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Abstract

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

Cancer that arises from the ovarian surface epithelium (OSE) accounts for approximately 90% of human ovarian cancer, and is the fourth leading cause of cancer-related deaths among women in developed countries. The pathophysiology of epithelial ovarian cancer is still unclear because of the poor understanding of the complex nature of its development and the unusual mechanism(s) of disease progression. Recent studies have reported epithelial–mesenchymal transition (EMT) in cultured OSE and ovarian cancer cell lines in response to various stimuli, but our understanding of the importance of these observations for normal ovarian physiology and cancer progression is not well established. This review highlights the current literature on EMT-associated events in normal OSE and ovarian cancer cell lines, and discusses its implication for normal ovarian function as well as acquisition of neoplastic phenotypes. The pathological changes in OSE in response to EMT during neoplastic transformation and the contribution of hormones, growth factors, and cytokines that initiate and drive EMT to sustain normal ovarian function, as well as cancer development and progression are also discussed. Finally, emphasis is placed on the clinical implications of EMT and potential therapeutic opportunities that may arise from these observations have been proposed. J. Cell. Physiol. 213:581–588. © 2007 Wiley-Liss, Inc.

Epithelial–mesenchymal transition (EMT), postulated as a versatile mechanism for allowing cellular movement required during embryonic development, tissue regeneration after injury/pathological conditions and cancer progression, is a complex process and perhaps least well understood in relation to normal ovarian physiology and the development/progression of ovarian cancer. During early embryogenesis ovarian surface epithelium (OSE) is derived from the celomic epithelium which covers the gonadal ridge. This epithelium proliferates and condenses with the underlying mesenchyme of the cortex and forms cortical cords giving rise to the granulosa cells in the primordial follicles (Auersperg et al., 2001). The Mullerian ducts develop as invaginations of celomic epithelium and are located dorsolaterally from the gonadal ridges (Auersperg et al., 2001).

Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

In an adult woman, OSE is a single layer of epithelium lining the ovarian cortex. These cells have either a flat or cuboidal appearance and are often referred to as mesothelial type of epithelial cells since they share a common embryological origin with the peritoneum and have characteristics in common with peritoneal mesothelial cells (Sundfeldt, 2003). OSE is a less differentiated monolayer with an “uncommitted” phenotype but retains the capacity to differentiate into different types of cells in response to environmental signals. Stationary uncommitted OSE expresses a keratin complement typical of epithelial cells (e.g., keratin types 7, 8, 18, and 19) and also expresses mucin antigen MUC1 and 17β-hydroxysteroid dehydrogenase typical for mesothelial cells (Auersperg et al., 2001). In addition, OSE cells constitutively express vimentin, N-cadherin, and α smooth muscle actin which are common mesenchymal markers, shown to be expressed in fibroblasts and cultured cells in response to wounding and other pathological conditions (Auersperg et al., 2001). OSE cells produce proteolytic enzymes such as urokinase plasminogen activator (uPA, Ahmed et al. unpublished data), matrix metalloproteases 2 and 9 (Ahmed et al., 2006) and also secrete collagen type III, an additional mesenchymal characteristic (Salamanca et al., 2004). The proteolytic activity of OSE might contribute to the remodeling, as well as the breakdown of OSE and the ovarian cortex during ovulation. In short, normal OSE is a stationary mesothelium which retains the capacity to alter its state of differentiation along stromal or epithelial phenotypes in response to environmental cues. During the postovulatory repair process and in culture, OSE cells undergo EMT and attain fibroblast-like characteristics reflecting their developmental relationship to stromal cells (Salamanca et al., 2004). This capacity of OSE to undergo EMT in response to postovulatory stimuli has been proposed to confer advantage to the postovulatory repair of the OSE by altering the motility and proliferative response requisite for extracellular matrix remodeling (Salamanca et al., 2004). In this scenario, EMT might facilitate the release of trapped OSE cells within the ovary during ovulation into the ovarian stroma as stromal fibroblasts (Ahmed et al., 2006). As an alternative hypothesis, it has been proposed that failure to undergo EMT under such conditions may render the aggregation of epithelial cells within the stroma resulting in the formation of inclusion cysts, a common site of ovarian cancer initiation and often seen in the “normal” ovaries of mutation carriers. EMT inducers of OSE such as epidermal growth factor (EGF) and collagen are present at the site of ovulatory rupture (Salamanca et al., 2004). In addition, transforming growth factor (TGF) β, which is an autocrine regulator of OSE growth (Berchuck et al., 1992) has been shown to induce EMT in a number of epithelial cells (Han et al., 2005). Moreover, OSE has been shown to undergo EMT in collagen gels (Kruk and Auersperg, 1992; Ohtake et al., 1999) and in other three-dimensional matrices (Kruk et al., 1994; Dyck et al., 1996), a scenario commonly experienced by displaced OSE during the ovulatory rupture and repair process. Hence, EMT is a part of normal OSE physiology and failure to undergo such process may be one of the reasons of initiation of ovarian cancer.

Currently, there is no definite explanation for the formation of these inclusion cysts, the initiation of EMT in those sites or why they may be the most likely site of neoplastic transformation. However, two hypotheses have been suggested that may promote mesenchymal transformation of OSE within the inclusion cyst in either a paracrine or an autocrine fashion (Auersperg et al., 2001). According to the paracrine hypothesis, stromal-derived growth factors, cytokines, and other bioactive agents (e.g., TGFβ, fibroblast growth factor (FGF), interleukin (IL)-6, etc.) which have an open access to OSE in the inclusion cysts may promote EMT and neoplastic transformation of OSE within the inclusion cysts. Neoplastic progression in OSE-lined cysts may also be promoted by autocrine mechanisms by OSE-derived cytokines, growth factors, and other bioactive molecules that accumulate in the confined sites of the inclusion cysts and impede their action in a localized fashion as an immediate growth promoter or act as secondary agents to promote changes in gene expression that facilitate neoplasia. The paracrine hypothesis is supported by the observations that OSE-lined cysts adjacent to the stroma undergo pronounced metaplastic and dysplastic changes compared to the ones further away from the stroma (Scully, 1995). The autocrine hypothesis on the other hand, is supported by the reports demonstrating the capacity of OSE to secrete cytokines and growth factors including interleukin (IL)-1 and IL-6 and by other reports signifying that these cytokines change the phenotype of ovarian carcinoma cells (Wu et al., 1992; Ziltener et al., 1993). The fact that OSE lining the inclusion cysts is two to three times more metaplastic in women with epithelial ovarian tumors than in women without such cancers (Scully, 1995), and ovarian carcinoma markers, for example CA 125 and CA19-9 are significantly more expressed in the epithelium of inclusion cysts than on the OSE suggests that early malignant changes occur more frequently in OSE-lined inclusion cysts than on the ovarian surface itself (Auersperg et al., 2001).

Cell–cell adhesion molecules in normal ovaries

The epithelial integrity of OSE is maintained by desmosomes, junctional proteins (Siemens and Auersperg, 1988), integrins (Kruk et al., 1994; Cruet et al., 1999), and cadherins (Sundfeldt et al., 1997). Among the cadherins, OSE expresses N-cadherin which maintains intracellular adhesion mechanism and inhibits apoptosis (Peluso et al., 1996). In peri-menopausal and postmenopausal women the ovary changes contour and decreases in size with the formation of OSE-lined epithelial inclusion cysts expressing distinct epithelial markers such as CA125 and E-cadherin (Auersperg et al., 2001). N-cadherin expression has also been detected in the inclusion cysts and it has been suggested that N-cadherin-mediated cell–cell adhesion in OSE may prevent the cells from entering apoptosis (Trolice et al., 1997). SV40 large T antigen immortalized human OSE cells transfected with a mouse E-cadherin gene have been reported to closely resemble ovarian carcinoma cells (Wong et al., 1999). Whether the expression of E-cadherin in the inclusion cysts or the induction of E-cadherin in OSE contributes to or results in the neoplastic transformation of ovarian epithelium is still unknown. However, recent literature suggests E-cadherin as the “master gene” of epithelial phenotype induction, as conversion of mesenchymal to epithelial transition (MET) has been shown to occur in E-cadherin cDNA transfected fibroblast cell lines (Nagafuchi et al., 1987) and tumor cell lines (Frixen et al., 1991). These studies describe endowment of epithelial characteristics in cells by E-cadherin expression such as the expression of junctions and desmosomes, basal polarity, assembly into epitheloid aggregates, and decreased invasiveness (Frixen and Nagamine, 1993). Parallel to that, loss of E-cadherin expression in several tumors has been shown to result in de-differentiation and invasiveness (Schipper et al., 1991; Behrens et al., 1992). Contrary to that, expression of N-cadherin in cell lines induces fibroblast-like morphology and increased migration and invasion (Islam et al., 1996; Kim et al., 2000). Considering the literature on E-cadherin and its suppression of tumor initiation and progression, loss of E-cadherin rather than its expression or upregulation would be considered as a cause of tumor development (Morton et al., 1993). Hence, early malignant change occurring in OSE-lined inclusion cysts concomitant with the expression of E-cadherin is puzzling. However, it can be suggested that the ovarian inclusion cysts comprise two groups of OSE cells; the first group consisting of flat metaplastic squamous OSE cells lacking E-cadherin expression and associated with ovulation and prone to EMT during the ovulatory rupture and repair process, and the second made of cuboidal cells lining the cyst and expressing E-cadherin with a propensity towards epithelial differentiation and neoplastic progression. One would assume that the latter group of cells expressing E-cadherin to be resistant to EMT in response to ovulatory stimuli, and hence likely to be more susceptible to neoplastic transformation as described below in OSE of women with a family history of ovarian cancer (FHO). This counter-intuitive polarity dependence for transformation is somewhat unique to OSE, and likely holds important clues to our understanding of malignant transformation in general.

