Ovarian cancer, consisting predominantly of carcinomas, is the most lethal gynecologic cancer and is the fifth leading cause of cancer-related death among women worldwide (Siegel et al., 2013). The poor survival of patients diagnosed with this malignancy is attributed to diagnosis at advanced stage (stages III/IV), when the tumor has metastasized (Siegel et al., 2013).
Epithelial–mesenchymal transition (EMT), a physiological process by which mesenchymal cells are formed and migrate to target organs during embryogenesis, is involved in cancer cell invasion and metastasis (Guo et al., 2014). During EMT, cells undergo a morphological switch from the epithelial polarized phenotype to a highly motile fibroblastic or mesenchymal phenotype; thus, the characteristic changes of EMT are the downregulation of epithelial markers such as E-cadherin and the upregulation of mesenchymal markers such as vimentin (Mendez et al., 2010).
E-cadherin is a transmembrane glycoprotein involved in calcium-dependent cell–cell adhesion. Ovarian surface epithelial cells do not express E-cadherin. However, E-cadherin is present in ovarian surface epithelial cells covering deep clefts, inclusion cysts, and ovarian tumors (Auersperg et al., 1999). Vimentin is a type III intermediate filament protein expressed in tissues of normal mesenchymal origin. It plays a predominant role in the changes in shape, adhesion, and motility of epithelial cells that occur during the EMT process (Mendez et al., 2010). Downregulation of E-cadherin and upregulation of vimentin are correlated with tumor invasion and metastasis in the majority of carcinoma cells (LV et al., 2013; Mima et al., 2013), including ovarian carcinomas (Wang et al., 2009; Koensgen et al., 2010; Ryabtseva et al., 2013).
EMT can be regulated by many signaling pathways and regulatory transcriptional networks including transforming growth factor-β (TGF-β) (Guo et al., 2014). TGF-β is a superfamily of peptide growth factors that play major roles in tumorigenesis by regulating cell growth, EMT, invasion, and metastasis (Gao et al., 2014). There are three isotypes of TGF-β (-β1, -β2, and -β3). TGF-β1 is the most prevalent form, whereas the other isoforms are expressed in a more limited spectrum of cells and tissues (Inan et al., 2006). TGF-β1 mediates EMT by inducing Smad signaling (Do et al., 2008; Mima et al., 2013). TGF-β1 is downregulated in ovarian cancer and can induce ovarian cancer cell death in high concentrations, supporting the tumor suppressor role (Ween et al., 2011). However, marked elevation of TGF-β1 in ovarian cancer cells and peritoneal cells (Bartlett et al., 1997; Ween et al., 2011), involvement of TGF-β1 in the progression from noninvasive borderline tumors to invasive carcinomas (Inan et al., 2006), and its ability to induce an EMT and invasion (Do et al., 2008; Ween et al., 2011; Cheng et al., 2012; Gao et al., 2014) suggest its tumor-promoting role.
As the changes in the EMT process do not fully occur in ovarian carcinomas (Davidson et al., 2012), and TGF-β1 seems to have a dual function in these tumors (Ween et al., 2012), the aim of the present study is to investigate the immunohistochemical expression of the EMT-related markers, E-cadherin and vimentin, in ovarian serous carcinomas (OSCs) in relation to TGF-β1 expression and clinicopathological factors and evaluate the regulatory role of TGF-β1 in the metastatic process of OSCs.
Materials and methods
Forty-two formalin-fixed paraffin-embedded blocks of OSC cases were collected from the archives of the Pathology Departments of Zagazig and Tanta Universities and some private laboratories from January 2005 to December 2013. The clinical data of the patients were obtained from medical files. The age of the patients ranged from 47 to 64 years, with a median age of 54 years. All tumor samples were obtained at the time of primary tumor debulking surgery before chemotherapy. There was no contact with patients and hence informed consent was not required.
Paraffin sections (5-μm-thick) stained with hematoxylin and eosin were re-examined to confirm the diagnosis of serous adenocarcinomas (n=15), serous cystadenocarcinomas (n=4), or serous papillary carcinomas (n=23). Tumors were graded as low grade and high grade by the two-tier grading system (Vang et al., 2009). Tumor stage (I–IV) was assigned according to the International Federation of Gynecology and Obstetrics classification (FIGO) (Benedet et al., 2000).
The immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue sections using standard streptavidin–biotin–peroxidase complex (ABC) methods. Briefly, after routine deparaffinization, the 5-μm-thick sections mounted on positively charged adhesive slides (Biogenex Co.) were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. Sections were then washed in PBS (pH=7.4) and Tris-buffer solution (pH=7.6). Thereafter, the slides were microwaved for 20 min for antigen retrieval and incubated overnight at 4°C with optimal dilutions of primary antibodies purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA): anti-E-cadherin (1 : 100 dilution), anti-vimentin (1 : 50 dilution), and anti-TGF-β1 (SC-146, 1 : 100 dilution). Sections were then treated with avidin–biotin–peroxidase reagent for 30 min (Dako, Japan Ltd, Kyoto, Japan). Finally, they were incubated with diaminobenzidine, counterstained with hematoxylin, and then cleared and mounted. A 0.01 mmol/l PBS or mouse serum (500 dilutions) was applied instead of primary antibodies as a negative control.
Assessment of E-cadherin, vimentin, and transforming growth factor-β1 immunoreactivity
Membranous E-cadherin, cytoplasmic vimentin, and TGF-β1 expression was analyzed according to the methods described by Mima et al. (2013). E-cadherin, vimentin, and TGF-β1 immunoreactivity was graded into categories from 0 to 3+ as follows: 0, no staining; weak, 1-25% staining; moderate, 26-50% staining; and strong, >50% staining. For E-cadherin and TGF-β1, the 2+ and 3+ samples were defined as positive immunohistochemistry results. For vimentin, the 3+ specimens were defined as positive immunohistochemistry results.
Statistical analysis was performed using SPSS version 20 (Statistical Package for Social Sciences; SPSS Inc., Chicago, Illinois, USA). Statistical analysis of data for labeling indices was performed using Spearman’s correlation coefficient and the χ2-test. P values less than 0.05 were considered statistically significant.
E-cadherin expression and its correlation with clinicopathological factors
E-cadherin expression was predominantly absent in 22 of 42 cases of OSCs (52%) and immunoreactive in the remaining 20 cases (48%). The pattern of E-cadherin expression in immunopositive tumors showed either focal weak or intense membranous staining (35 and 15% of cases, respectively), or focal moderate to intense membranous and cytoplasmic staining (30 and 20% of cases, respectively). Its expression was detected in solid zones and associated benign lesions and was consistently negative in stromal cells. The cytoplasmic expression of E-cadherin was observed frequently at the invasive margin (Figs 1a,b and 2a,b). Loss of E-cadherin immunoreaction was significantly detected in stage III/IV (61.7%) more than in stage I/II (12.5%) OSCs (P=0.012) with negative correlation (r=−0.387). Although loss of E-cadherin immunostaining was observed more frequently in high-grade (61%) than in low-grade (36%) OSCs, no statistical significance was found between E-cadherin loss and tumor grade (P=0.126). Also, no significant differences were found between E-cadherin immunostaining and other clinicopathological factors (P>0.05), as shown in Table 1.
Vimentin expression and its correlation with clinicopathological factors
Positive vimentin immunostaining was detected in 33 cases of 42 OSCs (79%). Diffuse cytoplasmic expression with paranuclear concentration was found in the majority of immunopositive cases (76%), whether weak (12%) or moderate (64%), whereas strong cytoplasmic vimentin expression was detected in 24% of tumors. It was also expressed in stromal cells, vessels, and in solid zones of tumor cells. Vimentin expression with increased intensity was frequently observed at the invasive margin of positive tumors (Figs 1c,d and 2c,d). Also, its expression was detected in OSCs of high rather than low grades (89 and 57% of cases, respectively) and in most OSC cases of stage III/IV (91%) than cases in stage I/II (25%). Vimentin expression was significantly correlated with tumor grade (P=0.017) (r=0.369) and FIGO stage (P<0.001) (r=0.633). No significant differences were found between vimentin expression and other clinicopathological factors (P>0.05), as shown in Table 1.
Expression of epithelial–mesenchymal transition-related marker and its correlation with transforming growth factor-β1 expression
Half of 42 OSCs (50%) exhibited an E-cadherin-negative/vimentin-positive expression profile (high ability of EMT), whereas a minority of cases (19%) had an E-cadherin-positive/vimentin negative expression profile (low ability of EMT). Also, out of the 42 OSC cases, 12 (29%) showed E-cadherin-positive/vimentin-positive expression profile, whereas both E-cadherin-negative/vimentin-negative expression was detected in one case (2%). TGF-β1 was detected in 29 of 42 OSC cases (69%). Immunopositive tumors had either focal moderate (83%) or strong (14%) cytoplasmic TGF-β1 expression with foci of increased uptake at the invasive margin and frequent expression in stromal cells. Only one case (3%) showed diffuse intense cytoplasmic expression of TGF-β1 (Figs 1e,f and 2e,f). Among the 21 E-cadherin-negative/vimentin-positive cases, 19 (90%) cases exhibited positive TGF-β1 immunostaining, whereas only two cases (25%) with E-cadherin-positive/vimentin-negative expression exhibited positive immunostaining of TGF-β1. TGF-β1 expression in OSCs was significantly correlated with E-cadherin-negative/vimentin-positive cases compared with E-cadherin-positive/Vimentin-negative tumors (P=0.0001, r=0.655) (Table 2).
