In the course of neoplastic progression the simple epithelial layer of OSE overlying the stroma undergoes changes in differentiation resulting in the loss of stromal components with the acquisition of differentiation characteristics of Mullerian duct-derived epithelia, for example, the oviduct, endometrium, and uterine cervix. The OSE and the epithelium of the Mullerian ducts share common origins and OSE frequently undergoes Mullerian type differentiation characteristic of metaplasia during adult life. This change in the differentiation pattern is important in our understanding of the origin of ovarian cancer subtypes and forms the basis for the classification of these cancers as serous (fallopian tube-like), mucinous (endocervical-like), endometrioid (endometrium-like), and less commonly clear cell carcinoma (resembling clear cells). About 80% of all epithelial ovarian carcinomas are of serous prototype most commonly seen in inclusion cysts that have undergone Mullerian metaplasia (Scully, 1995). At the cellular level Mullerian differentiation is followed by the expression of epithelial membrane antigens such as E-cadherin, CA 125 and mucins (MUC1, MUC2, MUC3, and MUC4; Van Niekerk et al., 1993; Sundfeldt et al., 1997). The high frequency of Mullerian differentiation may be required to provide survival and growth signals in transforming OSE (Karlan et al., 1995) and may form the basis for the initiation of ovarian cancer.
EMT in ovarian carcinoma—a cellular perspective
The initial process that regulates EMT is the disruption of the E-cadherin mediated cell–cell interaction that maintains the epitheloid network. Primary ovarian carcinomas have been reported to express E-cadherin and its expression is reduced in many advanced tumors (Sundfeldt, 2003). Higher levels of soluble E-cadherin have been reported in the cyst fluid of malignant ovarian tumors than in benign cyst (Sundfeldt et al., 2001), suggesting proteolytic mechanisms resulting in the cleavage of surface bound E-cadherin. This confirms that the prerequisite of EMT that is closely associated with the loss of E-cadherin expression and the acquisition of invasive phenotype exists in ovarian cancer. On the other hand, a recent study has demonstrated that E-cadherin expression was significantly increased in metastatic ovarian lesions compared to the respective primary tumors (Davidson, 2001), suggesting that the expression of E-cadherin occurs intermittently during the progression of ovarian carcinoma, and is required for the growth of both primary and secondary tumors. E-cadherin expressing cells have also been reported in the ascites of women with ovarian cancer and upregulation of E-cadherin expression has been reported in the carcinoma cells of ascites compared to primary carcinomas (Davidson, 2001). The upregulation of E-cadherin in the peritoneal and metastatic lesions of ovarian carcinomas can be marked as events in the progression of cancer possibly mediating survival signals for tumor cells at these sites through inhibition of anoikis and apoptosis or it may indicate facilitation of MET as shown in other cancers (Christiansen and Rajasekaran, 2006). An interesting parallel can be drawn to the re-epithelization of colon cancer metastases compared to mesenchymal attributes of invading cells and observation of epithelial attributes in bladder carcinoma selected for secondary site colonization in mice (Chaffer et al., 2006).
The loss of E-cadherin in advanced primary ovarian cancers is consistent with EMT propensity for actual metastatic spread. E-cadherin downregulation in some cases is accompanied by increased expression of N-cadherin, which promotes mesenchymal signaling through interaction with stromal cells. The expression of N-cadherin in ovarian carcinoma is not well documented. In a recent study of 54 tissues (including normal = 8, benign = 9, borderline = 9, grade 1 = 8, grade 2 = 9, and grade 3 = 11) performed in our laboratory the expression of E and N-cadherin was observed in both benign and malignant tumors (Fig. 1). The expression of E-cadherin was absent in normal ovarian tissues (n = 8) and weak expression of N-cadherin was observed in flat cells undergoing differentiation in only two out of eight normal ovarian tumors (Fig. 2). Enhanced expression of P-cadherin has been reported in ovarian tumor masses with progression to later stages (Patel et al., 2003). These studies suggest that multiple cadherin subtypes are expressed in ovarian tumors and their functional role in maintaining cell–cell and cell–peritoneal interaction is still not understood. Given the known role of N-cadherin in promoting malignant transformation, whether N-cadherin expression in normal OSE promotes malignant changes is not known.
Figure 1. Immunohistochemistry analysis of the expression of E- and N-cadherin in grades 1 and 3 endometrioid ovarian tumors. The same grade 1 and grade 3 tumors were analyzed. A,B: Expression of E-cadherin in grades 1 and 3 ovarian tumors; (C,D) expression of N-cadherin in grades 1 and 3 ovarian tumors. Arrows indicate membrane staining of E- and N-cadherin. Magnification 400×.
