Immunohistological Insight into the Correlation between Neuropilin-1 and Epithelial-Mesenchymal...

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published online 21 May 2014J Histochem CytochemOmar and Brenda L. Coomber

Sirin A. I. Adham, Ibtisam Al Harrasi, Ibrahim Al Haddabi, Afrah Al Rashdi, Shadia Al Sinawi, Abdullah Al Maniri, Taher BaTransition (EMT) Markers in Epithelial Ovarian Cancer (EOC)

Immunohistological Insight into the Correlation Between Neuropilin-1 and Epithelial Mesenchymal

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JHC Express Accepted May 10, 2014 This article can be cited as DOI: 10.1369/0022155414538821

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Copyright © 2014 The Author(s)

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Immunohistological Insight into the Correlation Between

Neuropilin-1 and Epithelial Mesenchymal Transition (EMT)

Markers in Epithelial Ovarian Cancer (EOC)

Sirin A. I. Adham1*, Ibtisam Al Harrasi1†, Ibrahim Al Haddabi2†, Afrah Al Rashdi2,

Shadia Al Sinawi2, Abdullah Al Maniri3, Taher Ba Omar1, Brenda L. Coomber4

1 Department of Biology, College of Science, Sultan Qaboos University, P. O. Box 36,

123 Muscat, Oman.

2 Department of Pathology, College of Medicine, Sultan Qaboos University, P.O.Box 35,

123 Muscat, Oman

3 The Research Council, P.O.Box 1422, 130 Muscat, Oman

4 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,

Guelph, ON, Canada N1G 2W1

*Corresponding author:

E-mail: [email protected]

Tel: +968-24146869

Fax: +968 2414 1437

†Ibrahim Al Haddabi and Ibtisam Al Harrasi contributed equally to this work.

by guest on May 30, 2014jhc.sagepub.comDownloaded from



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Abstract:

The mechanism by which neuropilin-1 (NRP-1) induces malignancy in Epithelial

Ovarian Cancer (EOC) is still unknown. This study is the first to demonstrate the

relationship between NRP-1 expression and EMT markers vimentin, N-cadherin, E-

cadherin and Slug. We used tissue microarrays containing the three main subtypes of

EOC tumors: serous, mucinous cystadenocarcinoma and endometrioid adenocarcinoma

and representative cases retrieved from our pathology archives. Immunohistochemistry

was performed to detect the expression levels and location of NRP-1 and the above EMT

proteins. NRP-1 was mainly expressed on cancer cells but not in normal ovarian surface

epithelium (OSE). The Immunoreactive Scoring (IRS) values revealed that the expression

of NRP-1, Slug and E-cadherin in the malignant subtypes of ovarian tissues was

significantly higher (5.18±0.64, 4.84±0.7, 4.98±0.68 respectively) than their expression

in the normal and benign tissues (1.04±0.29, 0.84±0.68, 1.71±0.66), there were no

significant differences among the studied subtypes. Vimentin was expressed in the cancer

cell component of 43% of tumors and it was exclusively localized in the stroma of all

mucinous tumors. The Spearman's rho value indicated that NRP-1 is positively related to

the EMT markers E-cadherin and Slug. This notion might indicate that NRP-1 is a

partner in the EMT process in EOC tumors.

Key words: Neuropilin-1, EMT, Biomarker, Slug, E-cadherin, Vimentin, OSE.




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Introduction:

Epithelial Ovarian Cancer (EOC) is the most common type of ovarian cancer

affecting women worldwide. Previous studies showed that about 75% of patients were

diagnosed at late stage of the disease (Hennessy et al. 2009; Siegel et al. 2011). EOC was

classified into five major subtypes which are serous, endometrioid, mucinous, clear cell

and transitional cell carcinomas (Brenner tumors) (Auersperg et al. 2001; Chauhan et al.

2009; Davidson et al. 2012; Soslow 2008; Vergara et al. 2010). These tumors are related

to epithelial fallopian tube, proliferative endometrioid, endocervical or intestinal,

gestational endometrioid and the urogenital tract respectively (Chauhan et al. 2009; Karst

and Drapkin 2010). Each of these subtypes has different prognosis as well as treatment

responses (Auersperg et al. 2001) due to their different genetic, phenotypic and

physiological features (Davidson et al. 2012). The serous subtype of ovarian carcinoma

accounts for about 60 to 80 percent of ovarian cancer cases and it is considered the most

aggressive subtype of ovarian neoplasms. High Grade Serous Ovarian Cancers (HGSOC)

arise in the absence of recognizable pre-existing conditions unlike the low-grade

adenocarcinomas of the ovary, specifically endometrioid and mucinous tumors which

normally follow the transformation from adenoma to carcinoma sequence (Levanon et al.

2008).

