Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas : an exception to the norm

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.

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

Vas Deferens Surface Epithelium of Agouti paca: Fine Structural Features

The surface epithelium of the vas deferens of Agouti paca, a wild and large South American rodent, was basically formed by principal and basal cells being only the principal cells related to endocytosis processes and also secretion taking base on their cytoplasmic ultrastructural features. Principal cell of vas deferens epithelium were characterized mainly by presence of vesicles with several shapes, sizes and internalized content at their apical cytoplasm occurring smaller pits and pale small vesicles seen next to the apical brush border of microvillus. Moreover, coated vesicles, smooth surface vesicles and great vesicles; multivesicular bodies, endosomes and lysosomes were seen. Presence of an apocrine secretory apparatus was also viewed, showing apical cytoplasmic expansions protruding into the vas deferens luminal compartment. The basal flattened cells, without luminal surface contact, occurred next to the basement membrane of the ductus, and did no exhibit special ultrastructural features.

Morphological investigations of the surface epithelium of ductuli efferentes of black isogenic mice (Mus musculus)

Morphological investigations of the epithelial cell types that line the ductuli efferentes (DE) of black isogenic mice confirm absorption of the luminal fluid phase by endocytosis as the main function of ductuli efferentes (DE) in this species. :Furthermore, all the histochemical and ultrastructural observations on the DE epithelial histoarchitecture indicate other cellular functions such as exocytosis and probably secretion: including an aprocrine secretory process.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Mutant p53 expression in fallopian tube epithelium drives cell migration

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

Tumorigenesis and peritoneal colonization from fallopian tube epithelium

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Étude du transcriptome des cellules non tumorales de l’épithélium de surface de l’ovaire des femmes porteuses d’une mutation des gènes BRCA1 et BRCA2

Nous avons étudié le transcriptome de neuf échantillons d'ARN extraits de cultures primaires de cellules non tumorales de l’épithélium de surface de l’ovaire (NOSE) provenant de quatre donneuses non porteuses de mutation, deux mutées sur BRCA1 et trois sur BRCA2, ainsi que de quatre échantillons d’ARN extraits de cultures primaires de cellules tumorales de l’ovaire (TOV) provenant de trois donneuses porteuses de mutation sur BRCA1 et une sur BRCA2. Nous avons identifié, pour la première fois, les signatures moléculaires associées à la présence d’une mutation de BRCA1 et BRCA2 dans les cellules NOSEs ainsi que la signature associée à la transformation tumorale des cellules NOSEs en TOVs chez les porteuses de mutation de BRCA1. Nous avons également localisé les domaines chromosomiques comportant des gènes corégulés en association avec la présence d’une mutation de BRCA1 dans les cellules NOSEs. Les allèles sauvage et muté de BRCA2 étaient exprimés dans les cellules TOVs provenant des porteuses de la mutation 8765delAG sur BRCA2. Nous avons observé que le niveau d’expression des transcrits de BRCA2 était plus élevé dans les cellules provenant des tumeurs ovariennes les plus agressives chez les femmes porteuses de la mutation 8765delAG sur BRCA2, les transcrits correspondants à l’allèle muté contribuant avec un pourcentage élevé du niveau d’expression total du gène. Le phénotype tumoral observé chez les Canadiennes Françaises porteuses de cette mutation pourrait résulter d’un effet de dosage de l’allèle muté.

Germ line Cysts and the Formation of the Germinal Epithelium During the Female Gonadal Morphogenesis in Cyprinus Carpio (Teleostei: Ostariophysi: Cypriniformes)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modelling the gastric epithelium for testing of new chemical entities

A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. A successful model should exhibit tight junction formation, maintenance of differentiation and polarity. Conditions for primary culture of guinea-pig gastric mucous epithelial cell monolayers on Tissue Culture Plastic (TCP) and membrane insects (Transwells) were established. Tight junction formation for cells grown on Transwells for three days was assessed by measurement of transepithelial resistance (TEER) and permeability of mannitol and fluorescein. Coating the polycarbonate filter with collagen IV, rather with collagen I, enhanced tight junction formation. TEER for cells grown on Transwells coated with collagen IV was close to that obtained with intact guinea-pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [3H] glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on TCP, but no major difference was found between cells grown on collagens I and IV. However, monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide. The proportion of cells, which stained positively for mucin with periodic Schiff reagent, was greater than 95% for all culture conditions. Gastric epithelial monolayers grown on Transwells coated with collagen IV were able to withstand transient (30 min) apical acidification to pH 3, which was associated with a decrease in [3H] mannitol flux and an increase in TEER relative to pH 7.4. The model was used to provide the first direct demonstration that an NSAID (indomethacin) accumulated in gastric epithelial cells exposed to low apical pH. In conclusion, guinea-pig epithelial cells cultured on collagen IV represent a promising model of the gastric surface epithelium suitable for screening procedures.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Role of Eda and Troy pathways in ectodermal organ development

Several organs of the embryo develop as appendages of the ectoderm, the outermost layer of the embryo. These organs include hair follicles, teeth and mammary glands, which all develop as a result of reciprocal tissue interactions between the surface epithelium and the underlying mesenchyme. Several signalling molecules regulate ectodermal organogenesis the most important ones being Wnts, fi broblast growth factors (Fgfs), transforming growth factor -βs (Tgf-βs) including bone morphogenetic proteins (Bmps), hedgehogs (Hhs), and tumour necrosis factors (Tnfs). This study focuses on ectodysplasin (EDA), a signalling molecule of the TNF superfamily. The effects of EDA are mediated by its receptor EDAR, an intracellular adapter protein EDARADD, and downstream activation of the transcription factor nuclear factor kappa-B (NF-кB). Mice deficient in Eda (Tabby mice), its receptor Edar (downless mice) or Edaradd (crinkled mice) show identical phenotypes characterised by defective ectodermal organ development. These mouse mutants serve as models for the human syndrome named hypohidrotic ectodermal dysplasia (HED) that is caused by mutations either in Eda, Edar or Edaradd. The purpose of this study was to characterize the ectodermal organ phenotype of transgenic mice overexpressing of Eda (K14-Eda mice), to study the role of Eda in ectodermal organogenesis using both in vivo and in vitro approaches, and to analyze the potential redundancy between the Eda pathway and other Tnf pathways. The results suggest that Eda plays a role during several stages of ectodermal organ development from initiation to differentiation. Eda signalling was shown to regulate the initiation of skin appendage development by promoting appendageal cell fate at the expense of epidermal cell fate. These effects of Eda were shown to be mediated, at least in part, through the transcriptional regulation of genes that antagonized Bmp signalling and stimulated Shh signalling. It was also shown that Eda/Edar signalling functions redundantly with Troy, which encodes a related TNF receptor, during hair development. This work has revealed several novel aspects of the function of the Eda pathway in hair and tooth development, and also suggests a previously unrecognized role for Eda in mammary gland development.

ISOLATION, LOCALIZATION, AND CARDIOVASCULAR ACTIVITY OF TACHYKININS FROM THE STOMACH OF THE BOWFIN AMIA-CALVA

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic-rich soil

Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is sequestered by the metalloprotein in certain target cells and tissues.

The role of the nuclear receptor Nr5a2 in ovulation in mice

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux.