Fatty acid synthase is a metabolic marker of cell proliferation rather than malignancy in ovarian cancer and its precursor cells.

Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitor-resistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated ß-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell lines­although resisting senescence­reveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth-inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug development.

The stem-cell profile of ovarian surface epithelium is reproduced in the oviductal fimbriae, with increased stem-cell marker density in distal parts of the fimbriae.

High-grade serous ovarian carcinomas are the most common and most lethal ovarian cancers, but their histologic origin is still controversial. Current evidence suggests that they may originate in the ovarian surface epithelium (OSE) and/or epithelium of oviductal fimbriae (FE). To further investigate this question we compared the stem-cell profiles of these epithelia. Formalin-fixed sections of normal FE (N=21) and ovaries (N=21) were stained immunohistochemically for the stem-cell markers NANOG, SFRP1, LHX9, ALDH1A1, and ALDH1A2. All markers were detected in both OSE and FE. A total of 75% to 100% of surface OSE expressed all markers except ALDH1A1, which occurred in about 25% of cells. Among epithelial inclusion cysts with flat-to-cuboidal epithelium, resembling OSE, ALDH1A1 was significantly increased, whereas SFRP1 was reduced compared with surface OSE, suggesting an increased trend towards malignant transformation. Similarly, among cysts lined by columnar cells resembling FE, SFRP1 expression was low, whereas ALDH1A1 approached 100% of the cysts. FE exhibited considerable variation between and within specimens. In about half of the samples, SFRP1 and NANOG were detected in ≤25% FE. The most widespread markers were ALDH1A1 and ALDH1A2. The highest proportion of all markers occurred in the distal parts of the FE, the site of the putative ovarian cancer precursors. Marker expression in tubal ampullae was low or absent except for ALDH1A1 and ALDH1A2. The results provide an explanation for the characteristic distal location of fimbrial high-grade serous ovarian carcinoma precursor lesions, and indicate that both OSE and FE have the capacity to undergo neoplastic transformation.

Ovarian surface epithelium as a source of ovarian cancers: unwarranted speculation or evidence-based hypothesis?

OBJECTIVES: There has been increasing evidence that high grade serous ovarian carcinomas (HGSOCs), the most common and most lethal of all ovarian cancers, originate in oviductal fimbriae and metastasize to the ovary. The alternate hypothesis, that ovarian carcinomas may originate within the ovarian stroma in inclusion cysts lined by ovarian surface epithelium (OSE), has been criticized and often dismissed on the basis of the OSE's embryonic origin, mesothelial phenotype, tissue-specific markers, questionable ability to undergo metaplasia, and the lack of identifiable precursor lesions. This review analyzes these criticisms and summarizes evidence indicating that OSE as a source of ovarian cancers cannot be ruled out. METHODS: The literature was reviewed and representative reports were chosen to evaluate the current criticisms of, and evidence in favor of, the OSE hypothesis. RESULTS: The close developmental relationship between the oviduct and OSE, both of which originate in the mesothelial coelomic epithelium, accounts for their capacity to produce similar tumors. Histopathologic and experimental data show that OSE does undergo serous metaplasia, and that transformation of pure OSE cultures produces aggressive neoplasms resembling high- and low-grade serous carcinomas, but never mesotheliomas. There is evidence of premalignant changes (e.g. p53 inactivation) in morphologically normal OSE and of rare but definitive dysplastic and early preinvasive lesions in OSE-lined inclusion cysts. Conclusions based on tissue-specific markers to identify origins of inclusion cysts usually disregard the changes in differentiation occurring when OSE is displaced to the stroma. Lastly, an explanation is offered for the rare detection of precursor lesions in OSE-lined cysts, based on the likelihood that the duration from initiation of malignant transformation to invasive growth is minimal and thus difficult to detect. CONCLUSION: The likelihood that HGSOCs originate both in fimbriae and in OSE should be considered in clinical decisions involving choices between prophylactic salpingo-oophorectomies and salpingectomies.

The origin of ovarian cancers--hypotheses and controversies.

Ovarian cancer is the prime cause of death from gynecological malignancies in the Western world. In spite of its importance, it is poorly understood and its prognosis remains poor. The most common and lethal of all ovarian cancer subtypes are the high grade serous ovarian carcinomas (HGSOCs). A major problem in their clinical management is the current uncertainty about their cell type of origin, which limits means of early detection and prevention. It has not been resolved whether all HGSOCs originate in oviductal fimbriae or in ovarian surface epithelium (OSE). This review summarises evidence for these two hypotheses and considers the alternative possibility that HGSOCs may arise at both sites. This concept is based on the common embryonic origin of OSE and fimbriae in the coelomic epithelium and evidence of overlapping differentiation between these epithelia in the adult, which suggests incomplete commitment and pleuripotentiality. This hypothesis would account for OSE and fimbriae giving rise to identical carcinomas, and for their susceptibility to neoplastic transformation that is absent in the adjacent extraovarian serosa and oviductal ampulla.