Epithelial ovarian carcinomas were initially thought to arise from the Ovarian

Surface Epithelium (OSE), therefore studies were focused in looking for closely related

biomarkers which are expressed specifically by the epithelial cells, such as mucins




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(Auersperg et al. 2001; Chauhan et al. 2006). Mucins are proteins found to be highly

expressed in EOC and they are related to dissemination and invasion of the ovarian

cancer cells due to their glycosylated extracellular domain which may overhang up to

200-2000 nm above the cell surface. Recently it has been shown that glycoprofiling of

CA125 improves differential diagnosis of ovarian cancer (Chauhan et al. 2009; Chen et

al. 2013). Previously identified ovarian cancer diagnostic biomarkers such as serum

MUC16 or CA125 concentration may not have the sensitivity or specificity to function

alone in EOC screening (Cannistra 2004). Regardless of its role in ovarian cancer,

CA125 does not exhibit an elevated serum level in over 50% of the patients diagnosed

with early stage tumors because this antigen is not expressed in most early stage ovarian

tumors (Jacobs et al. 1993). However, in a more recent clinical trial on advanced ovarian

cancer patients it was shown that CA125 was useful as a predictor of recurrence in

advanced ovarian cancer with a sensitivity of 91.5%, (Song et al. 2013).

Besides mucins, other molecular biomarkers are still needed to aid in designing

the proper therapy on a patient individual basis. In addition to origin from ovarian surface

epithelium, it is now apparent that EOC can also be derived from the fallopian tube,

potentially changing the criteria used to identify EOC. Thus the genes and or proteins

detected in the EOC cells might not be relevant markers for disease progression, but

rather their expression may reflect retention from the cell of origin (O'Shannessy et al.

2013). This is especially important given that the molecular makeup of tumor cells

changes dynamically during progression through the process of Epithelial Mesenchymal




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Transition (EMT) and its opposite Mesenchymal to Epithelial Transition (MET). It has

been shown that the EMT in ovarian cancer is controlled by different cytokines and

growth factors such as mucin (MUC4) and MUC16 (CA125) (Ponnusamy et al. 2010;

Theriault et al. 2011), bone morphogenetic protein 4 (BMP4), endothelin-1 (ET-1),

epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming

growth factor-β (TGF-β) (Vergara et al. 2010). More recently in an in vitro study,

thrombin showed an induction of the EMT proteins in EOC cells (Skov-3) and the use of

inhibitors for thrombin (hirudin) reversed this action which indicates that the use of such

anticoagulant might serve in the control of EOC progression (Zhong et al. 2013).

Neuropilins (NRPs) are a 130-140 kDa transmembrane glycoprotein family of

non-tyrosine kinase receptors (Ellis 2006; Hu et al. 2007; Lu et al. 2009; Yu et al. 2010).

They have a large extracellular region of three main domains (a1a2, b1b2 and c (MAM)),

a transmembrane domain, and a short intracellular (cytoplasmic) domain which lacks any

enzymatic activity (Ellis 2006; Herzog et al. 2011; Lu et al. 2009). They are expressed in

neurons, endothelial cells, mesothelial cells, bone marrow, and cancer cells (Kreuter et al.

2006; Stoeck et al. 2006). NRP-1 acts as a receptor for semaphorin 3A (SEMA), SEMA

3F, vascular endothelial growth factor (VEGF) members, such as VEGF-A165, VEGF-B

and VEGF-E, and it binds to latent and active TGF-β1 (Glinka et al. 2011; Smart et al.

2013; Yu et al. 2010). There are many studies confirming the correlation between

increased NRP-1 expression and induction of malignancy in different cancer types such

as breast (Stephenson et al. 2002), colorectal (Yu et al. 2010), myeloid leukemia (Kreuter




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et al. 2006), glioma (Hu et al. 2007), pancreatic (Wey et al. 2005), and prostate cancer

(Hu et al. 2007), and its silencing in hepatocellular carcinoma showed cellular growth

suppression in vitro and in vivo (Xu and Xia 2013). It has been reported that NRP-1

promotes ovarian cancer unlimited growth through evasion of contact inhibition (Wong

et al. 1999). Osada et al reported that ovarian carcinomas are characterized by a decrease

in semaphorin expression and an increase in NRP-1 and NRP-2 expression. The same

study showed that the expression of NRP-1 and NRP-2 was significantly higher in

ovarian carcinomas when compared with ovarian benign tumors (Osada et al. 2006).

NRP-1 is a multifunctional protein existing in two forms: soluble and

transmembrane receptor (Lu et al. 2009; Uniewicz et al. 2011). NRP-1 plays a critical

role in tumorigenesis, cancer invasion, and angiogenesis through VEGF, PI3K, and Akt

pathways (Hong et al. 2007; Pan et al. 2007). NRP-1 was found to have a heterotypic

association between cancer cells and (myo)-fibroblasts via N-cadherin through its binding

to L1 Cellular Adhesion Molecule (L1-CAM) in ovarian cancer (Bracke 2007). Anti-L1-

CAM monoclonal antibody demonstrated an inhibition of peritoneal growth and

dissemination of human ovarian carcinoma cells in vitro and in nude mice (Arlt et al.

2006). This later study confirms the association of NRP-1 with an important marker of

EMT, N-cadherin. It has been shown that L1-CAM was up-regulated in breast cancer

cells when the EMT process was induced by TGF-β (Kiefel et al. 2012).

Recently, in randomized phase III clinical trials, investigators found that

circulating VEGF-A and tumor NRP-1 expression can be used as potential predictive




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biomarkers for the decision whether to use bevacizumab (anti-VEGF antibody) or not in

the treatment plan of patients with breast, colorectal, and gastric cancers (Lambrechts et

al. 2013; Maru et al. 2013). For instance, in patients with metastatic breast cancer low

NRP-1 expression represents one of the most consistent and promising predictive

biomarker identified thus far (Jubb et al. 2011).