It is well established that cancer invasion and metastasis still represent the major causes of the failure of cancer treatment, and hence better understanding of the molecular mechanisms that govern ovarian cancer metastasis is a crucial step in controlling and treating this disease. TGF-β1-induced EMT is believed to play an important role in the regulation of cell invasion and metastasis in ovarian cancer (Do et al., 2008; Cheng et al., 2012). As OSCs comprise over half of all ovarian carcinomas and account for the majority of ovarian cancer-related deaths (Wang et al., 2009), this study aimed to investigate the immunohistochemical expression of the EMT-related markers (E-cadherin and vimentin) in relation to TGF-β1 and clinicopathological factors in 42 cases of OSCs.
Our study showed that E-cadherin expression was lost in 22 of 42 cases of OSCs (52%). Loss of E-cadherin expression was observed more often in OSCs of high rather than low grade (61 and 36% of cases, respectively) and in inpatients of stage III/IV (59%) than in patients of stage I/II (25%). There was a significant positive correlation between E-cadherin loss and tumor stage (P=0.012) (r=−0.266), with no significant correlation with other clinicopathological factors. Different studies reported decreased E-cadherin expression in invasive ovarian tumors in comparison with that in benign ovarian lesions, borderline, and well-differentiated tumors (Darai et al., 1997; Voutilainen et al., 2006; Yuecheng et al., 2006; Koensgen et al., 2010; Ryabtseva et al., 2013). Our results are consistent with that reported by Yuecheng et al. (2006) and Ryabtseva et al. (2013), who found significant association between E-cadherin loss and clinical stage in epithelial ovarian carcinomas and serous tumors. However, those authors also found significant association between E-cadherin loss and tumor grade. In contrast, Koensgen et al. (2010) and Voutilainen et al. (2006) reported no significant association between E-cadherin loss and clinicopathological parameters. The mechanism of E-cadherin gene inactivation along the progression of ovarian carcinoma may be attributed to E-cadherin mutation, or to two epigenetic mechanisms, including hypermethylation of the promoter and downregulation of the transcription factors, such as Snail, Slug, SIP1, PaK1, and miRNAs (Elloul et al., 2006; Elloul et al., 2010; Davidson et al., 2012).
The cytoplasmic reaction of E-cadherin that was observed in the majority of positive cancer cases and frequently at the invasive margin of positive tumors in this study was similar to that reported by LV et al. (2013) in their study on invasive breast carcinoma. This disturbance in E-cadherin expression has also been described in other epithelial tumors including breast and colorectal cancer (LV et al., 2013; Kanczuga-Koda et al., 2014). It may be hypothesized that the cytoplasmic location of E-cadherin in the neoplastic cells may result from the disturbances in β-catenin expression and the lack of formation of adhesive complexes, or disturbed connexin (Cx) gap junction proteins location, or it may have another biological function (Kanczuga-Koda et al., 2014).
In the current study, vimentin was detected in 33 of 42 cases of OSCs (79%). Its expression was detected in OSCs of high rather than low grade (89 and 57% cases, respectively), and in most OSC cases in stage III/IV (91%) than stage I/II (25%). Vimentin expression was significantly correlated with tumor grade and FIGO stage (P=0.017 and <0.001, respectively), with no significant association with other clinicopathological factors. This finding is consistent with that reported in other studies on ovarian carcinomas (Georgescu et al., 2004; Wang et al., 2009). Also, we found that vimentin expression with increased intensity was frequently observed at the invasive margin of positive tumors, a phenomenon that was previously reported by LV et al. (2013), suggesting epithelial cell deviation towards a mesenchymal transdifferentiation in ovarian cancer progression. However, the infrequent observation of spindle cells, which may indicate transformation from epithelial to mesenchymal phenotype in clinical specimens of ovarian carcinomas, and the fact that serous carcinomas frequently express EMT-related markers such as N-cadherin and vimentin, indicating its close histogenetic relationship to the mesothelium, are confounding matters (Davidson et al., 2012). The rapid acquisition of several important features of EMT in epithelial cells cultured in vitro – including the adoption of a mesenchymal shape, increased motility, and significant increase in focal adhesion dynamics after microinjection of vimentin or transfection with vimentin cDNA (Mendez et al., 2010), and increased intensity of vimentin expression at the invasive margin in our study and another study on invasive breast cancer described by LV et al. (2013) – provide the proof of principle for the process of EMT. However, the situation is complicated by the fact that EMT is inherently difficult to detect in vivo, as it may be a transient event limited to the invasive front. Also, epithelial tumors are often surrounded by mesenchymal cells, which may be either tumor cells after EMT or mesenchymal cells of nontumor origin. Nevertheless, there is increasing evidence for the validity of the concept of EMT in tumor progression (Mendez et al., 2010). It is possible that the expression of VIFs in epithelial cells influences the transition to a mesenchymal phenotype by the binding of vimentin to the actin-bundling protein fimbrin in adherent cells, modulating fimbrin–actin interactions (Correia et al., 1999), or that VIFs induce changes in desmosomal localization and the rate of paxillin turnover (Voutilainen et al., 2006; Mendez et al., 2010), or participating in signal transduction (Phua et al., 2009). Finally, VIF expression may also facilitate the ability of epithelial cells to withstand a variety of external mechanical forces. This may be a critical factor for the survival of metastatic cells, which are exposed to abnormal physical stresses as they navigate from primary to secondary tumor sites (Suresh, 2007).
