Immunohistological Insight into the Correlation between Neuropilin-1 and Epithelial-Mesenchymal Transition Markers in Epithelial Ovarian Cancer

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 aforementioned 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, respectively), with 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.

Keywords

neuropilin-1 EMT biomarker slug E-cadherin vimentin OSE

Introduction

Epithelial Ovarian Cancer (EOC) is the most common type of ovarian cancer affecting women worldwide. Previous studies have shown that about 75% of patients are diagnosed at the late stage of the disease (Hennessy et al. 2009; Siegel et al. 2011). EOC is classified into five major subtypes: 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 60–80% 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 sequence from adenoma to carcinoma (Levanon et al. 2008).

EOCs were initially thought to arise from the Ovarian Surface Epithelium (OSE); therefore, studies previously focused on identifying closely related biomarkers expressed specifically by epithelial cells, such as mucins (Auersperg et al. 2001; Chauhan et al. 2006). Mucins are proteins found to be highly expressed in EOCs and their glycosylated extracellular domain, which may overhang up to 200-2000 nm above the cell surface, has been associated with the dissemination and invasion of ovarian cancer cells. Recently, it has been shown that glycoprofiling of mucin 16 (MUC16), also known as CA125, improves the differential diagnosis of ovarian cancer (Chauhan et al. 2009; Chen et al. 2013). However, although CA125 was previously identified as an ovarian cancer diagnostic biomarker, it 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 patients diagnosed with early stage tumors because this antigen is not expressed in most early stage ovarian tumors (Jacobs et al. 1993). More recently though, in a clinical trial on advanced ovarian cancer patients it was shown that CA125 is 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 the design of appropriate, patient-specific therapy. In addition to ovarian surface epithelium, it is now apparent that EOC can also derive from the fallopian tube, potentially changing the criteria used to identify EOC. Thus, the genes and/or proteins detected in cells from EOC 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-to-Mesenchymal Transition (EMT) and its reverse process, Mesenchymal-to-Epithelial Transition (MET). It has been shown that EMT in ovarian cancer is controlled by different cytokines and growth factors, such as mucin (MUC4) and 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 induced the expression of EMT proteins in EOC cells (Skov-3), which could be reversed with the use of inhibitors against thrombin (hirudin); this indicates that the use of anticoagulants might serve in the control of EOC progression (Zhong et al. 2013).

Neuropilins (NRPs) are a transmembrane glycoprotein family of non-tyrosine kinase receptors ranging from 130 to 140 kDa (Ellis 2006; Hu et al. 2007; Lu et al. 2009; Yu et al. 2010). They have a large extracellular region comprising three main domains (a1a2, b1b2 and c (MAM)), a transmembrane domain, and a short, intracellular (cytoplasmic) domain that lacks any enzymatic activity (Ellis 2006; Herzog et al. 2011; Lu et al. 2009). NRPs are expressed in neurons, endothelial cells, mesothelial cells, bone marrow cells 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, including 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 the induction of malignancy in different cancer types, such as breast (Stephenson et al. 2002), colorectal (Yu et al. 2010), myeloid leukemia (Kreuter et al. 2006), glioma (Hu et al. 2007), pancreatic (Wey et al. 2005), and prostate (Hu et al. 2007) cancers, and its silencing in hepatocellular carcinoma led to cellular growth suppression in vitro and in vivo (Xu and Xia 2013). NRP-1 has also been reported to promote unlimited growth in ovarian cancer through evasion of contact inhibition (Wong et al. 1999). Osada et al. (2006) 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 benign ovarian tumors.

NRP-1 is a multifunctional protein existing in two forms: soluble and transmembrane receptors (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 facilitates 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). The use of an anti-L1-CAM monoclonal antibody inhibited peritoneal growth and the dissemination of human ovarian carcinoma cells in vitro and in nude mice (Arlt et al. 2006). This latter study confirmed 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 EMT 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 biomarkers for the decision as to 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 tumorigenesis is still not well defined. Here, 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 of 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 (as represented by the Immunoreactive Score, IRS) 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 pave the way 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.