In a previous study we showed that the ratio of NRP-1 to VEGFR2 expression

increases proportionally with tumor grade in 80 cases of EOC (Adham et al. 2010). The

mechanism by which NRP-1 influences tumorogenesis is still not well defined. In this

work we hypothesized that NRP-1 might be involved in the EMT pathway in EOC,

which is important for tumor metastasis and progression (Davidson et al. 2012). We

found that NRP-1 was only expressed in the epithelial (cancer cell) component of all the

tumors tested. Vimentin was also expressed in the cancer cell component of 43% of

tumors besides its usual localization in the stroma. NRP-1 expression (represented by

immunoreactive score) was positively correlated with Slug and E-cadherin expression.

Mucinous cystadenocarcinoma possessed differences in the tissue and cellular

localization of vimentin and NRP-1 respectively when compared with the other two EOC

subtypes. These in situ observations open the doors for more functional analyses to

investigate the use of NRP-1 as a potential biomarker for EOC. Further evaluation linking

cancer characteristics with patient outcome data will give stronger evidence about this

correlation.




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Materials and Methods

Tissue Microarrays (TMA) and tissue inclusion criteria

Human ovarian tissue arrays were used for immunohistochemical staining (Cat#

OVC1501; Pantomics, Richmond, CA, USA). These arrays contained tissues fixed in

10% formalin (pH 7.0) for 24 h and processed with identical standard operating

procedures. Each tissue array had 150 cores collected from 75 individuals in which two

cores represented one tumor tissue or biopsy from the same patient. Among the 75 cases

there were 2 normal, 3 benign (mucinous, serous cystadenoma, and 1 thecoma) and 73

EOC cases provided with pathological, grading and staging data (Table 1S). The regions

of each block chosen for inclusion in the TMA were reviewed by at least three

pathologists who examined the H&E slides. The main inclusion criteria were to select

the region of the tissue depending on getting sufficient and representative target cells

which was marked on the slides. The marked areas on the slides were then transferred to

corresponding re-marked blocks (formalin fixed paraffin embedded FFPE blocks) by a

pathologist. To ensure the representative nature of a case, for most of TMAs, duplicated

cores/per case were taken randomly from two marked areas of a block or from two blocks

of the same case (normally multiple areas containing representative target cells were

marked on a block by a pathologist). After a TMA block was made, a section was cut and

stained with H&E. Each core of the stained TMA slide was examined by a pathologist.

Patient specimens, and Ethics statement




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The clinical samples consisting of 5 normal, 4 benign (2 mucinous and 2 serous

cystadenoma and 4 neoplasia (2 mucinous and 2 serous cystadenocarcinoma) were

collected from the pathology archives at Sultan Qaboos University Hospital SQUH

Oman, and the paraffin blocks were cut into 4 μm thick sections for

immunohistochemistry staining (described below). Routinely, the patients were fully

consented for the donation of the tissue prior to any surgical procedure and their

information was kept confidential and used for research purposes only.

Immunohistochemistry

TMA slides and patient's individual ovary sections were deparaffinized in xylene,

rehydrated in a series of ethanol (100%, 95% and 75%) and tap water, then antigen

retrieval was performed using 1 mM Ethylenediaminetetraacetic acid (EDTA) (pH 9.0),

in 95°C water bath for 30-40 min. The activity of endogenous peroxidases was blocked

by 2% hydrogen peroxide for 15 min. The slides were washed twice in phosphate buffer

saline (PBS) then in PBS + 0.05% Triton X-100 for 5 min each. They were incubated

with a blocking solution of 5% normal goat serum for 30 min at room temperature, then

incubated overnight at 4°C with the appropriate primary antibody solutions contained one

of the following monoclonal antibodies: Neuropilin-1 1:250, vimentin 1:250 and N-

cadherin 1:500, (Epitomics, USA cat # 2621-1, 2707-1and 2019-1 respectively). E-

cadherin and Slug antibodies were used at 1:200 and were purchased from Cell Signaling




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Technologies USA (cat # 3195 and 9585 respectively). All other chemicals used were

purchased from Sigma Aldrich, Germany.

After the incubation with the primary antibodies the tissues were washed twice in PBS

and incubated with biotinylated secondary goat anti-rabbit antibody (1:100; Vector

Laboratories, Switzerland). The signal was enhanced by one-step incubation with

Vectastain Elite ABC reagent (Vector, Switzerland) for 30 min at room temperature.

Colorimetric detection was achieved by incubation with 3,3’-diaminobenzidine (DAB)

(Dako, Warrington, PA, USA) for 5 min. This was followed by counterstaining with

Mayer’s hematoxylin solution, which was added for approximately 1-2 min. The tissues

were dehydrated in a series of ethanol (75%, 95% and 100%) then in xylene. Finally, the

slides were mounted using DPX (Di-n-butyl phthalate in Xylene) (Sigma- Aldrich,

Germany). Tissues were visualized using an Olympus (BX 40) light microscopy with

digital camera (DP50). The images of cores were captured by 40X using OLYSIA Bio-

report and Twin Viewfinder Light (Version 1.0) software. A negative primary antibody

control slide was stained simultaneously to confirm staining specificity (Figure 1 S).