EMT and OSE from women with a family history of ovarian cancer

There have been reports on the histological and proteomic changes in the OSE of women with FHO compared with the ovaries of healthy women (NFH). A non-blind study in 1996 reported an increase in inclusion cysts in ovaries from women with FHO (Salazar et al., 1996). In another study, nuclear changes were reported in the OSE of women with FHO compared to those of NFH (Werness et al., 1999). Increased telomeric instability and reduced growth rate has also been demonstrated in FHO-OSE compared to NFH-OSE suggesting susceptibility for malignant transformation at an early age in the heritable ovarian cancer group (Kruk et al., 1999). Recently, a proteomic study on the OSE of women with FHO reported a significant change in the protein profile compared with those of NFH (He et al., 2005; Smith-Beckerman et al., 2005). The most striking finding from these studies was the upregulation in FHO-OSE of proteins involved in protein synthesis and the chaperone family, and downregulation of proteins associated with cytoskeletal arrangement. This suggests suppression of cytoskeletal remodeling and microfilament assembly in the FHO group, consistent with the genomic instability described above (He et al., 2005). Moreover, the expression of E-cadherin and CA 125 were detected more frequently and for longer duration in cultured OSE from women with FHO compared to OSE from control women (Auersperg et al., 1995). In these women mutations in the breast related carcinoma gene (BRCA1) were found in 10/14 cases. The increased expression of E-cadherin and CA125 in FHO-OSE compared to NFH-OSE suggests that these cells might retain an epithelial phenotype and may be less prone to undergo EMT in response to external stimuli, consistent with the early cytoprotective effect of EMT mentioned above. This hypothesis is supported by observations that FHO-OSE has an increased tendency to retain an epithelial phenotype and is less capable of undergoing mesenchymal changes, such as the capacity to secrete collagen type III in response to stimuli and to contract in response to three-dimensional matrices (Dyck et al., 1996). These studies suggest that OSE from women with FHO maintain complex epithelial phenotypes, are committed to that phenotype and remain unresponsive to environmental signals initiating mesenchymal changes presumably required for normal ovarian functions. Such unresponsiveness to normal environmental signals is consistent with the loss of normal control mechanisms characteristic of malignant transformation.

Another epithelial differentiation marker that persists longer in FHO-OSE in culture is the Met receptor for the hepatocyte growth factor (HGF) ligand. Met receptor was downregulated in prolonged cultures of NFH-OSE but was retained in FHO-OSE and ovarian carcinoma cell lines (Auersperg et al., 2001). HGF is a common EMT inducer in epithelial cells (Grotegut et al., 2006). Over expression of Met receptor has been reported in ovarian tumors (Di Renzo et al., 1994) and HGF activated downstream signaling molecules such as PI3 kinase and its downstream effectors Akt and p70 S6 kinase have been shown to be constitutively activated in many tumors, including ovarian (Nakayama et al., 2006). Consistent with that, activation of AKT and p70 S6 kinase is present in FHO-OSE but not in NFH-OSE (Auersperg et al., 2001), suggesting that FHO-OSE are independent of normal growth control mechanisms, a common feature demonstrated in cells undergoing malignant transformation. In summary, FHO-OSE possesses enhanced epithelial characteristics together with autocrine growth regulatory mechanisms providing a basis for the tendency to undergo neoplastic transformation and supporting the observations implicating epithelial features in sporadic ovarian cancer.

Epithelial Ovarian Carcinoma

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

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.

Spread of ovarian cancer

Spread of ovarian cancer characteristically involves local pelvic and abdominal organs. Tumors that disrupt the ovarian capsule are able to shed malignant cells into the peritoneal cavity which can be transported by normal peritoneal fluid. Malignant cells in the peritoneum often aggregate and form spheroid-like structures which subsequently implant on the walls of the peritoneal cavity with a varying extent of peritoneal invasion. In many cases lymph node metastases and peritoneal spread co-exist. Serous and undifferentiated carcinomas have been shown to metastasize most often to other organs through lymph nodes (Sakai et al., 1997). Distant spread of ovarian carcinoma can involve any organ, including the brain (Cormio et al., 1995). Liver involvement is highest, followed by the lung (Munkarah et al., 1997). The presence of ascites is thought to be the combined result of lymphatic obstruction and increased production of peritoneal fluid by cells lining the peritoneal cavity (Feldman et al., 1972). The presence of large volume of ascites correlates with poor prognosis (Dembo et al., 1990).

