RESUMEN
DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic 'hotspot' mutations at the four metal-binding sites within the RNase IIIb domain of DICER1 were identified in ovarian sex cord-stromal tumours and have since been described in other paediatric tumours. In this study, we screened the RNase IIIb domain of DICER1 in 290 endometrial tumours and identified six cases with hotspot mutations, including two cases affected by an atypical G1809R mutation directly adjacent to a metal-binding site. Using Illumina and Sanger targeted resequencing, we observed and validated biallelic DICER1 mutations in several cases with hotspot mutations. Through in vitro DICER1 cleavage assays, small RNA deep sequencing and real-time PCR, we demonstrated that mutations adding a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production to mutations at the metal-binding sites. As expected, 5p miRNAs were globally reduced in tumours and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes de-repressed due to loss of 5p miRNAs are strongly associated with pathways regulating the cell cycle. Using a Dicer1-null mouse cell line model, we found that expression of DICER1 hotspot mutants promoted cell proliferation, whereas wild-type (WT) DICER1 inhibited cell proliferation. Furthermore, targets of let-7 family miRNAs are enriched among the up-regulated genes, suggesting that loss of let-7 may be impacting downstream pathways. Our results reveal that DICER1 hotspot mutations are implicated in common malignancies and may constitute a unique oncogenic pathway.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/genética , MicroARNs/biosíntesis , Mutación , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/deficiencia , Análisis Mutacional de ADN , Neoplasias Endometriales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones Noqueados , MicroARNs/genética , Fenotipo , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasa III/deficiencia , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Germline truncating mutations in DICER1, an endoribonuclease in the RNase III family that is essential for processing microRNAs, have been observed in families with the pleuropulmonary blastoma-family tumor and dysplasia syndrome. Mutation carriers are at risk for nonepithelial ovarian tumors, notably sex cord-stromal tumors. METHODS: We sequenced the whole transcriptomes or exomes of 14 nonepithelial ovarian tumors and noted closely clustered mutations in the region of DICER1 encoding the RNase IIIb domain of DICER1 in four samples. We then sequenced this region of DICER1 in additional ovarian tumors and in certain other tumors and queried the effect of the mutations on the enzymatic activity of DICER1 using in vitro RNA cleavage assays. RESULTS: DICER1 mutations in the RNase IIIb domain were found in 30 of 102 nonepithelial ovarian tumors (29%), predominantly in Sertoli-Leydig cell tumors (26 of 43, or 60%), including 4 tumors with additional germline DICER1 mutations. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for microRNA interaction and cleavage, and were somatic in all 16 samples in which germline DNA was available for testing. We also detected mutations in 1 of 14 nonseminomatous testicular germ-cell tumors, in 2 of 5 embryonal rhabdomyosarcomas, and in 1 of 266 epithelial ovarian and endometrial carcinomas. The mutant DICER1 proteins had reduced RNase IIIb activity but retained RNase IIIa activity. CONCLUSIONS: Somatic missense mutations affecting the RNase IIIb domain of DICER1 are common in nonepithelial ovarian tumors. These mutations do not obliterate DICER1 function but alter it in specific cell types, a novel mechanism through which perturbation of microRNA processing may be oncogenic. (Funded by the Terry Fox Research Institute and others.).
Asunto(s)
ARN Helicasas DEAD-box/genética , Mutación Missense , Neoplasias Ováricas/genética , Ribonucleasa III/genética , Tumor de Células de Sertoli-Leydig/genética , Carcinosarcoma/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Mutación de Línea Germinal , Humanos , MicroARNs/metabolismo , Neoplasias de Células Germinales y Embrionarias/genética , Rabdomiosarcoma/genética , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Adenocarcinoma Mucinoso/sangre , Cistadenocarcinoma Seroso/sangre , Glicoproteínas/sangre , Neoplasias Ováricas/sangre , Adenocarcinoma Mucinoso/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Antígeno Ca-125/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Cistadenocarcinoma Seroso/metabolismo , Femenino , Glicoproteínas/metabolismo , Humanos , Menopausia/sangre , Persona de Mediana Edad , Neoplasias Ováricas/metabolismo , Adulto JovenRESUMEN
E-cadherin expression is unusually regulated in epithelial ovarian carcinoma. It is not expressed in poorly cohesive ovarian surface epithelial (OSE) target cells, but is expressed in cohesive pre-malignant lesions and in highly cohesive, well-differentiated tumors where it is membrane associated, presumably in adherens junctions. E-cadherin expression is subsequently suppressed, or its function is disrupted, in late-stage invasive tumors. Here, we observed that increased E-cadherin expression in ovarian carcinoma cells was associated with increased E-cadherin promoter activity, increased adherens junction formation, decreased beta-catenin signaling-dependent LEF-1 activity, and the generation of cohesive spheroids in basement membrane gel culture. Forced expression of wild-type E-cadherin in immortalized OSE cells initiated adherens junction formation, decreased LEF-1 activity, decreased the mesenchymal migration that is a characteristic of OSE cells that have been maintained in monolayer culture, and induced the formation of cohesive spheroids in basement membrane gels. Conversely, forced expression of a dominant-negative E-cadherin mutant in ovarian carcinoma cells disrupted adherens junctions, increased mesenchymal cell migration, and prevented spheroidal morphogenesis without altering LEF-1 signaling. Therefore, in addition to suppressing late-stage tumor progression, E-cadherin-mediated adherens junctions may also contribute to the initial emergence of a cohesive morphogenic phenotype that is a hallmark of differentiated epithelial ovarian carcinoma.
Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Carcinoma/patología , Neoplasias Ováricas/patología , Carcinoma/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/citología , Femenino , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Neoplasias Ováricas/metabolismoRESUMEN
OBJECTIVE: Ovarian surface epithelium (OSE), the precursor of the epithelial ovarian carcinomas, has limited growth potential in culture. Epidermal growth factor+hydrocortisone (EGF+HC) enhances its growth but induces epitheliomesenchymal transition (EMT). This study was undertaken to define the effects of EGF+HC and their reversibility, to optimize growth-promoting media, and to relate OSE phenotypes in vitro to physiologic states in vivo. METHODS: OSE was cultured in media 199/MDCB105 or EBM (Clonetics) with 2% or 10% fetal bovine serum with or without 10 ng/mL EGF, 1.0 microg/mL HC, and 1.0 microg/mL bovine brain extract. Growth rates and growth potentials (population doublings [PD] to senescence) were defined, and growth patterns and expression of keratin and collagen types III and IV were compared with the ovarian cancer cell lines OVCAR3 and SKOV3. RESULTS: EGF+HC increased growth potentials from 12-14 PD to 40-42 PD and reduced PD time from 53 hours to 20 hours. Without EGF+HC, OSE cells remained uniformly epithelial. EGF+HC induced EMT (mesenchymal shapes, reduced keratin, and production of collagenous extracellular matrix), but the EMT response varied greatly among OSE from different women. EMT was reversed over 1-2 weeks by subculture into EGF+HC-free medium in passage 1, but inconsistently thereafter. EGF+HC had no effect on the differentiation of ovarian carcinoma lines. CONCLUSION: The phenotype of intact OSE in vivo is most closely reproduced in media without EGF+HC. EGF+HC enhances growth but initiates EMT, which likely mimics a repair response. Variations in EGF+HC-induced phenotypes point to the existence of OSE subpopulations with differing responsiveness to growth factors or steroids, which may relate to their susceptibility to malignant transformation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Hidrocortisona/farmacología , Ovario/citología , Adulto , Células Cultivadas , Colágeno Tipo III/biosíntesis , Colágeno Tipo III/genética , Colágeno Tipo IV/biosíntesis , Colágeno Tipo IV/genética , Células Epiteliales/citología , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Queratinas/biosíntesis , Queratinas/genética , Microscopía Fluorescente , Fenotipo , Células Tumorales CultivadasRESUMEN
BACKGROUND: A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers. METHODOLOGY/PRINCIPAL FINDINGS: Herein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative. CONCLUSIONS/SIGNIFICANCE: In addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.
Asunto(s)
Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Tumor de Células de la Granulosa/genética , Mutación , Alelos , Línea Celular Tumoral , Cisteína/genética , Citogenética , Femenino , Proteína Forkhead Box L2 , Genómica , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Neoplasias Ováricas/genética , Distribución Tisular , Triptófano/genéticaRESUMEN
The transcription factor ZNF217 is often amplified in ovarian cancer, but its role in neoplastic progression is unknown. We introduced ZNF217-HA by adenoviral and retroviral infection into normal human ovarian surface epithelial cells (OSE), i.e., the source of ovarian cancer, and into SV40 Tag/tag expressing, p53/pRB-deficient OSE with extended but finite life spans (IOSE). In OSE, ZNF217-HA reduced cell-substratum adhesion and accelerated loss of senescent cells, but caused no obvious proneoplastic changes. In contrast, ZNF217-HA transduction into IOSE yielded two permanent lines, I-80RZ and I-144RZ, which exhibited telomerase activity, stable telomere lengths, anchorage independence and reduced serum dependence, but were not tumorigenic in SCID mice. This immortalization required short-term EGF treatment near the time of crisis. The permanent lines were EGF-independent, but ZNF217-dependent since siRNA to ZNF217 inhibited anchorage independence and arrested growth. Array CGH revealed genomic changes resembling those of ovarian carcinomas, such as amplicons at 3q and 20q, and deletions at 4q and 18, associated with underexpressed annexin A10, N-cadherin, desmocollin 3 and PAI-2, which have been reported as tumor suppressors. The lines overexpressed EEF1A2, SMARA3 and STAT1 and underexpressed other oncogenes, tumor suppressors and extracellular matrix/adhesion genes. The results implicate ZNF217 as an ovarian oncogene, which is detrimental to senescing normal OSE cells but contributes to neoplastic progression in OSE with inactivated p53/RB. The resemblance of the genomic changes in the ZNF217-overexpressing lines to ovarian carcinomas provides a unique model to investigate interrelationships between these changes and ovarian neoplastic phenotypes.