Immunohistochemical reactivity evaluation and scoring categories

The microscopic examination was done independently in a blinded fashion by two

different scientists and a pathologist, and the average score was considered for the final

analysis. The total 150 cores in the TMA obtained from 75 patient specimens (2 cores per

specimen) and another 13 representative patient ovarian tissue sections (5 normal, 4




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benign, and 4 malignant) were studied and analyzed for each biomarker. They were

visualized individually under the light microscope (400X) and given the appropriate

category. Finally, the scores of each case (2 cores) were joined together and given a

specific code for statistical analysis. The staining intensity (SI) for the epithelial

expressed proteins E- Cadherin, NRP-1 and Slug was given a grade which represents the

degree of the DAB color deposited in the tissue. The grading system used was: absence

of staining (negative), 0; weak staining, 1; moderate staining, 2; strong staining, 3. The

score for the distribution of the positively stained cells (percentage of positive cells; PP)

was based on the average score observed in ten random fields at 400X (five fields from

each TMA duplicated cores). Based on the cell staining proportion, all cases were

classified as 0, no positive cells; 1, 1-20% positive cells; 2, 21-50% positive cells; 3, 51-

100% positive cells. IRS value was calculated according to previously published method

as follows: IRS = SI x PP (Chui et al. 1996) the average IRS of each marker (NRP-1,

Slug, E-cadherin) for each case was calculated as the final value used for statistical

analysis .

..

Since N-cadherin and vimentin expression (intensity and distribution) was homogenous

in all TMA studied they were categorized based on tissue localization: group 1, mainly

stromal; group 2, mainly epithelial; group 3, both epithelial and stromal. In addition to

IRS score, Slug was also grouped for its tissue location as follows: 0 negative staining; 1

epithelial staining; 2 stromal staining, and for its cellular localization as: 0 negative



https://www.researchgate.net/publication/14570845_Immunohistochemical_expression_of_the_c-kit_proto-oncogene_product_in_human_malignant_and_non-malignant_breast_tissues?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


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staining; 1 nuclear staining; 2 cytoplasmic staining. Similarly, NRP-1 was expressed in

both the cytoplasm and nucleus; this localization was categorized as: 1 nuclear staining; 2

cytoplasmic staining; 3 nuclear and cytoplasmic staining.

Statistical Analysis

Statistical differences between tested variables were determined by Pearson Chi-Square

and Fisher's Exact Test (2 sided). Spearman’s rank correlation was used to determine

whether there was a positive or negative correlation between variables. Post Hoc test was

used to analyze the multiple comparisons of the measured IRS values with grade, stage

and pathology. To account for multiple testing, we used Bonferroni correction test,

considering α of 0.05, the Bonferroni's adjustment p-value was 0.0071. This p-value was

used as a cut point for significance level.. SPSS for Windows (19.0) was used to analyze

the data.




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Results

NRP-1, slug and E-cadherin expression in normal, benign and malignant ovarian tissue

A total of 7 normal ovarian tissues were used in which 2 (4 cores) were included

within the TMA and another 5 were normal ovaries retrieved from pathology archives.

Similarly, another 7 benign cases were examined, 3 were included in TMA slides (1

mucinous cystadenoma, 1 serous cystadenoma and 1 thecoma) and another 4 cases

retrieved from the pathology archives which were 2 serous and 2 mucinous cystadenoma.

The normal and benign cases were used as a control to track changes in protein

expression and compare them to the neoplasia cases (Fig 1 A). While E-cadherin and slug

were weakly expressed in all normal cases (TMA & individual specimens) represented by

the mean IRS value which was equal to 0.7143 (±0.19 SEM) and 0.1714 (±0.1749)

respectively. NRP-1 was not detected in any of those specimens so its IRS value was 0.0

(Fig 1 A & table 1). NRP-1, Slug and E-cadherin were detected in all benign cases with

different intensities. However, the univariate analysis of the variance did not show any

statistical differences between the normal and benign cases, and the Bonferroni adjusted

p value for IRS-NRP-1, IRS-E-cadherin and IRS-slug was > 0.05 (Fig 1A&B). Slug

expression was detected only in one of the two normal ovarian tissue cases included in

the TMA slide (Fig 1C) and was not detected in any of the other individual patient cases

studied. As shown in Fig 1A and Table 1 the mean IRS values of the three markers NRP-

1, Slug and E-cadherin were significantly higher in the three subtypes of EOC when

compared with the normal and benign cases, however there were no statistical differences




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among the three subtypes, The Bonferroni adjusted p value between the three

pathologies was > 0.05 (Fig 1A& Table 1).

Localization and tissue type expression of markers.

Three pathological subtypes of Epithelial Ovarian Cancers (EOC) were studied

and statistically analyzed for their expression of the five proteins (E-cadherin, N-

cadherin, vimentin, Slug and NRP-1). The studied pathologies were: serous

cystadenocarcinoma, endometrioid adenocarcinoma and mucinous cystadenocarcinoma,

since they had higher frequencies among the tested cases compared to others. These three

pathological subtypes represented 35.8 %, 40.3% and 17.9% of the 67 carcinoma cases

respectively, with the other subtypes accounting for 3% or less of cases evaluated (Table

1S). These percentages represent the random distribution of the different pathologies on

the TMA slides and do not reflect actual statistical frequencies of the different subtypes

of ovarian carcinomas.