In this study, half (50%) of the 42 OSC cases exhibited an E-cadherin-negative/vimentin-positive expression profile (high ability of EMT), whereas a minority (19%) had an E-cadherin-positive/vimentin-negative expression profile (low ability of EMT). Some authors described an overexpression of E-cadherin and vimentin in disseminated lesions and in ovarian carcinoma effusion in comparison with the primary tumor (Elloul et al., 2006; Elloul et al., 2010), suggesting that ovarian carcinoma cells undergoing partial EMT in the primary tumor enables them to become motile and invasive. These cells either go through full EMT indicated by overexpression of vimentin in disseminated lesions to intravasate a lymph/blood vessel and form solid metastases or undergo mesenchymal–epithelial transition indicated by overexpression of E-cadherin in disseminated lesions to survive and proliferate in the peritoneal cavity as spheroids (Elloul et al., 2010), or suggest that epithelial ovarian carcinomas spread in the abdomen through collective migration of cancer cells rather than as single cells (Rorth, 2009). Cells that undergo partial EMT but fail to go through one of these two processes probably die through anoikis (Kang et al., 2007).
Regarding TGF-β1, positive expression was detected in 29 of 42 cases of OSCs (69%). TGF-β1 expression in OSCs was significantly correlated with E-cadherin-negative/vimentin-positive OSCs, than in E-cadherin-positive/vimentin-negative carcinomas (P=0.001, r=0.655). By using different methods of analysis, different studies showed that TGF-β1 was markedly elevated in ovarian cancer cells and peritoneal cells (Bartlett et al., 1997; Ween et al., 2011), was involved in the progression from noninvasive borderline tumors to invasive carcinomas (Inan et al., 2006; Cheng et al., 2012), and is able to induce an EMT, MMP secretion, and invasion (Do et al., 2008; Ween et al., 2011), suggesting a tumor-promoting role for TGF-β1. However, TGF-β may act as a tumor suppressor in the early stages of cancer progression, and it becomes a tumor promoter in later stages (Ween et al., 2011). The molecular mechanism of this switch is complicated, perhaps context dependent, and remains largely unknown (Cheng et al., 2012). Science pathway components, the TGF-β receptors, and Smad proteins are rarely mutated or lost in ovarian cancer (Bast et al., 2009); other mechanisms such as the level of other endogenous transcription factors, mutation of p53, modulation of cancer-associated fibroblasts by TGF-β, or induction of isoform switching of FGFRs may be involved in the TGF-β switching mechanism (Shirakihara et al., 2011; Yeung et al., 2013; hAinmhire et al., 2014; Guo et al., 2014). Other EMT regulatory molecules may include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, hepatocyte growth factor, stem cell factor, bone morphogenetic proteins, the Wnt signaling pathway, integrins, Notch transcription factors, prostaglandin E2 and cyclooxygenase-2, parathyroid hormone, UV, nicotine, bile acids, and estrogens (Guo et al., 2014).
Our results indicated that loss of E-cadherin and expression of vimentin are associated with progression and invasion of serous ovarian carcinomas. TGF-β1 may promote the invasion and metastasis of OSCs through induction of EMT. The TGF-β1 pathway is a potent target for chemotherapy in serous ovarian carcinomas. However, further analysis of EMT-related markers and their regulatory molecules and signaling pathway may facilitate the identification of candidate prognostic markers, which is a crucial step in controlling and treating epithelial tumors, including ovarian carcinomas.
Conflicts of interest
There are no conflicts of interest.
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