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Figure 2. Immunohistochemistry analysis of the expression of N-cadherin in normal ovarian tissues. A: No expression of N-cadherin was seen in six of the eight normal ovaries. B: Expression of N-cadherin in the flat cells of the ovarian epithelium observed in two out of eight normal ovarian tissues. Arrows indicate membrane staining of N-cadherin. Magnification 400×.
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Loss of E-cadherin gene expression which is the hallmark of EMT is mainly due to an upregulation of Snail, Slug, TWIST, SIP-1, and other transcription factors repressing E-cadherin expression (Elloul et al., 2006). Ectopic expression of Snail or Slug has resulted in EMT-associated enhanced motility, invasiveness, and tumorigenicity in a SKOV3 ovarian cancer cell line (Kurrey et al., 2005). Snail suppresses expression of adheren junction components (E-cadherin and β catenin) and tight junction components (occludin and ZO-1), while Slug suppresses expression of both components and also desmosomal junction components (Dsg2). Further activation of these transcriptional factors by hypoxia revealed immediate upregulation of Slug expression in ovarian cancer cells with consequent downregulation of Snail and E-cadherin expression (Kurrey et al., 2005). Moreover, hypoxia-induced upregulation of Snail expression resulted in the upregulation of hypoxia-inducible factor 1α (HIF-1α), downregulation of E-cadherin and invasiveness of ovarian cancer cells (Imai et al., 2003). These observations suggest that hypoxia associated tumor microenvironment may facilitate EMT and transformation into metastatic phenotype of ovarian tumors. Further evidence of EMT in ovarian cancer is supported by a recent study which demonstrated that 17β-estradiol increased Snail expression with subsequent increase in the MMP-2 expression and decrease in E-cadherin expression in estrogen receptor positive and estrogen receptor negative ovarian cancer cell lines (Ding et al., 2006). These studies suggest that the Slug–Snail–cadherin systems may be possible candidates for molecular targeting in the future treatment of ovarian cancer.
The endothelin family of peptides which constitutes of ET-1, -2, -3 are potent mitogens for several human tumors (Rosano et al., 2003). Compared to normal ovaries, ET-1 and its receptor (ETAR) are over expressed in primary and metastatic ovarian carcinomas and high levels of ET-1 are detectable in patient ascites (Bagnato et al., 1999). ET-1 induces EMT in ovarian cancer cells (Rosano et al., 2005) and indirectly modulate tumor–host interaction by modulating the proliferation, migration, invasion, protease secretion, tube formation of endothelial cells, and stimulating neovascularization in vivo through upregulation of VEGF and HIF-1 α expression (Salani et al., 2000). ET-1 induced EMT in ovarian cancer cells involves changes to fibroblast-like morphology, loss of E-cadherin and β-catenin and gain in N-cadherin, vimentin, and Snail expression (Rosano et al., 2005). Activation of ETAR by ET-1 triggers an integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase 3 β (GSK3β) inhibition (Rosano et al., 2005). This pathway is crucial for EMT-inducing effects in other carcinomas (D'Amico et al., 2000) and renal interstitial fibrogenesis (Li et al., 2003). We have recently demonstrated that EGF-induced EMT in OSE is also dependent on this pathway (Ahmed et al., 2006). ET-1 stimulation of ovarian cancer cells has also been demonstrated to confer resistance to taxol-induced apoptosis (Del Bufalo et al., 2002). Consistent with taxol being an epithelial specific chemotherapeutic agent (Markman, 2007), ET-1 stimulated mesenchymal ovarian cancer cells are chemoresistant to taxol. In addition, ETAR activation in ovarian cancer cells results in EGF receptor transactivation, another known inducer of EMT in ovarian cancer cells (Vacca et al., 2000). Recent studies from our laboratory have demonstrated that EGF-induced EMT in epithelial ovarian cancer cells result in downregulated expression of neutrophil gelatinase activated lipocalin (NGAL), an established inducer of epithelial phenotypes (Lim et al., 2007). Collectively, these findings suggest that there is a coexistence of ET-1 and EGF-induced EMT signaling in ovarian carcinomas representing potential targets for anticancer therapy.