Slug tissue localization was pathology subtype dependent

As an EMT inducer slug was detected in both epithelial and stromal parts of the tissue

(Fig. 2C). The majority of tumors, 52 out of the 73 tumors (72.2%), expressed slug in the

epithelium compared to only 19.4% with expression in the stroma (Table 1S). Comparing

the three studied pathologies, we found a significant difference among them in terms of

their tissue localization (epithelial\stromal) (p=0.001) (Fig. 2C; Table 2). Twenty-one out

of 24 serous cystadenocarcinomas (87.5%) and 23 out of 27 endometrioid

adenocarcinomas (85.2%) displayed higher percentages of epithelial slug expression




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when compared to the mucinous cystadenocarcinoma tumors in which only 8 out of 12

cases (61.5%) had slug localized in the epithelium (Table 2).

Neuropilin-1 (NRP-1) cellular localization was pathological subtype dependent

NRP-1 was expressed exclusively in the epithelial parts of the EOC tumors. Therefore,

we investigated its expression by two categories: immunoreactive score and cellular

localization. We found that NRP-1 was not differentially expressed among the different

pathologies (p>0.05) (Fig 1A, Table 1 and Fig. 3A). However its cellular localization

(nuclear, cytoplasmic or both) showed a significant difference among the pathological

subtypes (p=0.004) (Table 2). The highest percentage of NRP-1 localization (expression)

in both sites was observed in 12 out of 27 endometrioid adenocarcinomas (44.4%), 5 out

of 24 serous cystadenocarcinomas (20.8%) and 4 out of 13 mucinous

cystadenocarcinomas (30.80%) (p=0.004). Cytoplasmic only NRP-1 localization was not

detected in any of the 12 mucinous cystadenocarcinomas (0.0%). However, The number

of patients with mucinous tumours and NRP-1 localized in the nucleus represented the

highest percentage, (6 out of 13; 46.20%), compared with the other two pathologies. The

patients with serous tumors had higher nuclear NRP-1 percentage compared to

endometrioid (8 out of 24 serous; 33.3% and 5 out of 27 endometrioid; 18.5% carcinomas

respectively (Fig. 3; Table 2).

Vimentin was expressed in both stroma and epithelia in the majority of the ovarian




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tissues

Descriptive statistics was used to find the association between vimentin tissue

distribution (stromal, epithelial and both stromal and epithelial) in the normal, benign and

three subtypes of ovarian carcinomas (cross tabulation 3X5). Vimentin was expressed

either in the stroma or both stroma and epithelia of both normal and benign tissues (Table

4). Vimentin was exclusively expressed in the epithelial cancer cells of two endometrioid

cases only (Fig. 2). The rest of the tumors expressed vimentin either only in the stroma

(38 cases) or in both the stroma and epithelia (27 cases), Fig. 2; Table 1S). Vimentin was

exclusively expressed in the stroma of all mucinous tumors present in TMA & the

additional 2 pathology retrieved individual cases with no expression in the epithelial

compartment (Fig. 2B), while its expression in both stroma and epithelia was detected in

14 out of 24 serous carcinomas (61.5%) and 10 out of 27 endometrioid carcinomas

(37.0%) (Table 3).

Unlike E-cadherin, N-cadherin was expressed in both stroma and epithelium. Its

tendency to be expressed in both epithelium and stromal was higher than its expression in

only the stroma and or epithelia (in 50 out of the 70 EOC cases N-cadherin was expressed

in both locations) (Fig. 4; Table 1S). Multiple comparisons showed that N cadherin tissue

distribution was associated with the different ovarian tissue types as shown in Table 4.

N-cadherin was related significantly with tumor stage




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Although none of the proteins studied showed a significant relationship with

tumor grade (Table 2S), N-cadherin tissue distribution (stromal, epithelial and both)

showed a significant relationship with tumor stage (p>0.001 and Table 4). N-cadherin

was observed in 27 out of 35 stage I tumors (56.3%), 5 out of 11 stage II tumors (50.0%),

5 of 5 stage III tumors (100.0%) and 10 out of 16 stage IV tumors (66.7 %) (table 4).

Figure 4 shows the staining pattern observed in four representative tissues from four

different stages

Slug, E-cadherin and NRP-1 expression are significantly related

Spearman’s rank correlation test showed that the expression of both E-cadherin and NRP-

1 had a significant positive correlation with slug (ρ=0.861, p<0.001) and (ρ=0.602,

p<0.001, respectively). (Table 3S).

Discussion

Histopathological examination is the first method used in the diagnosis and

differentiation of EOC heterogeneous tumors and in the separation of closely related ones

(Ichigo et al. 2012). EOC tumors are generally classified into histological subgroups

depending on their aggressive behavior and their malignant potential (D'Andrilli et al.

2008; Lalwani et al. 2011). EOC classification becomes of interest for planning of

treatment and management since it has been found that the different subtypes respond

differently to treatment regimens (Bamias et al. 2010). Further investigation for new



https://www.researchgate.net/publication/23135796_Epithelial_Ovarian_Cancer_The_Role_of_Cell_Cycle_Genes_in_the_Different_Histotypes?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/23135796_Epithelial_Ovarian_Cancer_The_Role_of_Cell_Cycle_Genes_in_the_Different_Histotypes?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/228082941_Transitional_cell_carcinoma_of_the_ovary_Review?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/41167306_Mucinous_But_Not_Clear_Cell_Histology_Is_Associated_With_Inferior_Survival_in_Patients_With_Advanced_Stage_Ovarian_Carcinoma_Treated_With_Platinum-Paclitaxel_Chemotherapy?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


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predictive biomarkers identifying subpopulations of patients who are most likely to

respond to a given therapy should aid in EOC disease control.