Materials & Methods

Tissue Microarrays (TMAs) and Tissue Inclusion Criteria

Human ovarian tissue arrays were used for immunohistochemical staining (Cat# OVC1501; Pantomics, Richmond, CA). These arrays contained tissues fixed in 10% formalin (pH 7.0) for 24 hr and were processed with identical standard operating procedures. Each tissue array had 150 cores collected from 75 individuals, with two cores representing one tumor tissue or biopsy from the same patient. Among the 75 cases there were 2 normal, 3 benign (mucinous, serous cystadenoma and thecoma) and 73 EOC cases, each provided with pathological, grading and staging data (Supplemental Table S1). The regions of each block chosen for inclusion in the TMA were reviewed by at least three pathologists who examined the H&E-stained slides. The main inclusion criteria were to select a region of the tissue that provided sufficient and representative target cells, and this region was marked on the slides. The marked areas were then transferred to corresponding re-marked blocks (formalin-fixed paraffin-embedded blocks) by a pathologist. To ensure the representative nature of a case, duplicated cores per case for most of the TMAs 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

Clinical samples, consisting of five normal, four benign (two mucinous and two serous cystadenoma) and four neoplasia (two mucinous and two 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 (described below). Patients routinely provide their full consent for the donation of the tissue prior to any surgical procedure and their information is kept confidential and used for research purposes only.

Immunohistochemistry

TMA slides and patient individual ovary sections were deparaffinized in xylene, rehydrated in a series of ethanol (100%, 95% and 75%) followed by tap water, and then subjected to antigen retrieval using 1 mM EDTA (pH 9.0), in 95C 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-buffered saline (PBS), followed by PBS containing 0.05% Triton X-100, for 5 min each wash. The slides were then incubated with a blocking solution of 5% normal goat serum for 30 min at room temperature, then incubated overnight at 4C with appropriate primary antibody solutions containing one of the following monoclonal antibodies: Neuropilin-1, 1:250; vimentin, 1:250; and N-cadherin, 1:500 (Epitomics Inc.; Burlingame, CA; cat # 2621-1, 2707-1 and 2019-1, respectively). E-cadherin and Slug antibodies were used at 1:200 and were purchased from Cell Signaling Technologies (Beverly, MA) (cat #3195 and #9585, respectively). All other chemicals used were purchased from Sigma Aldrich (Munich, Germany).

After 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 Laboratories) for 30 min at room temperature. Colorimetric detection was achieved by incubation with 3,3’-diaminobenzidine (DAB) (Dako; Warrington, PA) 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%) and then immersed into xylene. Finally, the slides were mounted using DPX (Di-n-butyl phthalate in Xylene) (Sigma- Aldrich). Tissues were visualized using an Olympus (BX 40; Olympus Optical Co. Ltd; Tokyo, Japan) light microscopy with digital camera (DP50). The images of cores were captured by 40× using OLYSIA Bio-report and Twin Viewfinder Light (Version 1.0) software (Olympus). A negative primary antibody control slide was stained simultaneously to confirm staining specificity (Supplementary Fig. S1).

Immunohistochemical Reactivity Evaluation and Scoring Categories

Microscopic examination was performed independently in a blinded fashion by two scientists and one 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 benign, and 4 malignant) were studied and analyzed for each biomarker. Each specimen was visualized individually under the light microscope (400×) and assigned to an appropriate category. Finally, the scores of each case (2 cores) were combined and given a specific code for statistical analysis. The staining intensity (SI) for the expression of epithelial proteins—E- Cadherin, NRP-1 and Slug—was graded based on DAB staining intensity. 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 400× (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. The IRS value was calculated according to a previously published method, as follows: IRS = SI × PP (Chui et al. 1996); the mean IRS for each marker (NRP-1, Slug, E-cadherin) for each case was calculated as the final value used for statistical analysis.

Because N-cadherin and vimentin expression (intensity and distribution) was homogenous in all TMA studied, these two proteins were categorized based on tissue localization: group 1, mainly stromal; group 2, mainly epithelial; group 3, both epithelial and stromal. In addition to the IRS score, Slug was also grouped for its tissue location (0, negative staining; 1, epithelial staining; 2, stromal staining) and for its cellular localization (0, negative 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 (two-sided). The Spearman’s rank correlation was used to determine whether there was a positive or negative correlation between variables. Post hoc tests were 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 tests, considering α of 0.05; the Bonferroni’s adjustment p value was 0.0071. This p value was used as a cut-off point for significance. SPSS for Windows (v19.0; Chicago, IL) was used to analyze the data.