Bone morphogenetic proteins (BMPs) are expressed in many adult tissues including ovaries (Theriault et al., 2007). In rats BMP4 is upregulated in OSE adjacent to the site of ovulation (Fathalla, 1971), suggesting a role of BMP4 in OSE repair through promoting cell proliferation and migration. Recently, it has been reported that there is an inherent difference in BMP4 signaling in normal OSE and ovarian cancer cells (Theriault et al., 2007). BMP4-treated ovarian cancer cell lines undergo EMT-associated changes such as elevated levels of Snail and Slug expression, downregulation of E-cadherin expression with enhanced cellular motility, invasiveness, ECM remodeling with reorganization of the actin cytoskeleton, and activation of Rho GTPases (Theriault et al., 2007). On other hand, OSE cells do not respond to BMP4 by altering cell motility but undergo cytoskeletal rearrangement as evidenced in cancer cells. This suggests that OSE and ovarian cancer cells possess distinct regulatory mechanisms to respond to exogenous stimuli. In addition to the cytokines described above, VEGF and lysophosphatidic acid which are present in high abundance in the ascites and serum of ovarian cancer patients have been shown to induce invasiveness in cultured ovarian cancer cell lines (Ren et al., 2006; Wang et al., 2006). In a recent study, we have shown that ascites from ovarian cancer patients induce distinct changes associated with invasiveness in ovarian cancer cells (Ahmed et al., 2005). A recent study has demonstrated that gonadotropins, such as follicle stimulating hormone and luteinizing hormone, activate proteolysis, and invasiveness of ovarian cancer cells (Choi et al., 2006), consistent with the epidemiological studies demonstrating an increased occurrence of ovarian cancer in women exposed to excess gonadotrophins during menopause or infertility treatment (Shoham, 1994; Shushan et al., 1996). However, whether this facilitation of invasiveness by hormones and growth factors is EMT-dependent is still not known.
The studies described above provide convincing evidence that EMT plays an essential role in modulating the motility and invasiveness of ovarian cancer cells growing in a two-dimensional situation. However, no data exits on the EMT status of ovarian cancer cells shed into the peritoneum. Few recent studies have demonstrated that carcinoma cells floating in the ascites have the ability to adhere and invade the mesothelial lining (Burleson et al., 2006). Upregulation of MMP-2 expression with concomitant downregulation of TIMP-2 expression has been demonstrated in malignant cells in peritoneal ascites compared to primary tumors (Davidson, 2001). Recently, the expression of Smad interacting protein 1 (Sip1) which regulates E-cadherin and MMP-2 expression has been demonstrated in the carcinoma cells of ascites of patients with ovarian carcinoma (Elloul et al., 2005). Moreover, peritoneal ovarian carcinoma aggregates or spheroids have been shown to be protected from apoptosis induced by radiation and therapeutic drugs such as taxol (Makhija et al., 1999). These studies suggest that the shed ovarian tumor cells that survive as free floating cellular aggregates in the ascites are invasive and capable of undergoing EMT. Moreover, ascites has an abundance of growth factors and cytokines which can induce EMT in floating tumor cellular aggregates. As malignant cellular aggregates in ascites are difficult to separate, we have adapted a tissue culture model of ovarian cancer cellular aggregates or spheroid by using a liquid overlay technique (Santini and Rainaldi, 1999). Using that technique, we have recently demonstrated that ovarian cancer cell lines grown as cellular aggregates can sustain growth for 10 days, while the normal ovarian cell line failed to grow beyond 2 days (Shield et al., 2007a). Cancer cellular aggregates expressed enhanced levels of mesenchymal markers such as α2β1 integrin (Valles et al., 1996), N-cadherin, vimentin, and secreted pro-MMP2/MMP9 as well as activated MMP2/MMP9, with no such activation of MMP's observed in monolayer cells (Shield et al., 2006, 2007b). A recent study reported the invasive characteristics of ascites spheroids isolated from ovarian cancer patients and correlated that invasiveness with the shortened survival of patients by 16–17 months (Burleson et al., 2006). The same study also reported retraction of the mesothelial layer at the site of spheroid attachment. This effect, however, disappeared by day 7, upon complete spheroid cell dispersal, indicating that ascites tumor spheroids possess the capacity to degrade the mesothelial monolayer but once disaggregated lose the capacity to do so. Based on these preliminary observations one can propose that ovarian carcinoma in a “spheroid mode” in the peritoneum facilitates EMT, endowing cells with properties that favor metastasis. However, metastatic progression on the omentum might ultimately require the reverse, MET, representing metastases to closely resemble their corresponding primary tumors. Based on these observations, we propose a working model of ovarian cancer progression that provides a framework for the development of EMT/MET as a mechanistic process for the localized spread of ovarian carcinoma (Fig. 3).
Figure 3. A working model of ovarian cancer progression. During ovarian cancer progression, epithelial ovarian cancer cells growing on the surface of the ovary undergo EMT to attain motile functions required for cancer metastasis. Rupture of the ovarian tumors result in shedding of tumor cells into the peritoneum where they survive as cellular aggregates/spheroids. These spheroids undergo changes into invasive mesenchymal phenotype to sustain survival and motility. Cancer spheroids and the surrounding mesothelial and infiltrating blood cells secrete cytokines and growth factors (e.g., VEGF, TNF-α, IL-6, IL-8, bFGF, lysophosphatidic acid, etc.) in the form of ascites in the peritoneum. The secreted factors form an autocrine/paracrine loop that initiate and sustain EMT to facilitate the invasiveness of carcinoma spheroids until they find a secondary attachment site. Growth on the omentum however, requires MET to sustain cancer growth.
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