TGF-beta induces serous borderline ovarian tumor cell invasion by activating EMT but triggers apoptosis in low-grade serous ovarian carcinoma cells.

Apoptosis in ovarian surface epithelial (OSE) cells is induced by transforming growth factor-beta (TGF-ß). However, high-grade serous ovarian carcinomas (HGC) are refractory to the inhibitory functions of TGF-ß; their invasiveness is up-regulated by TGF-ß through epithelial-mesenchymal transition (EMT) activation. Serous borderline ovarian tumors (SBOT) have been recognized as distinct entities that give rise to invasive low-grade serous carcinomas (LGC), which have a relatively poor prognosis and are unrelated to HGC. While it is not fully understood how TGF-ß plays disparate roles in OSE cells and its malignant derivative HGC, its role in SBOT and LGC remains unknown. Here we demonstrate the effects of TGF-ß on cultured SBOT3.1 and LGC-derived MPSC1 cells, which express TGF-ß type I and type II receptors. TGF-ß treatment induced the invasiveness of SBOT3.1 cells but reduced the invasiveness of MPSC1 cells. The analysis of apoptosis, which was assessed by cleaved caspase-3 and trypan blue exclusion assay, revealed TGF-ß-induced apoptosis in MPSC1, but not SBOT3.1 cells. The pro-apoptotic effect of TGF-ß on LGC cells was confirmed in another immortalized LGC cell line ILGC. TGF-ß treatment led to the activation of Smad3 but not Smad2. The specific TßRI inhibitor SB431542 and TßRI siRNA abolished the SBOT3.1 invasion induced by TGF-ß, and it prevented TGF-ß-induced apoptosis in MPSC1 cells. In SBOT3.1 cells, TGF-ß down-regulated E-cadherin and concurrently up-regulated N-cadherin. TGF-ß up-regulated the expression of the transcriptional repressors of E-cadherin, Snail, Slug, Twist and ZEB1. In contrast, co-treatment with SB431542 and TßRI depletion by siRNA abolished the effects of TGF-ß on the relative cadherin expression levels and that of Snail, Slug, Twist and ZEB1 as well. This study demonstrates dual TGF-ß functions: the induction of SBOT cell invasion by EMT activation and apoptosis promotion in LGC cells.

EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells?

In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.

HOXA4 protein levels and localization in the aorta and in human abdominal aortic aneurysms.

This report presents evidence for the specificities of select commercially available HOXA4 antibodies in regards to concerns about the specificity of the HOXA4 antibody used by Lillvis et al. (Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms. BMC Physiol 2011, 11:9). Using an antibody characterized extensively by us, Lillvis et al. report detecting HOXA4 at a size of 33 kDa despite our previous reports that HOXA4 is detected at ~37-39 kDa and that the ~30-33 kDa band is non-specific. Using small interfering RNA targeting HOXA4, forced expression of full-length HOXA4 and HOXA4-positive and -negative ovarian cancer cell lines, we confirm our previous findings that the ~30-33 kDa band is non-specific and that HOXA4 is detected at ~37-39 kDa. Moreover, we demonstrate that HOXA4 small interfering RNA reduces the ~37-39 kDa HOXA4 band, but not the ~30-33 kDa non-specific band, in a human acute monocytic leukemia cell line used by Lillvis et al. Western blot analysis performed with two additional commercially available HOXA4 antibodies also detected HOXA4 at ~37-39 kDa. Lastly, immunofluorescent staining of a HOXA4-negative ovarian cancer cell line with the antibody used by Lillvis et al. yields strong perinuclear staining, similar to that observed by Lillvis et al., which cannot be attributed to HOXA4. Our results highlight and briefly discuss the importance of careful antibody validation and selection for use in various applications.

The origin of ovarian carcinomas: a unifying hypothesis.