Results

NRP-1, Slug and E-cadherin Expression in Normal, Benign and Malignant Ovarian Tissue

A total of seven normal ovarian tissues were used, in which two (four cores) were included within the TMA and another five were normal ovaries retrieved from pathology archives. Similarly, of another seven benign cases that were examined, three were included in the TMA slides (one each of mucinous cystadenoma, serous cystadenoma and thecoma) and another four cases were retrieved from the pathology archives (two serous and two mucinous cystadenoma). The normal and benign cases were used as controls to track changes in protein expression and compare them to the neoplasia cases (Fig. 1A). E-cadherin and Slug were weakly expressed in all normal cases (TMA and individual specimens), as represented by the mean IRS values of 0.7143 ± 0.19 (SEM) and 0.1714 ± 0.1749, respectively. NRP-1 was not detected in those specimens (IRS value, 0.0; Fig. 1A, 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 p>0.05 (Fig. 1A, 1B). 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, no statistical differences were detected among the three subtypes. The Bonferroni-adjusted p value between the three pathologies was p>0.05 (Fig. 1A, Table 1).

Figure 1.

Biomarker expression in normal, benign and malignant ovarian tissues. (A) The graphs from left to right represent the mean IRS values for NRP-1, Slug and E-cadherin in 7 normal and 7 benign ovarian tissues, and 24 serous, 27 endometrioid (Endom) and 12 mucinous (Mucin) ovary carcinomas. The error bars represent the SEM; * indicates significance as compared with normal tissues; p<0.05. (B) The top images show two different normal ovaries and negative staining of NRP-1 on the surface epithelium. The bottom panel shows images for two different benign ovarian tissues, with positive NRP-1 surface epithelium staining. (C) Normal tissue stained positively for Slug. Scale bars = 50 µm.

Table 1.

Expression of NRP-1, Slug and E-cadherin in Normal, Benign and Three Subtypes of Epithelial Ovarian Carcinoma.

Tissue	IRS NRP-1	IRS Slug	IRS E-cadherin
Normal (7 cases)	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
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

Data are the mean IRS value ± SEM.

Localization and Tissue Type Expression of Markers

Three pathological subtypes of 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, as these pathologies had higher frequencies of the proteins among the test cases. 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 (Supplemental Table S1). 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 Is Pathology Subtype Dependent

The EMT inducer Slug was detected in both epithelial and stromal parts of the tissue (Fig. 2C). Furthermore, 52 of the 73 tumors (72.2%) expressed Slug in the epithelium as compared to only 19.4% with expression in the stroma (Supplemental Table S1). 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 of 24 serous cystadenocarcinomas (87.5%) and 23 of 27 endometrioid adenocarcinomas (85.2%) displayed higher percentages of epithelial slug expression when compared with mucinous cystadenocarcinoma tumors, for which only 8 of 12 cases (61.5%) showed slug localization in the epithelium (Table 2).

Figure 2.

Vimentin and Slug tissue localization in the different EOC subtypes. (A) Slug expression was located mainly in the cytoplasm of serous and endometrioid carcinomas, however, it was mainly nuclear in mucinous tumors. (B) Top images, endometrioid carcinoma tissues grade II and III stained with vimentin in their epithelial components. (C) Bottom images, vimentin was only positive in the stroma of all mucinous carcinomas and adenomas. Scale bars = 50 µm.

Table 2.

Profiling of Slug and NRP-1 Tissue Localization among the Three Pathological Types of Epithelial Ovarian Carcinoma.

