Cancer-associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse-strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage.

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non-epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal-binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3' end of the 5p miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated 'hotspot' mutations, we engineered mouse DICER1-deficient ES cells to express wild-type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal-binding site mutations were compared to each other and to wild-type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation-carrying cells were distinct from both wild-type and DICER1-deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p-derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal-binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations.

Germline mutations in CDH1 are infrequent in women with early-onset or familial lobular breast cancers.

BACKGROUND: Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer; lobular breast cancer also occurs excessively in families with such condition. METHOD: To determine if CDH1 is a susceptibility gene for lobular breast cancer in women without a family history of diffuse gastric cancer, germline DNA was analysed for the presence of CDH1 mutations in 318 women with lobular breast cancer who were diagnosed before the age of 45 years or had a family history of breast cancer and were not known, or known not, to be carriers of germline mutations in BRCA1 or BRCA2. Cases were ascertained through breast cancer registries and high-risk cancer genetic clinics (Breast Cancer Family Registry, the kConFab and a consortium of breast cancer genetics clinics in the United States and Spain). Additionally, Multiplex Ligation-dependent Probe Amplification was performed for 134 cases to detect large deletions. RESULTS: No truncating mutations and no large deletions were detected. Six non-synonymous variants were found in seven families. Four (4/318 or 1.3%) are considered to be potentially pathogenic through in vitro and in silico analysis. CONCLUSION: Potentially pathogenic germline CDH1 mutations in women with early-onset or familial lobular breast cancer are at most infrequent.

Culture and characterization of human ovarian surface epithelium.

The ovarian surface epithelium (OSE) is the part of the pelvic mesothelium that covers the ovary. It comprises only a minute fraction of the ovary-however, it is the source of the epithelial ovarian carcinomas which are the prime cause of death from gynecological malignancies in North American and European women. The implication that OSE is the source of the epithelial ovarian cancers is based mainly on the demonstration of histopathological changes (1). The early stages of epithelial ovarian car-cinogenesis are poorly understood because these carcinomas are usually diagnosed in late stages and there are no adequate animal models for their study.

Increased E-cadherin expression in ovarian surface epithelium: an early step in metaplasia and dysplasia?

Ovarian surface epithelium (OSE), the source of common epithelial carcinomas, is a simple mesothelium that during carcinogenesis acquires complex epithelial characteristics normally found in tubal, endometrial, and endocervical epithelia. These characteristics include the intercellular adhesive molecule E-cadherin. We examined cryostat sections of 12 normal ovaries by immunofluorescence and immunocytochemistry to determine whether E-cadherin is a component of normal OSE that is retained in ovarian cancers or whether it, like many other epithelial characteristics, is acquired in the course of metaplasia and neoplastic progression. E-cadherin expression by OSE varied with its location within the ovary and with cell shape. It was present inconsistently on the ovarian surface but was increased in surface invaginations and particularly in epithelial inclusion cysts. Independent of location, E-cadherin was most prominent in columnar, variable in cuboidal, and absent in flat OSE. This relationship to cell morphology accounts for the increased E-cadherin expression in inclusion cysts, which are sites of frequent metaplastic and dysplastic changes. These results suggest that the morphologic variation of OSE reflects differences in E-cadherin-mediated intercellular adhesion. Thus, the appearance of this adhesion molecule in columnar OSE may represent an early step in the increased commitment to epithelial phenotypes that accompanies metaplasia and neoplastic progression of OSE.

Simian virus 40-transformed human ovarian surface epithelial cells escape normal growth controls but retain morphogenetic responses to extracellular matrix.

OBJECTIVE: We aimed to improve the availability of experimental models for the study of human ovarian surface epithelium. STUDY DESIGN: Low-passage cultures of human ovarian surface epithelium were transfected with SV40 large- T antigen and the resulting lines were characterized. RESULTS: Three immortalized lines were obtained. They formed epithelial monolayers resembling ovarian surface epithelium in serum-free medium, expressed large-T antigen and overexpressed p53, produced laminin, and were CA 125 negative. Two lines expressed keratin. On plastic surfaces, the growth rate and growth potential of immortalized ovarian surface epithelium were increased over the growth of ovarian surface epithelium, but on extracellular matrices their growth and morphologic features resembled ovarian surface epithelium. The lines were not tumorigenic in Nu/Nu mice. CONCLUSION: The immortalized ovarian surface epithelium lines resemble cells early in neoplastic progression and should be useful to study ovarian carcinogenesis.