It is currently a controversial issue whether epithelial ovarian cancers arise in the ovarian surface epithelium (OSE) or the fimbrial epithelium of the oviduct. The hypothesis presented here aims to reconcile these 2 views and provides a possible explanation for 2 questions arising: first, why tumors originating in the fimbriae and OSE, which are parts of different organs, express common features; second, why these epithelia are prone to neoplastic transformation whereas the remaining oviduct and the extraovarian mesothelium are not. We hypothesize that these questions relate to the common origin of the OSE and fimbriae in that region of the embryonic coelomic epithelium, which will eventually link the extraovarian mesothelium to the epithelium of the oviductal ampulla. OSE and fimbriae become separated during embryonic development but, like other transitional, interepithelial junctions in adults, this region might remain incompletely committed and thus prone to neoplastic progression. To define differentiation at the OSE-tubal junction, salpingo-oophorectomy specimens were stained immunohistochemically for mesenchymal differentiation markers of OSE and for epithelial markers and Pax8, characterizing oviductal fimbriae and ampullae. OSE and ampullae were distinctly different, but there was no sharp boundary between OSE and fimbriae. Rather, both mesenchymal and epithelial markers overlapped, and Pax8 and fimbrial epithelial markers diminished distally, near the OSE. The results support the hypothesis that the OSE and fimbriae are parts of a transitional epithelium of common origin rather than 2 independent sources of ovarian cancer, and suggest that their immature, incompletely determined phenotype contributes to their propensity to neoplastic transformation.

Oviductal glycoprotein (OVGP1, MUC9): a differentiation-based mucin present in serum of women with ovarian cancer.

INTRODUCTION: Epithelial ovarian carcinomas are highly lethal because most are detected at late stages. A previous immunohistochemical analysis showed that oviductal glycoprotein 1 (OVGP1), a secretory product of the oviductal epithelium under estrogen dominance, is produced predominantly by borderline and low-grade malignant epithelial ovarian tumors. In the present study, we investigated OVGP1 as a possible serum marker for the detection of ovarian cancer. METHODS: We generated a highly specific monoclonal antibody, clone 7E10, to human OVGP1. Using 7E10 and a polyclonal antibody, a sandwich enzyme-linked immunosorbent assay was developed to assay OVGP1 levels in 135 normal sera, and sera from 21 benign tumors, 12 borderline tumors, and 87 ovarian cancers (18, grade 1-2 serous; 44, grade 3 serous; 10, mucinous; 10, clear cell; and 5, endometrioid). RESULTS: Using a 95% confidence interval cutoff from the mean of normal postmenopausal sera, median OVGP1 levels were elevated in the sera from 75% of the women with borderline tumors and 80% of the women with mucinous, 60% with clear cell, 59% with grade 1 and 2 serous, 22% with grade 3 serous, and 0% with endometrioid carcinomas. By stage, OVGP1 levels were highest in the sera from the borderline tumors, stage I and II serous carcinomas, and mucinous carcinomas. OVGP1 levels varied independently of cancer antigen 125 (CA125). CONCLUSIONS: Increases in OVGP1 serum levels vary with ovarian tumor histotypes and stages. Being differentiation based, OVGP1 seems to detect a different spectrum of ovarian epithelial cancers than other markers and thus should be a useful adjunct for more accurate detection, particularly of early serous ovarian cancers and mucinous carcinomas, which tend to lack increased CA125.

Induction of papillary carcinoma in human ovarian surface epithelial cells using combined genetic elements and peritoneal microenvironment.

Papillary differentiation is one of the most common histological features of ovarian cancer, although the underlying mechanism that leads to such differentiation is not known. We hypothesized that human ovarian surface epithelial cells can be transformed into carcinoma with papillary differentiation by overexpressing HER2/neu in these cells. Mice were injected either subcutaneously or intraperitoneally with two immortalized human ovarian surface epithelial cell lines after enforced expression of HER-2/neu. Mice subcutaneously injected with tumor cells from either the T29Nt or T80Nt developed undifferentiated carcinomas. In contrast, mice injected intraperitoneally with T29Nt cells developed papillary carcinoma, and those injected intraperitoneally with T80Nt cells developed undifferentiated carcinoma. Our results demonstrate that ovarian surface epithelial cells can develop into papillary carcinoma in mice, and that the induction of papillary differentiation depends not only on specific genetic modifications but also on the tumor microenvironment and epithelial cell type from ovary from different patients.

Cell motility and spreading are suppressed by HOXA4 in ovarian cancer cells: possible involvement of beta1 integrin.