	Histological Subtypes
Biomarkers & Categories	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%)

NRP-1 Cellular Localization Is Pathological Subtype Dependent

NRP-1 was expressed exclusively in the epithelial parts of EOC tumors. Therefore, we investigated its expression through IRS 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 of 27 endometrioid adenocarcinomas (44.4%), 5 of 24 serous cystadenocarcinomas (20.8%) and 4 of 13 mucinous cystadenocarcinomas (30.80%) (p=0.004). Cytoplasmic-restricted NRP-1 localization was not detected in any of the 12 mucinous cystadenocarcinomas (0.0%). However, the number of patients with NRP-1 localized in the nucleus was highest in patients with mucinous tumors (6 of 13, 46.20%) as compared with the other two pathologies. Patients with serous tumors had higher nuclear NRP-1 expression as compared with endometrioid (8 of 24 serous, 33.3%; and 5 of 27 endometrioid; 18.5%) (Fig. 3, Table 2).

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 in samples with a (A) cellular distribution, (B) nuclear localization, and C) nuclear and cytoplasmic distribution. Scale bars = 50 μm.

Vimentin Is Expressed in Both Stroma and Epithelia in the Majority of Ovarian Tissues

Descriptive statistics was used to ascertain an association for Vimentin tissue distribution (stromal, epithelial and both stromal and epithelial) in normal, benign and three subtypes of ovarian carcinomas (cross tabulation 3×5). 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 remaining tumors expressed vimentin either only in the stroma (38 cases) or in both the stroma and epithelia (27 cases) (Fig. 2, Supplemental Table S1). Vimentin was exclusively expressed in the stroma of all mucinous tumors present in TMAs and in the additional two retrieved pathologies, with no expression in the epithelial compartment (Fig. 2B). In contrast, Vimentin expression in both stroma and epithelia was detected in 14 of 24 serous carcinomas (61.5%) and 10 of 27 endometrioid carcinomas (37.0%) (Table 3).

Table 3.

Tissue Localization of Both Vimentin and N-cadherin in Normal, Benign and the Three Different Subtypes of Epithelial Ovarian Carcinoma.

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%)

Unlike E-cadherin, N-cadherin was expressed in both stroma and epithelium. Its tendency to be expressed in both epithelium and stroma was higher than its expression in only the stroma and/or epithelia (Fig. 4; Supplemental Table S1). (In 50 of the 70 EOC cases, N-cadherin was expressed in both locations.) Multiple comparisons showed that N-cadherin tissue distribution was associated with the different ovarian tissue types, as shown in Table 4.

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) epithelial ovarian cancer samples.

Table 4.

Changes in N-cadherin Significantly Correlate with Changes in the Stage of Epithelial Ovarian Carcinoma.

	Stage I	Stage II	Stage III	Stage IV
Biomarkers & Categories	n (%)	n (%)	n (%)	n (%)	P value
N-cadherin
Mainly stromal	5 (15.2%)	4 (40%)	0	5 (33.3%)	p < 0.001
Mainly epithelial	1 (9.1%)	1 (10.0%)	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%)

N-cadherin Is Related Significantly with Tumor Stage

Although none of the proteins studied showed a significant relationship with tumor grade (Supplemental Table S2), the N-cadherin tissue distribution (stromal, epithelial and both) was significantly related to tumor stage (p>0.001 and Table 4). N-cadherin was observed in 27 of 35 stage I tumors (56.3%), 5 of 11 stage II tumors (50.0%), 5 of 5 stage III tumors (100.0%) and 10 of 16 stage IV tumors (66.7 %) (Table 4). Figure 4 shows the staining patterns 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). (Supplemental Table S3).

Discussion

Histopathological examination is the first method used in the diagnosis and differentiation of EOC heterogeneous tumors and in the separation of closely related cases (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 during the planning of treatment and management as studies show that the different subtypes respond differently to treatment regimens (Bamias et al. 2010). Further investigations into new predictive biomarkers that can identify 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 normal ovary tissue samples; however, it was detected in the benign tissue specimens. This finding is fully consistent with the previous study by Hall et al. (2005) and partially consistent with the study reported by Baba et al. (2007). Baba’s group showed that NRP-1 was neither expressed in the normal ovary surface epithelia nor in the benign cases. Yet, in this study, we showed that the cellular localization of NRP-1 was significantly different among the three subtypes of EOC. The nuclear localization of NRP-1 was more prevalent in mucinous cystadenocarcinoma tumors than in endometrioid adenocarcinoma or 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 the samples expressed cytoplasmic NRP-1.