Asunto(s)
Neoplasias Ováricas/etiología , Transactivadores/fisiología , Adenoviridae/genética , Células Cultivadas , Progresión de la Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Femenino , Humanos , Hidrocortisona/farmacología , Hibridación de Ácido Nucleico , Neoplasias Ováricas/patología , Fenotipo , Transactivadores/genéticaRESUMEN
The ovarian surface epithelium (OSE) is the precursor of common epithelial ovarian carcinomas. In the present study, we examined the molecular mechanisms and possible physiological basis for the propensity of OSE cells to undergo epithelio-mesenchymal transition (EMT) in response to environmental influences. We hypothesized that EMT may be a homeostatic mechanism that permits displaced OSE to assume a stromal phenotype within the ovarian cortex. We report that EGF in conjunction with hydrocortisone is the EMT-inducing factor of OSE as shown by changes to a fibroblast-like morphology and growth pattern. EGF increased cell motility, enhanced the activities of secreted pro-matrix metalloproteinase (MMP)-2 and -9, and enhanced expression and activation of Erk and integrin-linked kinase (ILK). Increased ILK expression correlated with the activation of PKB/Akt, the phosphorylation of GSK-3beta, and the increased expression of cyclin E and cdk2 kinase. EGF withdrawal resulted in a more epithelial morphology and reversal of the EGF-induced activation of signaling pathways and pro-MMP activity. In contrast, treatment of EGF-treated cells with specific inhibitors of phosphatidylinositol 3-kinase, Mek, or ILK inhibited the inhibitor-specific pathways. The inhibitors caused suppression of EGF-induced migration and pro-MMP-2/-9 activities but did not lead to any change in EGF-induced mesenchymal morphology. ILK small interfering RNA inhibited Akt phosphorylation and reduced pro-MMP-2/-9 activities but had no effect on Erk activation or cell morphology. These results indicate that the EGF-induced morphological and functional changes in OSE cells are controlled by distinct signaling mechanisms working in concert. EMT of OSE cells displaced by ovulation likely permits their survival and integration with a fibroblast-like identity within the stroma. Failure to do so may lead to the formation of epithelium-derived inclusion cysts, which are known preferential sites of malignant transformation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Epitelio/metabolismo , Mesodermo/metabolismo , Ovario/fisiología , Adulto , Western Blotting , Movimiento Celular , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , Ovario/patología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño , Transducción de Señal/fisiologíaRESUMEN
A hybrid cell line, IOSE-Ov29, was created through fusion of cells from the human ovarian adenocarcinoma line OVCAR3 and the non-tumorigenic SV40 Tag-transfected human ovarian surface epithelial line IOSE-29. OVCAR3 cells exhibit a differentiated epithelial phenotype, whereas line IOSE-29 expresses mesenchymal characteristics that were acquired in culture by epithelio-mesenchymal transition. Microsatellite analysis, comparative genomic hybridization (CGH), and MFISH showed the genotype of the IOSE-Ov29 cells to contain components of both parent cell lines, but to be predominantly OVCAR3 derived. IOSE-Ov29 resembled OVCAR3 and differed from IOSE-29 as shown by its unlimited life span, tumorigenicity, epithelial morphology, keratin, occludin, E-cadherin and CA125 expression, increased expression of kinases of the PI3K pathway, and loss of cGMP-dependent protein kinase expression. IOSE-29-derived properties included SV40 Tag expression, growth inhibition by activin, collagen type III secretion, increased adhesion and spreading on tissue culture plastic, and increased growth rate. Proliferation of all three lines was stimulated by FSH and ATP and inhibited by GnRH I and GnRH II. Interestingly, IOSE-Ov29 was more anchorage independent than either parent line and was the only line that invaded Matrigel in Boyden chambers and formed invasive branches in collagen gels. The results indicate that IOSE-Ov29 is an IOSE-29/OVCAR3 hybrid, which differs from both parent lines genetically and phenotypically. Unexpectedly, fusion with the non-tumorigenic IOSE-29 cells enhanced malignancy-associated characteristics of OVCAR3, presumably as a result of the expression of IOSE-29-derived mesenchymal properties that are usually acquired by carcinoma cells through epithelio-mesenchymal transition during metastatic progression.