HOX genes are transcription factors that control morphogenesis, organogenesis and differentiation. Increasing evidence suggests that HOX genes play a role in ovarian cancer progression; however few studies have defined functional roles and mechanisms of action. We showed previously that HOXA4 expression is increased in invasive, compared to noninvasive, epithelial ovarian tumors. However, HOXA4 suppressed cell migration suggesting that elevated HOXA4 expression in invasive tumors constitutes a homeostatic response. In the present study, we used siRNA and forced-expression in multiple cell lines to define the role of HOXA4 in the regulation of transwell migration/invasion and cellular/colony morphology. Knockdown of endogenous HOXA4 increased migration, but not Matrigel invasion, of OVCAR-8 and OVCAR-3 cells. HOXA4 knockdown also increased cell spreading on plastic or fibronectin, reduced cell-cell adhesion, and increased filopodia in two- and three-dimensional cultures. These changes were not associated with significant changes in alphaV or beta3 integrin and E- or N-cadherin. However, down-regulation of HOXA4 significantly reduced beta1 integrin protein levels within cell colonies and cell aggregates, but not of single, nonadherent cells. It had no effect on beta1 integrin, alpha5 integrin, or fibronectin mRNA levels. Conversely, overexpression of HOXA4 in CaOV-3 cells suppressed transwell migration and increased beta1 integrin protein levels. Our results confirm that HOXA4 inhibits cell motility, show that it suppresses cell spreading and filopodia formation while enhancing cell-cell adhesion, and suggest a role for beta1 integrin in mediating these changes. These observations support the hypothesis that overexpression of HOXA4 in invasive ovarian tumors is a homeostatic, invasion-suppressive response.

Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation.

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.

Expression and function of HOXA genes in normal and neoplastic ovarian epithelial cells.

We studied the roles of three HOXA genes in cultured normal ovarian surface epithelial (OSE) cells and ovarian cancer cells. They included HOXA4 and HOXA7 because, by cDNA microarray analysis, these were more highly expressed in invasive ovarian carcinomas than in benign or borderline (noninvasive) ovarian tumors, and HOXA9 because it characterizes normal oviductal epithelium, which resembles ovarian serous adenocarcinomas. The three HOXA genes were more highly expressed when OSE cells were dividing and motile than when they were confluent and stationary, and also when they dispersed in response to EGF treatment or to reduced calcium concentrations in culture media. The expression of the HOXA genes varied among ovarian cancer cell lines, but was highest in lines with compact epithelial morphologies. We focused on HOXA4 as the most highly expressed in the ovarian carcinoma array. HOXA4 expression did not parallel proliferative activities of either OSE or ovarian cancer lines. Moreover, modifying HOXA4 expression in ovarian cancer cell lines did not alter either E-cadherin expression or CA125 secretion. However, HOXA4 downregulation enhanced EGFR phosphorylation and migration in serum-starved OSE and ovarian cancer cells in response to EGF, and enhanced migration of all ovarian cancer lines in 5% serum even without EGF treatment. Thus, HOXA4 expression does not correlate with proliferation or with epithelial differentiation, but it increases in response to OSE cell dispersion and negatively regulates EGFR activation and the motility of OSE and of ovarian cancer cells. HOXA4 expression was highest in cancer lines with compact epithelial growth patterns, suggesting, again, an anti-dispersion function. In summary, increased HOXA4 expression in ovarian cancer appears to constitute a tumor-suppressive, homeostatic response to aberrant cell behavior, and, in particular, to cell dispersion and migration.

HOX cofactors expression and regulation in the human ovary.

BACKGROUND: HOX cofactors enhance HOX binding affinities and specificities and increase HOX's unique functional activities. The expression and the regulation of HOX cofactors in human ovaries are unknown. METHODS: In this study, the expression of HOX cofactors, PBX1, PBX2, and MEIS1/2, were examined by using RT-PCR, immunofluorescence in cultured immortalized human granulosa (SVOG) cells. The distribution of these HOX cofactors in human ovaries was examined by immunohistochemistry. The effects of growth differentiation factor-9 (GDF-9) and follicle-stimulating hormone (FSH) on PBX2 in SVOG cells were investigated by western blot analysis. Binding activities of HOXA7 and PBX2 to the specific sequences in granulosa cells were determined by electrophoretic mobility shift assay (EMSA). RESULTS AND CONCLUSION: In SVOG cells, PBX1, PBX2 and MEIS1/2 were expressed during cell culture. In normal human ovaries, PBX1 and MEIS1/2 were expressed in granulosa cells at essentially all stages of follicular development. These cofactors were expressed in the nuclei of the granulosa cells from the primordial to the secondary follicles, whereas beyond multilayered follicles was observed in the cytoplasm. The co-expression of PBX1 and MEIS1/2 in granulosa cells in normal human ovaries suggested that MEIS1/2 might control PBX1 sublocalization, as seen in other systems. PBX2 was not expressed or weakly expressed in the primordial follicles. From the primary follicles to the preovulatory follicles, PBX2 expression was inconsistent and the expression was found in the granulosa cell nuclei. The PBX2 expression pattern is similar to HOXA7 expression in ovarian follicular development. Furthermore, FSH down-regulated, GDF-9 did not change PBX2 expression, but co-treatment of the granulosa cells with FSH and GDF-9 up-regulated PBX2 expression. These results implicated a role for PBX2 expression in the steroidogenic activities of granulosa cells in humans. Moreover, PBX2 and HOXA7 bound together to the Pbx sequence, but not to the EMX2 promoter sequence, in SVOG cells. Our findings indicate that HOX cofactors expression in normal human ovary is temporally and spatially specific and regulated by FSH and GDF-9 in granulosa cells. HOX proteins may use different HOX cofactors, depending on DNA sequences that are specific to the granulosa cells.