In this study we found that NRP-1 might be a potential marker that is related to

the EMT pathway. NRP-1 was not detected in the normal ovary tissue, however it was

detected in the benign tissue specimens. This finding is consistent with the previous study

by Hall et al, (Hall et al. 2005) and partially consistent with the study reported by Baba et

al, (Baba et al. 2007) where they showed that NRP-1 was not expressed in the normal

ovary surface epithelia. However they reported that NRP-1 was also not expressed in the

benign cases and they explained their results by the specificity of the antibody. Yet in

this study we showed that the cellular localization of NRP-1 was significantly different

among the three subtypes of EOC. Nuclear localization of NRP-1 was more prevalent in

mucinous cystadenocarcinoma tumors than in endometrioid adenocarcinoma and serous

cystadenocarcinoma. The mucinous cystadenocarcinomas in our study did not express

vimentin in the epithelial components of the tumors, Slug was less expressed in the

epithelium when compared with the other two subtypes and none of them expressed

cytoplasmic NRP-1.

Generally, the stromal expression of vimentin in the three pathologies is due to its

nature as a mesenchymal derived intermediate filament (Satelli and Li 2011). The

epithelial expression of vimentin in serous and endometrioid carcinomas can be

interpreted by the tendency of some carcinoma cells to undergo EMT. These events lead

to increased mitotic activity of cancer cells and induce their invasion and metastasis




19


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(Vergara et al. 2010). Vimentin expression was detected in all tumors studied, however,

mucinous cystadenocarcinoma tumors did not show any detectable epithelial vimentin

expression. Serous cystadenocarcinoma and endometrioid adenocarcinoma tumors

expressed vimentin on both stroma and epithelia tissue sites. This later result agrees with

a previous study that found vimentin co-expression with cytokeratin which was more

prevalent in serous and endometrioid tumors and only one out of 29 mucinous tumors

exhibited vimentin expression (Viale et al. 1988). However, the study did not indicate

whether vimentin expression in this single tumor was in the stromal or epithelial part

(Viale et al. 1988).

The expression of vimentin in the epithelium of the studied normal tissues is

consistent with the previous report by Auersperg et al., 2001 who found expression of the

mesenchymal marker vimentin in the Ovarian Surface Epithelium (OSE) of normal

tissues. These cells are considered a mesothelial type of epithelial cell since they share a

common embryological origin with the peritoneum and have characteristics in common

with peritoneal mesothelial cells (Ahmed et al. 2007; Auersperg et al. 2001; Sundfeldt

2003).

Thus, the mucinous cystadenocarcinoma tumors studied exhibited a spatial pattern

in their expression of the different EMT markers which might be related to their response

to treatments. For instance, mucinous adenocarcinoma are considered to be refractory

cancers that are biologically distinct from serous adenocarcinoma and some studies

suggested that mucinous tumors present a poorer outcome from platinum-based first-line



https://www.researchgate.net/publication/12038906_Ovarian_Surface_Epithelium_Biology_Endocrinology_and_Pathology_1?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


https://www.researchgate.net/publication/20701516_Coexpression_of_cytokeratins_and_vimentin_in_common_epithelial_tumours_of_the_ovary_an_immunocytochemical_study_of_eighty-three_cases?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==



20


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chemotherapy when compared to non-mucinous epithelial ovarian cancers (Bamias et al.

2010; Sugiyama et al. 2009).

. Generally, the relationship between E-cadherin and slug in the overall

pathologies of EOC studied was strong positive (rho value =0.861). Although it has been

shown that E-cadherin was repressed by slug (Kim et al. 2012; Peinado et al. 2007;

Vergara et al. 2010; Yoshida et al. 2009). some studies showed that EOC cells do not

always undergo full and classical EMT (Davidson et al. 2012; Peinado et al. 2007). For

instance, E-cadherin was found to be up-regulated in some ovarian neoplasm cells

(Davidson et al. 2012; Rodriguez et al. 2012; Sundfeldt et al. 1997).

In this present study we showed that N-cadherin expression was significantly related to

tumor stages. Cadherin switch is a main hallmark for the EMT process (Vergara et al.

2010). Most EMT-related studies in ovarian cancer, including EOC, confirmed that both

E-cadherin down-regulation and N-cadherin up-regulation are responsible for inducing

the EMT process (Cheng et al. 2012; Theriault et al. 2011). However, N-cadherin

expression rather than E-cadherin down-regulation might be more important in metastasis

and invasion (Nakajima et al. 2004; Nieman et al. 1999). Indeed, during ovarian tumor

progression the expression of E-cadherin and N-cadherin was found to follow two

different routes. The first is when N-cadherin expression is high in the primary tumors; it

is retained in the poorly differentiated metastatic tumors while E-cadherin expression is

lost. The other route is when the metastatic tumors are strongly stained for both cadherins