Generally, the stromal expression of vimentin in the three pathologies is due to its role 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 an increased mitotic activity of cancer cells and induce their invasion and metastasis (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 latter result agrees with a previous study that found vimentin co-expression with cytokeratin to be more prevalent in serous and endometrioid tumors, whereas only 1 of 29 mucinous tumors exhibited vimentin expression (Viale et al. 1988). However, in that study, the authors did not indicate whether vimentin expression in this single tumor was in the stromal or epithelial part of the tissue (Viale et al. 1988).

The expression of vimentin in the epithelium of the studied normal tissues is consistent with that of a previous report by Auersperg et al. (2001), wherein the authors showed expression of the mesenchymal marker vimentin in Ovarian Surface Epithelium (OSE) of normal tissues. These cells are considered a mesothelial type of epithelial cell because 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).

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 suggest that mucinous tumors present a poorer outcome from platinum-based first-line 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 strongly positive (ρ=0.861). Although it has been shown that E-cadherin is repressed by Slug (Kim et al. 2012; Peinado et al. 2007; Vergara et al. 2010; Yoshida et al. 2009), some studies show that EOC cells do not always undergo a 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. A 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 expressions of E-cadherin and N-cadherin were found to follow two different routes. When N-cadherin expression is high in the primary tumors, it is retained in the poorly differentiated metastatic tumors whereas E-cadherin expression is lost. The other route is when the metastatic tumors stain strongly for both cadherins (Hudson et al. 2008). The actual role of both cadherins in inducing the EMT process in EOC requires further investigation to determine their association with other signaling molecules within the 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 have 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 binding with VEGF (Domingues et al. 2011). VEGF, the ligand for 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) (Supplemental Table S2), a finding that is inconsistent with our previous study (Adham et al. 2010). This controversial difference might be due to the different approach in data analysis used in the two studies. For example, in our previous study, the cases on the TMAs were not duplicated and we did not perform a 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 NRP-1 was recently 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 in our study, we found that three markers were positively correlated. The rho value between Slug and NRP-1 (ρ=0.602) is very similar to that between NRP-1 and E-cadherin (ρ=0.608) and the rho value between Slug and E-cadherin is also 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, which 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), two strong inducers of the EMT process.

Collectively, our observations indicate that NRP-1 is a candidate molecule that 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.

Footnotes

Acknowledgements

A special thanks to Dr. Charles Bekhit for revising the statistical analysis.

Supplementary material for this article is available on the Journal of Histochemistry & Cytochemistry Web site at .

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We thank Sultan Qaboos University for the generous Grant (# IG/Biol/12/01) to SA that supported this study.

References

Adham

Sher

Coomber

(2010). Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells. Lab Invest 90:709-723.

Ahmed

Thompson

Quinn

(2007). Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm. J Cell Physiol 213:581-588.

Arlt

Novak-Hofer

Gast

Gschwend

Moldenhauer

Grunberg

Honer

Schubiger

Altevogt

Kruger

(2006). Efficient inhibition of intra-peritoneal tumor growth and dissemination of human ovarian carcinoma cells in nude mice by anti-L1-cell adhesion molecule monoclonal antibody treatment. Cancer Res 66:936-943.

Auersperg

Wong

Choi

Kang

Leung

(2001). Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr Rev 22:255-288.

Baba

Kariya

Higuchi

Mandai

Matsumura

Kondoh

Miyanishi

Fukuhara

Takakura

Fujii

(2007). Neuropilin-1 promotes unlimited growth of ovarian cancer by evading contact inhibition. Gynecol Oncol 105:703-711.

Bamias

Psaltopoulou

Sotiropoulou

Haidopoulos

Lianos

Bournakis

Papadimitriou

Rodolakis

Vlahos

Dimopoulos

(2010). Mucinous but not clear cell histology is associated with inferior survival in patients with advanced stage ovarian carcinoma treated with platinum-paclitaxel chemotherapy. Cancer 116:1462-1468.

Blazquez

Cook

Micklem

Harris

Gatter

Pezzella

(2006). Phosphorylated KDR can be located in the nucleus of neoplastic cells. Cell Res 16:93-98.

Bracke

(2007). Role of adhesion molecules in locoregional cancer spread. Cancer Treat Res 134:35-49.