SV40 early genes induce neoplastic properties in serous borderline ovarian tumor cells.

OBJECTIVE: Serous borderline ovarian tumors (SBOT) are slow growing, noninvasive ovarian epithelial neoplasms, which tend to recur as low-grade invasive carcinomas (LGC) with a much worse prognosis. We investigated the molecular basis of this progression. METHODS: We established cultures of three SBOTs and one LGC from tumor biopsies, and inactivated p53, Rb and PP2A in the cells with SV40 large T (LT) and small T (ST) antigen. They were examined for cadherins by immunofluorescence and immunoblotting, invasiveness in Boyden chambers, motility by scratch-wound healing assay, anchorage independence by growth in agarose, and protease activity by gelatin zymography, immunoassay and colorimetry. Cells were overexpressed with N-cadherin using an adenovirus. RESULTS: Inactivation of p53, Rb and PP2A by SV40 LT/ST antigen resulted in greatly enhanced growth potential, invasiveness, motility and anchorage independence, and in epithelio-mesenchymal transition, as indicated by morphology and substitution of N-cadherin for E-cadherin. Overexpressed N-cadherin did not induce invasiveness of SBOT cells and there was no consistent change in protease activities, suggesting that these were not primary effectors of the enhanced neoplastic characteristics. Low passage LGC cells were more invasive than SBOT cells, but this difference disappeared with the introduction of LT/ST into the two cell types. CONCLUSION: Downregulation or inactivation of p53, Rb and/or PP2A plays a role in the progression from SBOT to invasive ovarian carcinomas.

The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas.

Ovarian epithelial carcinomas (OECs) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In our study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

Role of PELP1/MNAR signaling in ovarian tumorigenesis.

Emerging evidence suggests that nuclear receptor (NR) coregulators have potential to act as master genes and their deregulation can promote oncogenesis. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is a novel NR coregulator. Its expression is deregulated in hormone-driven cancers. However, the role of PELP1/MNAR in ovarian cancer progression remains unknown. Analysis of serial analysis of gene expression data suggested deregulation of PELP1/MNAR expression in ovarian tumors. Western analysis of PELP1/MNAR in normal and serous ovarian tumor tissues showed 3- to 4-fold higher PELP1/MNAR expression in serous tumors compared with normal ovarian tissues. To examine the significance of PELP1/MNAR in ovarian cancer progression, we have generated model cells that overexpress PELP1/MNAR and ovarian cancer cells in which PELP1/MNAR expression is down-regulated by stable expression of PELP1/MNAR-specific shRNA. Down-regulation of PELP1/MNAR in cancerous ovarian model cells (OVCAR3) resulted in reduced proliferation, affected the magnitude of c-Src and protein kinase B (AKT) signaling, and reduced tumorigenic potential of ovarian cancer cells in a nude mouse model. PELP1/MNAR overexpression in nontumorigenic immortalized surface epithelial cells (IOSE cells) promoted constitutive activation of c-Src and AKT signaling pathways and promoted anchorage-independent growth. Immunohistochemical studies using human ovarian cancer tissue arrays (n = 123) showed that PELP1/MNAR is 2- to 3-fold overexpressed in 60% of ovarian tumors, and PELP1/MNAR deregulation occurs in all different types of ovarian cancer. Collectively, these results suggest that PELP1/MNAR signaling plays a role in ovarian cancer cell proliferation and survival, and that its expression is deregulated in ovarian carcinomas.