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.

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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).

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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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Clinical Perspective of EMT in Ovarian Cancer

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

The literature on EMT in ovarian cancer is limited compared to other human cancers with most based on the evaluation of EMT-associated changes in response to external stimuli in cultured ovarian cancer cell lines. Few studies have evaluated cancer markers using tissue arrays and have not implicated any known EMT markers with increasing tumor stage (Hibbs et al., 2004). However, immunohistochemical studies and those using cell lines provide evidence that epithelial ovarian tumors undergo EMT during disease progression. One can argue that this transition is not complete and tumors may retain both the epithelial and mesenchymal phenotypes or they may undergo MET as described above and shown for other cancers (Chaffer et al., 2006; Christiansen and Rajasekaran, 2006).

The current chemotherapy regime for the treatment of ovarian cancer consists of a combination of taxol and carboplatin. This is designed to treat mainly epithelial neoplasia. In this perspective, one can argue that epithelial and mesenchymal cells would have different sensitivity to chemotherapeutic drugs, and cytotoxic treatment of one population of cells may contribute to the dominance of another population in recurrent disease. This usually results in drug resistance and failure of first line treatment, very commonly observed in ovarian cancer. It is known that the transition towards a mesenchymal dominated population is induced by the selective elimination of epithelial cells (Mobus et al., 2001), but it still remains to be evaluated if the basis of that mesenchymal population initially consisting of an epithelial phenotype is due to selective eradication of epithelial cells or due to physiologically induced EMT. A tumor which consists of both epithelial and mesenchymal populations (resembling carcinosarcoma of the ovary) would not benefit from standard treatment protocols for epithelial ovarian cancer. These patients would benefit more from combination therapy using agents against both epithelial and mesenchymal cells, such as those used for sarcomas (Ferrari and Palmerini, 2007). However, considering drug cytotoxicity, it is not clear if the inclusion of these agents in a combination therapy would be optimal for a patient with tumors consisting of both epithelial/mesenchymal cells. Hence, a detailed histopathological examination may be essential to assess the phenotype of a tumor before selecting treatment protocols. Selection of drugs against mesenchymal tumors in combination with carboplatin and paclitaxel might be beneficial for recurrent disease which might have a dominance of mesenchymal population. Hence, a knowledge of EMT or MET in the diagnosis of ovarian cancer is essential to enable choice of the right treatment protocol.

Conclusion

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

Studies described above suggest EMT as a very likely candidate regulating the ovulatory physiology of OSE and metastasis of ovarian cancer cells either on the surface of the ovary or in the peritoneum. Whatever the scenario, cells undergo a series of alterations in response to the environmental changes and also due to the differences in the nature of interacting cells in the new environment. As it is the population of cells that is most responsive to change that survive, alteration in the changed microenvironment may result in the adjustment of signals, triggering a whole set of transcriptional changes which may enhance the expression of a set of molecules and/or dampen the expression of another set, resulting in EMT or MET. No matter what the underlying mechanism, the consequences of EMT as well as MET have a far reaching therapeutic effects as we are dealing with two very different populations of cells. Hence, histopathological examination of the tumor specimen for EMT or MET markers may provide opportunities for better diagnosis, with improved treatment options and prognosis for ovarian cancer patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited

The authors thank Prof. Jock Findlay, Director of Research, Royal Women's Hospital and Mr. Clyde Riley, Women's Cancer Research Centre for the critical appraisal of the manuscript. The technical help of Mr. Clyde Riley with immunohistochemistry is also appreciated.

Literature Cited

  1. Top of page
  2. Abstract
  3. Relation Between Cellular Origin and the EMT Process—Neoplastic Initiation and Progression
  4. Epithelial Ovarian Carcinoma
  5. Clinical Perspective of EMT in Ovarian Cancer
  6. Conclusion
  7. Acknowledgements
  8. Literature Cited
  • Ahmed N, Maines-Bandiera S, Quinn MA, Unger WG, Dedhar S, Auersperg N. 2006. Molecular pathways regulating EGF-induced epithelio–mesenchymal transition in human ovarian surface epithelium. Am J Physiol Cell Physiol 290: C15321542.
  • Ahmed N, Riley C, Oliva K, Rice G, Quinn M. 2005. Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. Br J Cancer 92: 14751485.
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