression modulates basal

apoptosis as measured by percentages of subG1 cells. Shown are data from three or more

independent biological experiments performed in triplicates in Caov-3 and OVCAR-3 cells (A, B, D).

Figure 7: SOX2 expression induces apoptosis resistance. Elevated SOX2 expression reduces

apoptotic responses after treatment with (A) staurosporine (STS), (B) TRAIL and (C)

chemotherapeutic drugs (cisplatin, carboplatin, paclitaxel). (D) SOX2 overexpression restores

apoptosis resistance in SOX2-knockdown cells. (E, 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 subG1 cells and caspase-3/7-activities performed 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, F). The results of each panel

represent mean values ± SEM from three independent biological experiments performed in triplicates.




A

2D-culture

Spheres

ALDH+ CD133+ ALDH+/CD133+0

10

20

30

40

50

60

P=0.0481

P<0.0001

% p

os

itiv

e c

ells

Patient #1

C

Cao

v-3

OVCAR-3

OVCAR-5

OVCAR-8

SK-O

V-3

0

1

2150

250

350

450

e-04

rela

tiv

e S

OX

2 g

en

e e

xp

res

sio

n

#1 #2 #3 #40

0.1

0.20.5

2.5

4.5

e-03

cell lines patient samples

B

SOX2 ALDH1 Lin28 Oct4

0

1

2

35

15

25

rela

tiv

e g

en

e e

xp

res

sio

n

SOX2 ALDH1 Lin28 Nanog Oct401

3

5

7

9

11

rela

tiv

e g

en

e e

xp

res

sio

n i

n

sp

he

res

ve

rsu

s 2

D

Patient #2

***

******

***

***


3

5

7

9

11

n.s.

** * n.s.

***

OVCAR-3 Caov-3

Figure 1




A B

Protein expression

Actin

SOX2

control SOX2

40 kDa

35 kDa

SOX2 overexpression

Caov-3

control SOX20

25

50

75

100

P=0.0127

rela

tiv

e S

OX

2g

en

e e

xp

res

sio

n

SOX2 knockdown

OVCAR-3

control shSOX20.00

0.25

0.50

0.75

1.00

P<0.0001

rela

tiv

e S

OX

2g

en

e e

xp

res

sio

n

Actin

SOX2

40 kDa

35 kDa

control shSOX2

Figure 2




A BCaov-3

0

0.5

1.05.0

205220

600


***

***

***

***

**control

SOX2

0

1

2

3

4


Patient #3

n.s.

***

* *

***control

SOX2

0

1

2

15

25


Patient #4

n.s.n.s.

*** ***

***

control

SOX2

Patient #1


0

1

3

5

7 ***

******

***

***

control

SOX2

fold

re

lative g

ene

exp

ressio

n

in S

OX

2 o

ve

rexp

ressin

g v

ers

us c

ontr

ol ce

llsFigure 3




C

SOX2 overexpressionCaov-3

A

0.0

2.5

5.0

7.5

10.0

12.5

P=0.0161

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

control SOX2

SOX2 overexpression - single spheresCaov-3

control SOX2

0

5

10

15

nu

mb

er

of

sp

he

res

fro

m o

ne

dis

so

cia

ted

sin

gle

sp

he

re

secondary spheres

P=0.0021

control SOX2

0

5

10

15

nu

mb

er

of

sp

he

res

pe

r

20

pla

ted

ce

lls

tertiary spheres

P<0.001

control SOX2

0

5

10

15

20

25

30

nu

mb

er

of

sp

he

res

pe

r 1

00

pla

ted

sin

gle

ce

lls

P=0.0372

primary spheres

control SOX2 0

5

10

15

quaternary spheres

P=0.0085

nu

mb

er

of

sp

he

res

pe

r

20

pla

ted

ce

lls

0

5

10

15

control shSOX2

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

SOX2 knockdownOVCAR-3

B

D

SOX2 positive

0

20

40

60

80

100

SOX2 negative

P<0.001

0

5

10

15

20

25

30

SOX2 positive

SOX2 negative

P<0.001

0

5

10

15

20

25

30

SOX2 positive

SOX2 negative

P=0.0202

0

5

10

15

20

25

30

SOX2 positive

SOX2 negative

P=0.0167

secondary spheres tertiary spheres quaternary spheresprimary spheres

SOX2 reporter cellsOVCAR-3

SOX2 overexpression

control SOX2

0

5

10

15

20

25

30

35

GFP+ GFP-

secondary spheres

P=0.0016 (GFP+)

primary spheres

control SOX2

0

5

10

15P=0.0165 (all)

P<0.001 (GFP+)P<0.001 (all)

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

control SOX2

0

5

10

15

control SOX2

0

5

10

15

20

25

30

35

P<0.001 (GFP+)P<0.001 (all)

P=0.0166 (GFP+)P=0.0292 (all)

secondary spheresprimary spheres

Patient #1 Patient #2

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

nu

mb

er

of

sp

he

res

pe

r 1

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ce

lls

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

nu

mb

er

of

sp

he

res

pe

r 1

00

ce

lls

P=0.0185

Figure 4




C H&E 12.5x SOX2 12.5x Ki-67 12.5x

Co

ntr

ol

SO

X2

B

500.000 cellsA 100.000 cells

ControlSO

X2

0 2 4 60

1

2

3

4

5Control

SOX2

weeks

nu

mb

er

of

pa

lpa

ble

tu

mo

rs

0 2 4 6 8 10 12 140

1

2

3

4

5Control

SOX2

weeksn

um

be

r o

f

pa

lpa

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tu

mo

rs

p.i. 15 weeks p.i.V

olu

metr

y [

mm

3]