Cannistra

(2004). Cancer of the ovary. N Engl J Med 351:2519-2529.

10.

Chauhan

Kumar

Jaggi

(2009). Mucins in ovarian cancer diagnosis and therapy. J Ovarian Res 2:21.

11.

Chauhan

Singh

Ruiz

Johansson

Jain

Smith

Moniaux

Batra

(2006). Aberrant expression of MUC4 in ovarian carcinoma: diagnostic significance alone and in combination with MUC1 and MUC16 (CA125). Mod Pathol 19:1386-1394.

12.

Chen

Gentry-Maharaj

Burnell

Steentoft

Marcos-Silva

Mandel

Jacobs

Dawnay

Menon

Blixt

(2013). Microarray Glycoprofiling of CA125 improves differential diagnosis of ovarian cancer. J Proteome Res 12:1408-1418.

13.

Cheng

Auersperg

Leung

(2012). EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells? PLoS One 7:e34071.

14.

Choi

Park

Joo

(2007). Transforming growth factor-beta1 represses E-cadherin production via slug expression in lens epithelial cells. Invest Ophthalmol Vis Sci 48:2708-2718.

15.

Chui

Egami

Yamashita

Kurizaki

Ohmachi

Yamamoto

Ogawa

(1996). Immunohistochemical expression of the c-kit proto-oncogene product in human malignant and non-malignant breast tissues. Br J Cancer 73:1233-1236.

16.

D’Andrilli

Giordano

Bovicelli

(2008). Epithelial ovarian cancer: the role of cell cycle genes in the different histotypes. Open Clin Cancer J 2:7-12.

17.

Davidson

Trope

Reich

(2012). Epithelial-mesenchymal transition in ovarian carcinoma. Front Oncol 2:33.

18.

Domingues

Rino

Demmers

de Lanerolle

Santos

(2011). VEGFR2 translocates to the nucleus to regulate its own transcription. PLoS One 6:e25668.

19.

Ellis

(2006). The role of neuropilins in cancer. Mol Cancer Ther 5:1099-1107.

20.

Fox

Turley

Cheale

Blazquez

Roberts

James

Cook

Harris

Gatter

(2004). Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus. J Pathol 202:313-320.

21.

Glinka

Stoilova

Mohammed

Prud’homme

(2011). Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta. Carcinogenesis 32:613-621.

22.

Hall

Turnbull

Bedford

Richmond

Helboe

Atkin

(2005). Neuropilin-1 and VEGF correlate with somatostatin expression and microvessel density in ovarian tumours. Int J Oncol. 27:1283-1288.

23.

Hennessy

Coleman

Markman

(2009). Ovarian cancer. Lancet 374:1371-1382.

24.

Herzog

Pellet-Many

Britton

Hartzoulakis

Zachary

(2011). VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation. Mol Biol Cell 22:2766-2776.

25.

Hong

Chen

Yuan

Chao

Chung

Yang

Pan

Shih

Chan

Yang

(2007). Targeting neuropilin 1 as an antitumor strategy in lung cancer. Clin Cancer Res 13:4759-4768.

26.

Guo

Bar-Joseph

Imanishi

Jarzynka

Bogler

Mikkelsen

Hirose

Nishikawa

Cheng

(2007). Neuropilin-1 promotes human glioma progression through potentiating the activity of the HGF/SF autocrine pathway. Oncogene 26:5577-5586.

27.

Chang

Cheng

Liu

(2010). Snail associates with EGR-1 and SP-1 to upregulate transcriptional activation of p15INK4b. FEBS J 277:1202-1218.

28.

Hudson

Zeineldin

Stack

(2008). Phenotypic plasticity of neoplastic ovarian epithelium: unique cadherin profiles in tumor progression. Clin Exp Metastasis 25:643-655.

29.

Ichigo

Takagi

Matsunami

Murase

Ikeda

Imai

(2012). Transitional cell carcinoma of the ovary (Review). Oncol Lett 3:3-6.

30.

Jacobs

Rivera

Oram

Bast

Jr. (1993). Differential diagnosis of ovarian cancer with tumour markers CA 125, CA 15-3 and TAG 72.3. Br J Obstet Gynaecol 100:1120-1124.