A simplified method to culture human ovarian surface epithelium.

The ovarian surface epithelium (OSE) is thought to give rise to over 85% of human ovarian carcinomas. In spite of its clinical importance, no animal models for the in vivo investigation of this tissue exist, and available culture methods have yielded limited success. In this study, OSE cells from 55 normal ovarian biopsy specimens were used to improve and simplify the methodology for OSE culture and to define the influence of clinical parameters on cultured OSE cells. An improved explanation method was developed which takes advantage of the tenuous attachment of OSE to underlying tissues: the surface epithelium was scraped off the ovarian surface with a rubber scraper, generating epithelial fragments which produced monolayers in culture, with little contamination by other cell types. The scrape method is superior to the explant method previously described (Siemens CH, Auersperg N: J Cell Physiol 134:347, 1988) in terms of speed, simplicity, higher purity of cultures, and increased cell yield. An improved nutrient medium (199/MCDB105/15%FBS) resulted in OSE lines that maintained the original epithelial phenotype for up to 12 population doublings. OSE, detached from the ovary, remained viable if frozen in liquid nitrogen either before culture or in primary culture on strips of plastic, providing OSE independently of the availability of surgical specimens. Growth was not influenced by diagnosis (nonmalignant gynecologic disorders), patient age (mean range: 40.5, 20 to 62 years), or the presence of inclusion cysts or large follicles in the biopsy specimen. This culture system provides conditions for in depth studies of OSE physiology and pathology.

Secretion of bioactive interleukin-1, interleukin-6, and colony-stimulating factors by human ovarian surface epithelium.

The ovarian surface epithelium (OSE) takes part in the lysis and repair of the ovulatory site. It also forms invaginations and cysts that give rise to the majority of ovarian epithelial carcinomas. In the present study, we investigated the capacity of cultured human OSE to secrete cytokines that may contribute to the regulation of ovarian functions and may influence ovarian carcinogenesis. Bioassays, combined with antibody neutralization experiments, showed that OSE cells in short-term culture secrete bioactive interleukin-1 (IL-1), interleukin-6 (IL-6), macrophage colony-stimulating factor (CSF-1), granulocyte colony-stimulating factor (G-CSF), and limited granulocyte-macrophage colony-stimulating factor (GM-CSF). There was a tendency for these factors to be absent or secreted in reduced amounts in SV40-immortalized OSE lines and in two ovarian carcinoma lines. No IL-2, IL-3, or IL-4 was detected. The results show that normal OSE cells secrete factors that are known to have regulatory effects on follicular growth and differentiation, ovulation, and the distribution of intraovarian cells of the immune system. In addition, the results suggest that the secretion of cytokines by ovarian carcinomas represents the retention of normal precursor cell properties, rather than new characteristics acquired as a result of neoplastic progression.

An ovarian adenocarcinoma line derived from SV40/E-cadherin-transfected normal human ovarian surface epithelium.

Epithelial ovarian carcinomas are thought to originate in the ovarian surface epithelium (OSE), i.e., the mesothelium covering the ovary, but experimental evidence for this origin has been lacking. Contrary to most epithelia, where neoplastic progression is associated with a reduction of E-cadherin, this cell-cell adhesion molecule is sparse in normal human OSE but its expression increases with the development of ovarian epithelial metaplasia and neoplasia. Concurrently, the tumors tend to acquire characteristics of the complex epithelia of the oviduct and uterus. The high proportion of ovarian cancers where such aberrant Mullerian differentiation occurs suggests that this change may confer a selective advantage on the transforming cells. We previously demonstrated that increased E-cadherin expression may be a cause, rather than a consequence, of such Mullerian differentiation. E-cadherin was transfected into SV40 large T antigen-immortalized, E-cadherin-negative cells derived from normal OSE. Constitutive expression of E-cadherin re-established normal epithelial markers that had been lost in culture, such as keratin, and induced markers of metaplasia and neoplasia, such as CA125. In the present study, SV40-immortalized, E-cadherin-transfected cells, but not the E-cadherin-negative controls, were found to be anchorage-independent and to form transplantable, invasive s.c. and i.p. adenocarcinomas in 100% of injected SCID mice. Tumor cells injected i.p. seeded the mesenteries and omentum, invaded the liver and thigh musculature and produced ascites. The presence of SV40 large T antigen in the tumor cell nuclei confirmed their origin as transfected OSE cells. Our results demonstrate that ovarian adenocarcinomas can be derived by genetic manipulation of normal human OSE.

Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.

BACKGROUND: The ovarian surface epithelium (OSE) is a modified mesothelium that gives rise to most human ovarian carcinomas. In culture, OSE cells tend to assume atypical morphologies that make it difficult to accurately identify normal OSE cells and to recognize pathologic changes. The present study was undertaken to improve the accuracy of OSE identification and to distinguish phenotypic variations of normal OSE cells from early (pre)neoplastic changes. EXPERIMENTAL DESIGN: The expression of epithelial and stromal markers was compared between OSE cultures in low passage, three simian virus 40-immortalized OSE lines (IOSE lines) and two ovarian carcinoma lines, using immunofluorescence microscopy, immunocytochemistry, and Western blots, with fibroblasts and vascular endothelial cells as controls. RESULTS: Whereas keratin remained a convenient and specific epithelial marker for normal OSE, it was not expressed by all cells, and it diminished with passages in culture. E-cadherin and desmoplakins were absent in cultured OSE, mucin was detected in few cells, and microvilli diminished within one to two passages. Laminin and collagen IV were uniformly expressed and stable with time but were also found in endothelial cells. In contrast to endothelial cells, OSE lacked Factor VIII and did not bind Ulex Europaeus Lectin. The three IOSE lines were more stable than OSE morphologically, and keratin was expressed consistently in 100%, 90%, and 0% of the cells, respectively. All IOSE cells produced laminin and collagen IV but lacked E-cadherin. Microvilli persisted in 50% of the cells in one IOSE line and were lacking in the others. The antibody to breast/ovarian carcinoma, 2G3, reacted with few OSE cells but with significantly more IOSE cells. All fibroblast markers tested (vimentin, collagen types I and III, and prolyl-4-hydroxylase) were expressed in OSE and IOSE cultures, concurrently with the epithelial markers. There was no consistent relationship between any of the markers and cell morphology. CONCLUSIONS: Cultured OSE is more accurately identified if the demonstration of keratin is supplemented by 2G3, laminin, or the lack of endothelial markers. The modulation to a fibroblast-like morphology by OSE cells may reflect the expression of their dual epithelio-mesenchymal phenotype rather than epithelio-mesenchymal conversion. Possible indicators of early neoplastic change in immortalized OSE cells include reduced morphologic plasticity and increased 2G3 binding.

Expression of two mucin antigens in cultured human ovarian surface epithelium: influence of a family history of ovarian cancer.

OBJECTIVE: The genetic changes in hereditary ovarian cancer syndromes suggest that the phenotype of ovarian surface epithelium in women with familial ovarian cancer might be altered. To test this hypothesis, we compared two tumor markers, CA 125 and 2G3, in cultures of overtly normal epithelium from patients with and without familial ovarian cancer. STUDY DESIGN: Surface epithelia from 18 patients with no family history of ovarian cancer, five with family histories that were insufficient to be classified as familial, and seven with strong family histories were examined by immunofluorescence, immunocytochemistry, and radioimmunoassay. The influence of cell density, morphologic features, propagation in culture, and immortalization with SV40 on the expression of the markers was investigated. RESULTS: CA 125 occurred in cells with epithelial rather than atypical morphologic features. In cultures with no family history and minor family history, CA 125 was present in up to 45% cells in passage 1 but in only < 5% cells in 14 of 16 cultures by passages 3 to 4. In contrast, nine of 10 cultures with family history retained > 5% CA 125-positive cells in passages 3 to 4. This prolonged presence of CA 125 correlated with a persisting epithelial phenotype, whereas most cells with no family history and minor family history became atypical by passage 3. Immortalization eliminated CA 125 in all three types of cells. 2G3 bound to few cells in low passage, independent of family history and morphologic features. The proportion of 2G3-expressing cells increased significantly with immortalization in all cultures, independent of family history. Ovarian carcinoma lines expressed both markers. CONCLUSION: In cultures of ovarian surface epithelium 2G3 expression increases with immortalization, whereas CA 125 is lost with immortalization but correlates with epithelial cell morphologic features and with family history. The results suggest that there may be phenotypic changes in overtly normal ovarian surface epithelium of women with family histories of ovarian cancer.