(Hudson et al. 2008). The actual role of both cadherins in inducing the EMT process in



https://www.researchgate.net/publication/5453397_Hudson_LG_Zeineldin_R_Stack_MS_Phenotypic_plasticity_of_neoplastic_ovarian_epithelium_unique_cadherin_profiles_in_tumor_progression_Clin_Exp_Metastasis_25_643-655?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/6324472_Peinado_H_Olmeda_D_Cano_A_SNAI1_Zeb_and_bHLH_factors_in_tumour_progression_an_alliance_against_the_epithelial_phenotype_Nat_Rev_Cancer_7_415-428?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


https://www.researchgate.net/publication/50594859_MUC16_CA125_regulates_epithelial_ovarian_cancer_cell_growth_tumorigenesis_and_metastasis?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/38058499_Epithelial-mesenchymal_transition_in_ovarian_cancer_Cancer_Lett?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==



https://www.researchgate.net/publication/223961155_EGF-Induced_EMT_and_Invasiveness_in_Serous_Borderline_Ovarian_Tumor_Cells_A_Possible_Step_in_the_Transition_to_Low-Grade_Serous_Carcinoma_Cells?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==



https://www.researchgate.net/publication/228061149_Functional_regulation_of_SlugSnail2_is_dependent_on_GSK-3b-mediated_phosphorylation?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/221969774_E-cadherin's_dark_side_Possible_role_in_tumor_progression?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/13997404_E-cadherin_expression_in_human_epithelial_ovarian_cancer_and_normal_ovary_Int_J_Cancer?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/24021715_Treatments_of_epithelial_ovarian_cancer_by_histologic_subtype?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/222910997_Changes_in_the_expression_of_E-cadherin_repressors_Snail_Slug_SIP1_and_Twist_in_the_development_and_progression_of_ovarian_carcinoma_the_important_role_of_Snail_in_ovarian_tumorigenesis_and_progressio?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


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EOC requires further investigation to determine their association with other signaling

molecules within the cascade of EMT pathway.

The exclusive nuclear localization of NRP-1 in the mucinous tumors is interesting

and opens the doors to investigate its role as a transcriptional regulator. Many studies

demonstrated that VEGFR2, the main receptor to which NRP-1 binds, translocates to the

nucleus in different tumor cells (Blazquez et al. 2006; Fox et al. 2004; Zhang et al. 2005).

It has been reported that VEGFR2 can auto regulate its own transcription upon its binding

with VEGF (Domingues et al. 2011). VEGF, the ligand of both VEGFR2 and NRP-1, is

the major regulator of VEGFR2 auto-regulation, and Sp1 which is a well-known

regulator of NRP-1 expression was also found to be associated with VEGFR-2 in

regulating its own promoter (Domingues et al. 2011). Therefore, nuclear NRP-1 might

act similarly in regulating its own transcription in the mucinous carcinomas studied.

In the current study NRP-1 was not significantly related to tumor grade (p=0.051)

(Table 2S), a finding not consistent with our previous study (Adham et al. 2010). This

controversial difference might be due to the different approach in data analysis used in

both studies. For example, in the past study the cases on the tissue microarray used were

not duplicated and we did not perform the multivariate statistical analysis, as we only

evaluated the staining intensity. To date there are no reports indicating that NRP-1 can be

a partner in the EMT process of EOC tumors, but recently NRP-1 was reported to drive

EMT in High Gleason grade prostate carcinomas by promoting Snail1 nuclear

localization (Mak et al. 2010). According to the Spearman’s coefficient values obtained



https://www.researchgate.net/publication/41657324_Molecular_blockade_of_VEGFR2_in_human_epithelial_ovarian_carcinoma_cells?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==

https://www.researchgate.net/publication/7675124_Expression_and_cellular_localization_of_vascular_endothelial_growth_factor_A_and_its_receptors_in_acute_and_chronic_leukemias_An_immunohistochemical_study?el=1_x_8&enrichId=rgreq-32adaacc690093711fd8f9c7b6f99a90-XXX&enrichSource=Y292ZXJQYWdlOzI2MjY4OTgxNztBUzoxMDI2NTQ2ODAyNDAxMzRAMTQwMTQ4NjE5MjMwNA==


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in our study we found that three markers were positively correlated. The rho value

between slug and NRP-1 (ρ=0.602) which is very similar to the value between NRP-1

and E-cadherin (ρ=0.608) and the rho value between slug and E-cadherin is highly

positive (ρ=0.861). This similarity might indicate that slug is a transcriptional repressor

for E-cadherin, and possibly can control the expression of NRP-1 in a similar way. In

agreement with this, Sp1 that regulates NRP-1 transcription has been shown to share a

binding site in the promoter of both slug (Choi et al. 2007) and snail (Hu et al. 2010),

strong inducers of the EMT process.

Collectively our observations indicate that NRP-1 is a candidate molecule which might

be responsible for the progression of EOC through its potential involvement in EMT and

other aspects of metastasis. Confirming the role of NRP-1 in EOC, EMT and

tumorigenicity could establish this molecule as a potential target for cancer therapies and/

or diagnosis.

Acknowledgment




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We thank Sultan Qaboos University for the generous Grant (# IG/Biol/12/01) to SA that

supported this study. A special thanks for Dr. Charles Bekhit for revising the statistical

analysis.