31.

Jubb

Miller

Rugo

Harris

Chen

Reimann

Cobleigh

Schmidt

Langmuir

Hillan

Chen

Koeppen

(2011). Impact of exploratory biomarkers on the treatment effect of bevacizumab in metastatic breast cancer. Clin Cancer Res 17:372-381.

32.

Karst

Drapkin

(2010). Ovarian cancer pathogenesis: a model in evolution. J Oncol 2010:932371.

33.

Kiefel

Bondong

Pfeifer

Schirmer

Erbe-Hoffmann

Schafer

Sebens

Altevogt

(2012). EMT-associated up-regulation of L1CAM provides insights into L1CAM-mediated integrin signalling and NF-kappaB activation. Carcinogenesis 33:1919-1929.

34.

Kim

Yang

Cho

Lee

Cho

(2012). Functional regulation of Slug/Snail2 is dependent on GSK-3beta-mediated phosphorylation. FEBS J 279:2929-2939.

35.

Kreuter

Woelke

Bieker

Schliemann

Steins

Buechner

Berdel

Mesters

(2006). Correlation of neuropilin-1 overexpression to survival in acute myeloid leukemia. Leukemia 20:1950-1954.

36.

Lalwani

Prasad

Vikram

Shanbhogue

Huettner

Fasih

(2011). Histologic, molecular, and cytogenetic features of ovarian cancers: implications for diagnosis and treatment. Radiographics 31:625-646.

37.

Lambrechts

Lenz

de Haas

Carmeliet

Scherer

(2013). Markers of response for the antiangiogenic agent bevacizumab. J Clin Oncol 31:1219-1230.

38.

Levanon

Crum

Drapkin

(2008). New insights into the pathogenesis of serous ovarian cancer and its clinical impact. J Clin Oncol 26:5284-5293.

39.

Xiang

Liu

Tong

Watts

Koch

Sandoval

Damico

Wong

Meng

(2009). Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration. MAbs 1:364-369.

40.

Mak

Leav

Pursell

Bae

Yang

Taglienti

Gouvin

Sharma

Mercurio

(2010). ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading. Cancer Cell 17:319-332.

41.

Maru

Venook

Ellis

(2013). Predictive biomarkers for bevacizumab: are we there yet? Clin Cancer Res 19:2824-2827.

42.

Nakajima

Doi

Toyoda

Tsuji

Wada

Koizumi

Tulachan

Ito

Kami

Mori

Kawaguchi

Fujimoto

Hosotani

Imamura

(2004). N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma. Clin Cancer Res 10:4125-4133.

43.

Nieman

Prudoff

Johnson

Wheelock

(1999). N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J Cell Biol 147:631-644.

44.

Osada

Horiuchi

Kikuchi

Ohira

Ota

Katsuyama

Konishi

(2006). Expression of semaphorins, vascular endothelial growth factor, and their common receptor neuropilins and alleic loss of semaphorin locus in epithelial ovarian neoplasms: increased ratio of vascular endothelial growth factor to semaphorin is a poor prognostic factor in ovarian carcinomas. Hum Pathol 37:1414-1425.

45.

O’Shannessy

Jackson

Twine

Hoffman

Dezso

Agoulnik

Somers

(2013). Gene expression analyses support fallopian tube epithelium as the cell of origin of epithelial ovarian cancer. Int J Mol Sci 14:13687-13703.

46.

Pan

Chanthery

Liang

Stawicki

Mak

Rathore

Tong

Kowalski

Yee

Pacheco

Ross

Cheng

Le Couter

Plowman

Peale

Koch

Bagri

Tessier-Lavigne

Watts

(2007). Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell 11:53-67.

47.

Peinado

Olmeda

Cano

(2007). Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7:415-428.

48.

Ponnusamy

Lakshmanan

Jain

Das

Chakraborty

Dey

Batra

(2010). MUC4 mucin-induced epithelial to mesenchymal transition: a novel mechanism for metastasis of human ovarian cancer cells. Oncogene 29:5741-5754.

49.

Rodriguez

Lewis-Tuffin

Anastasiadis

(2012). E-cadherin’s dark side: possible role in tumor progression. Biochim Biophys Acta 1826:23-31.