Autonomy of the epithelial phenotype in human ovarian surface epithelium: changes with neoplastic progression and with a family history of ovarian cancer.

Epithelial ovarian carcinomas originate in the ovarian surface epithelium (OSE). In culture, OSE undergoes epithelio-mesenchymal conversion, an event mimicking a wound response, while ovarian carcinomas retain complex epithelial characteristics. To define the onset of this increased epithelial autonomy in ovarian neoplastic progression, we examined mesenchymal conversion in OSE from 25 women with no family histories (NFH-OSE) and 13 women with family histories (FH-OSE) of breast/ovarian cancer (including 8 with mutated BRCA1 or 17q linkage) and in 8 ovarian cancer lines. After 3-6 passages in monolayer culture, most NFH-OSE exhibited reduced keratin expression and high collagen type III expression. In contrast, keratin remained high but collagen expression was lower in p. 3-6 FH-OSE. This difference was lost in SV40-transformed lines, which all resembled FH-OSE. Most carcinoma lines remained epithelial and did not undergo mesenchymal conversion. In 3-dimensional (3-D) sponge culture, NFH-OSE cells dispersed and secreted abundant extracellular matrix (ECM). FH-OSE remained epithelial and did not secrete ECM. ECM production was also reduced in SV40-transformed lines. Carcinoma lines in 3-D formed epithelial cysts, aggregates and papillae and lacked ECM. Sponge contraction (a mesenchymal characteristic) was greater in NFH-OSE than in FH-OSE both before and after SV40 transformation and was absent in the cancer lines. Our results suggest that increased autonomy of epithelial characteristics is an early indicator of ovarian neoplastic progression and that phenotypic changes indicative of such autonomy are found already in overtly normal OSE from women with histories of familial breast/ovarian cancer.

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.

Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: influence of family history of ovarian cancer.

Epithelial ovarian carcinomas arise in a simple mesothelium (ovarian surface epithelium, OSE) but exhibit properties of oviductal and endometrial epithelia. Thus, during malignant progression, their differentiation proceeds from simple to complex, in contrast to carcinomas in other tissues. Related changes in OSE of women with a history of familial ovarian cancer indicate that this aberrant differentiation is initiated very early in neoplastic progression. The mechanisms underlying this process are not understood. Because cadherins are known regulators of differentiation, we investigated the relationship of the cadherins E, N and P to OSE morphology, growth patterns and differentiation in cultures of normal and metaplastic OSE from women with (FH-OSE) and without (NFH-OSE) a family history of ovarian cancer and in the ovarian carcinoma lines OVCAR-3 and CaOV3. We used immunofluorescence, RT-PCR, in situ hybridization and Western blotting. Our results define N-cadherin as the constitutively expressed cadherin of normal and metaplastic OSE and indicate that P-cadherin is undetectable while E-cadherin expression is conditional and related to genotype, stage of neoplastic progression and growth pattern. The altered expression of E-cadherin in apparently normal OSE of women with hereditary ovarian cancer syndromes in conjunction with the known capacity of E-cadherin to induce epithelial characteristics implicates this adhesion molecule as a possible inducer of the aberrant Mullerian differentiation which characterizes epithelial ovarian carcinomas. Abnormal differentiation in such (pre)-neoplastic tissues may represent an early, irreversible, non-mutational step in ovarian epithelial neoplastic progression.