24


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Figure 1

Biomarker expression in normal, benign and malignant ovarian tissues

A) The Graphs from the left to the right represent the mean IRS values for NRP-1,

Slug and E-cadherin in 7 normal, 7 benign ovarian tissues and in 24 serous, 27

endometrioid and 12 mucinous ovary carcinomas,.. The error bars represent the

±Standard Error of the Mean (SEM) among the different IRS values ; *

significantly different from normal; p < 0.05.. B)The top images show two

different normal ovaries and the surface epithelium was negatively stained for




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NRP-1. The bottom panel shows images for two different benign ovarian tissues,

with surface epithelium stained positively for NRP-1. C. The image shows the

only normal tissue stained positively for slug. Scale bars = 50 μm.




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Figure 2

Vimentin and Slug tissue localization in the different EOC subtypes

A) Top images endometrioid carcinoma tissues grade II and III stained with vimentin in

their epithelial components. Bottom images, vimentin was only positive in the stroma of

all mucinous carcinomas and adenomas. B) Slug expression was located mainly in the

cytoplasm of serous and endometrioid carcinomas however it was mainly nuclear in

mucinous tumors. Scale bars = 50 μm.




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Figure 3

Differential cellular localization of NRP-1 among the three EOC pathologies

Immunohistochemical detection of NRP-1 in the three different EOC pathological

subtypes.

A) shows the intensity of staining and positive cell distribution, B) shows NRP-1

localized in the nucleus and C) shows NRP-1 localized in both the nucleus and cytoplasm

simultaneously. Scale bars = 50 μm.




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Figure 4

Representative images for N-cadherin staining distribution in both tissue compartments

stromal and epithelial in stage I (A), stage II (B), stage III (C) and stage IV (D).




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Table 1. Expression of NRP-1, slug and E-cadherin in normal, benign and three subtypes of ovarian carcinoma represented by the mean IRS value

Tissue IRS NRP-1 IRS Slug IRS E-cadherin

Normal (7 cases) 0.00 (± 0.00) 0.17 (± 0.17) 0.71 (± 0.19)

Benign (7 cases) 2.08 (±0.58) 1.51(±1.20) 2.71 (±1.14) Total IRS mean 1.04 (±0.29) 0.84 (±0.68) 1.71 (±0.66) Serous (24 cases) 5.76 (±0.54) 5.69 (±0.59) 5.29 (±0.52) Endometrioid (27 cases) 5.97 (±0.54) 4.48 (±0.56) 5.03 (±0.53)




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Biomarkers & Categories

Histological Subtypes

Serous Endometrioid Mucinous P value Slug Locus (Tissue level)

Negative 1 (4.2%) 1 (3.7%) 3 (23.1%) 0.001* Epithelial 21 (87.5%) 23 (85.2%) 8 (61.5%) Stromal 2 (8.3%) 3 (11.1%) 2 (15.4%) Total 24 (100.0 %) 27 (100.0 %) 12 (100.0%) NRP-1 Locus (Cellular level)

Negative 2 (4.2%) 2 (7.5%) 2 (23%) 0.004* Nuclear 8 (33.3%) 5 (18.5%) 5 (46.2%) Cytoplasmic 10 (41.7%) 8 (29.6%) 0 (0.0%) Nuclear & Cytoplasmic

5 (20.8%) 12 (44.4%) 4 (30.8%)

Total 24 (100.0%) 27 (100.0%) 12 (100.0%) Table 2. Profiling of slug and NRP-1 tissue localization among the three pathological

types of Epithelial Ovarian Cancer (EOC)

Mucinous (12 cases) 3.83 (±0.84) 4.35 (±0.96) 4.62 (±1.00) Total IRS mean 5.18 (±0.64) 4.84 (±0.7) 4.98 (±0.68)




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Vimentin

Normal Benign Serous Endometrioid Mucinous p value

Stromal 3 (42.9%)) 4 (57.1%) 10 (38.5%) 15 (55.6%) 12 (100.0%) P=0.058

Epithelial 0 (0.00%) 0 (0.00%) 0 (0.00%) 2 (7.4%) 0 (0.00%)

Epithelial &

Stromal

4 (57.1%) 3 (42.9%) 14 (61.5%) 10 (37.0%) 0 (0.00%)

Total 7(100.0%) 7(100.0%) 24 (100.0%) 27 (100.0%) 12(100.0%)

N Cadherin

Stromal 2 (28.6%) 2 (28.6%) 2 (8.3%) 8 (29.6%) 4 (33.3%) P<0.001*

Epithelial 5 (71.4%) 4 (57.1%) 1(4.2%) 1 (3.7%) 0 (0.0%)

Epithelial &

stromal

0 (0.00%) 1 (14.3%) 21 (87.5%) 18 (66.7%) 8 (66.7%)

Total 7 (100.0%) 7 (100.0%) 24(100.0%) 27 (100.0%) 12(100.0%)

Table 3. Tissue localization of both vimentin and N-cadherin in normal, benign and the

three different subtypes of EOC




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Biomarkers & Categories

Stage I Stage II Stage III Stage IV P value

n (%) n (%) n (%) n (%) N-cadherin Mainly stromal

5 (15.2%) 4 (40%) 0 (0.0) 5 (33.3%) P < 0.001

Mainly epithelial

1 (9.1%) 1 (10.0%) 0 (0.0) 0 (0.0)

Both epithelium & Stromal

27 (56.3%) 5 (50.0%) 5 (100.0) 10 (66.7%)

Total 35 (100.0%) 11 (100.0%) 5 (100.0) 16 (100.0) Table 4. N-cadherin significantly changing as the stage of EOC changes



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