0

250

500

750

1000

1250

%ID

/cc [

18F

]FD

G

0

1

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4

n=1 n=4 n=4n=4

8 weeks

p.i. 15 weeks p.i.

n=1 n=4 n=4n=4

8 weeks

500.000

cells

100.000

cells

500.000

cells

100.000

cells

Control SOX2

Caspase-3 12.5x

0 1 3 5 7 9 110

1

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4

5Control

SOX2

weeks

nu

mb

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of

pa

lpab

le t

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ors

50.000 cells

E SOX2 Control SOX2 Control

H&E 50x H&E 630x

D

Cells injected

Immunohistological tumor

detection after 7 weeks

10.000

SOX2 control

1.000

100

10

4/5 3/5

4/5 1/5

0/5

0/50/5

0/5

Figure 5




SOX2 overexpression SOX2 knockdownA

D SOX2 knockdown

B SOX2 overexpression

C Primary SOC samples

Caov-3 OVCAR-3 Caov-3

OVCAR-3

SOX2 overexpression

Caov-3

OVCAR-3

SOX2 knockdown

Control SOX20

10

20

30

40

% B

rdU

po

sit

ive

cells

Control shSOX20

5

10

15

20

25

30

35

% B

rdU

po

sit

ive

ce

lls

Control SOX20

2

4

6

8

10

12

14

P=0.004

Su

b G

1 f

racti

on

%

Control shSOX20

2

4

6

8

10

12

Su

b G

1 f

racti

on

%

SOX2 positivity

negative low medium high0

10

20

30

40

50

60

70

80

90

% K

i-6

7 p

os

itiv

ity

G0-phase S/M/G2-phase

0

10

20

30

40

50

60

70

SOX2

Control

% P

I p

os

itiv

e c

ells

G0-phase S/M/G2-phase

0

10

20

30

40

50

60 Control

shSOX2

% P

I p

os

itiv

e c

ells

days

0 1 2 3 4 5 6 7 8 9 100

2.5

5.0

7.5Control

SOX2

nu

mb

er

of

via

ble

ce

lls

days

0

2.5

5.0

7.5

10.0

12.5

Control

shSOX2

P=0.0053

0 1 2 3 4 5 6 7 8 9 10

(x1

05)

nu

mb

er

of

via

ble

ce

lls

(x1

05)

Figure 6




A Staurosporine treatment

Chemotherapytreatment

C

B TRAIL treatment

SOX2 knockdownSOX2 overexpression SOX2 overexpression SOX2 knockdown

SOX2 overexpression

D Rescue of SOX2 expression in SOX2 knockdown OVCAR-3 cells

Staurosporine treatment TRAIL treatment

E SOX2 knockdownSOX2 overexpression F SOX2 overexpressionCaov-3 OVCAR-3

Caov-3 OVCAR-3 Caov-3 OVCAR-3

Caov-3

Caov-3

Control SOX2

0

50

100

150

200

250

anti-apoptotic

P=0.0042

rela

tiv

e g

en

e e

xp

res

sio

no

f B

CL

2

BBC3 PMAIP1

0

20

40

60

80

100

120

Control

SOX2P=0.0001

pro-apoptotic

rela

tiv

e g

en

e e

xp

res

sio

n

TRAILR1 TRAILR2

0

1000

2000

3000Control

shSOX2

Dif

fere

nce

of

med

ian

flu

ore

sce

nc

e in

ten

sit

y

TRAILR1 TRAILR2

0

200

400

600

800

1000

1200Control

SOX2

Dif

fere

nc

e o

f m

ed

ian

flu

ore

sc

en

ce

in

ten

sit

y

Cisplatin Carboplatin Paclitaxel

0

10

20

30

40

50

60

70

Control

SOX2

P=0.0073

P=0.0040

P=0.0232

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Control shSOX2 shSOX20

10

20

30

40

50

60

P=0.0014 P<0.0001

+ SOX2

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Control shSOX2 shSOX20.00

0.25

0.50

0.75

1.00

P<0.0001 P=0.0078

+ SOX2

rela

tiv

e S

OX

2g

en

e e

xp

ress

ion

Control shSOX2 shSOX20

10

20

30

40

50

60P=0.0120

+ SOX2S

ub

G1

fra

cti

on

%(s

pe

cif

ic a

po

pto

sis

)

Control SOX20

25

50

75

100

P<0.0001

rela

tiv

e R

LU

(%

)

Control shSOX20

25

50

75

100

125

150

175

rela

tiv

e R

LU

(%

)

Control shSOX20

100

200

300

400

500

600

rela

tiv

e R

LU

(%

)

Control SOX20

25

50

75

100

P=0.0001

rela

tiv

e R

LU

(%

)

Control SOX20

10

20

30

40

50

60P<0.0001

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Control SOX20

10

20

30

40

50

60

P=0.0073

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Control shSOX20

10

20

30

40

50

60

P=0.0036

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Control shSOX20

10

20

30

40

P=0.0060

Su

b G

1 f

rac

tio

n %

(sp

ec

ific

ap

op

tos

is)

Figure 7




Published OnlineFirst July 18, 2013.Cancer Res Petra M Bareiss, Anna Paczulla, Hui Wang, et al. ovarian carcinomaSOX2 expression associates with stem cell state in human

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