50.

Satelli

(2011). Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci 68:3033-3046.

51.

Siegel

Ward

Brawley

Jemal

(2011). Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin 61:212-236.

52.

Smart

Morrison

Saunus

Vargas

Keith

Reid

Wockner

Amiri

Sarkar

Simpson

Clarke

Schmidt

Reynolds

Lakhani

Lopez

(2013). In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity. PLoS One 8:e64388.

53.

Song

Lee

Choi

Son

Lee

Yoon

Park

Hur

Ryu

Lee

(2013). Diagnostic value of CA125 as a predictor of recurrence in advanced ovarian cancer. Eur J Gynaecol Oncol 34:148-151.

54.

Soslow

(2008). Histologic subtypes of ovarian carcinoma: an overview. Int J Gynecol Pathol 27:161-174.

55.

Stephenson

Banerjee

Saxena

Cherian

Banerjee

(2002). Neuropilin-1 is differentially expressed in myoepithelial cells and vascular smooth muscle cells in preneoplastic and neoplastic human breast: a possible marker for the progression of breast cancer. Int J Cancer 101:409-414.

56.

Stoeck

Schlich

Issa

Gschwend

Wenger

Herr

Marme

Bourbie

Altevogt

Gutwein

(2006). L1 on ovarian carcinoma cells is a binding partner for Neuropilin-1 on mesothelial cells. Cancer Lett 239:212-226.

57.

Sugiyama

Kumagai

Hatayama

(2009). [Treatments of epithelial ovarian cancer by histologic subtype]. Gan To Kagaku Ryoho 36:187-192.

58.

Sundfeldt

(2003). Cell-cell adhesion in the normal ovary and ovarian tumors of epithelial origin; an exception to the rule. Mol Cell Endocrinol 202:89-96.

59.

Sundfeldt

Piontkewitz

Ivarsson

Nilsson

Hellberg

Brannstrom

Janson

Enerback

Hedin

(1997). E-cadherin expression in human epithelial ovarian cancer and normal ovary. Int J Cancer 74:275-280.

60.

Theriault

Pinard

Comamala

Migneault

Beaudin

Matte

Boivin

Piche

Rancourt

(2011). MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis. Gynecol Oncol 121:434-443.

61.

Uniewicz

Cross

Fernig

(2011). Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis. J Biol Chem 286:12-23.

62.

Vergara

Merlot

Lucot

Collinet

Vinatier

Fournier

Salzet

(2010). Epithelial-mesenchymal transition in ovarian cancer. Cancer Lett 291:59-66.

63.

Viale

Gambacorta

Dell’Orto

Coggi

(1988). Coexpression of cytokeratins and vimentin in common epithelial tumours of the ovary: an immunocytochemical study of eighty-three cases. Virchows Arch A Pathol Anat Histopathol 413:91-101.

64.

Wey

Gray

Fan

Belcheva

McCarty

Stoeltzing

Somcio

Liu

Evans

Klagsbrun

Gallick

Ellis

(2005). Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells. Br J Cancer 93:233-241.

65.

Wong

Maines-Bandiera

Rosen

Wheelock

Johnson

Leung

Roskelley

Auersperg

(1999). Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: influence of family history of ovarian cancer. Int J Cancer 81:180-188.

66.

Xia

(2013). NRP-1 silencing suppresses hepatocellular carcinoma cell growth in vitro and in vivo. Exp Ther Med 5:150-154.

67.

Yoshida

Horiuchi

Kikuchi

Hayashi

Osada

Ohira

Shiozawa

Konishi

(2009). 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 progression. Med Mol Morphol 42:82-91.

68.

Waby

Chirakkal

Staton

Corfe

(2010). Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation. Mol Cancer 9:276.

69.

Zhang

Pillai

Gatter

Blazquez

Turley

Pezzella

Watt

(2005). Expression and cellular localization of vascular endothelial growth factor A and its receptors in acute and chronic leukemias: an immunohistochemical study. Hum Pathol 36:797-805.

70.

Zhong

Zhang

(2013). Thrombin promotes epithelial ovarian cancer cell invasion by inducing epithelial-mesenchymal transition. J Gynecol Oncol 24:265-272.

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