RESUMO
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.
Assuntos
Biomarcadores Tumorais/genética , Proliferação de Células/genética , Ácido Graxo Sintase Tipo I/genética , Neoplasias Ovarianas/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
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.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário , Epitélio/patologia , Feminino , HumanosRESUMO
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.
Assuntos
Carcinoma/metabolismo , Transformação Celular Neoplásica , Tubas Uterinas/patologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/patologia , Células-Tronco/patologia , Adolescente , Adulto , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Biomarcadores/metabolismo , Carcinoma/patologia , Carcinoma Epitelial do Ovário , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/metabolismo , Epitélio/patologia , Tubas Uterinas/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteína Homeobox Nanog , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Retinal Desidrogenase/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Carcinoma/patologia , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Neoplasias Ovarianas/patologia , Adulto , Antígenos de Neoplasias/metabolismo , Caderinas/metabolismo , Calbindina 2 , Carcinoma/metabolismo , Moléculas de Adesão Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Molécula de Adesão da Célula Epitelial , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Feminino , Proteínas Ligadas por GPI/metabolismo , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Mesotelina , Mucina-1/metabolismo , Neoplasias Ovarianas/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
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.
Assuntos
Aorta/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio/biossíntese , Animais , Feminino , Humanos , MasculinoRESUMO
High-density array comparative genomic hybridization (CGH) showed amplification of chromosome 1q22 centered on the RAB25 small GTPase, which is implicated in apical vesicle trafficking, in approximately half of ovarian and breast cancers. RAB25 mRNA levels were selectively increased in stage III and IV serous epithelial ovarian cancers compared to other genes within the amplified region, implicating RAB25 as a driving event in the development of the amplicon. Increased DNA copy number or RNA level of RAB25 was associated with markedly decreased disease-free survival or overall survival in ovarian and breast cancers, respectively. Forced expression of RAB25 markedly increased anchorage-dependent and anchorage-independent cell proliferation, prevented apoptosis and anoikis, including that induced by chemotherapy, and increased aggressiveness of cancer cells in vivo. The inhibition of apoptosis was associated with a decrease in expression of the proapoptotic molecules, BAK and BAX, and activation of the antiapoptotic phosphatidylinositol 3 kinase (PI3K) and AKT pathway, providing potential mechanisms for the effects of RAB25 on tumor aggressiveness. Overall, these studies implicate RAB25, and thus the RAB family of small G proteins, in aggressiveness of epithelial cancers.
Assuntos
Neoplasias da Mama/genética , Regulação da Expressão Gênica , Neoplasias Ovarianas/genética , RNA Mensageiro/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Cromossomos Humanos Par 1/genética , Feminino , Humanos , Immunoblotting , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteínas rab de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Adenocarcinoma Mucinoso/sangue , Cistadenocarcinoma Seroso/sangue , Glicoproteínas/sangue , Neoplasias Ovarianas/sangue , Adenocarcinoma Mucinoso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Antígeno Ca-125/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Cistadenocarcinoma Seroso/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Menopausa/sangue , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/fisiopatologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/fisiopatologia , Ovário/citologia , Ovário/metabolismo , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Carcinoma/patologia , Neoplasias Ovarianas/patologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/citologia , Feminino , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Neoplasias Ovarianas/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) expression correlates with microvessel density, stage, malignant ascites, metastasis, and survival in ovarian cancer. By transducing VEGF165 into a nontumorigenic rat ovarian surface epithelial cell line (ROSE199), we investigated the direct effect of an angiogenic phenotype on tumor development. The neu oncogene, which is overexpressed in >30% of ovarian cancers, was used in comparison. Neu-transfected ROSE199 cells showed phenotypic characteristics of transformation in vitro with an abundance of focus-forming units in monolayer cultures and anchorage-independent growth in soft agar. In contrast, VEGF-secreting ROSE199 cells (VR) retained normal morphology and in vitro growth characteristics (e.g., proliferation rate) compared with parental ROSE199 cells. Interestingly, injection of VR cells into athymic mice formed malignant ascites in 100% of the animals when injected into the peritoneum and developed vascularized tumors in 85% of the mice when injected s.c. Furthermore, blocking VEGF-mediated signaling by the Flk-1/KDR receptor kinase inhibitor SU5416 significantly inhibited the growth of VR tumors. To validate that the proangiogenic switch is responsible for tumor development, the angiogenic phenotype was balanced by the inducible coexpression of endostatin under the control of Tet-activated promoter. Coexpression of endostatin along with VEGF reversed the tumorigenic phenotype of VR cells. These studies show that alterations in the angiogenic characteristics of ovarian surface epithelium may play an important role in the etiology of ovarian cancer, and that inhibition of angiogenesis can be effective in the treatment of epithelial ovarian cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Inibidores da Angiogênese/farmacologia , Animais , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/genética , Endostatinas/biossíntese , Endostatinas/genética , Feminino , Indóis/farmacologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Pirróis/farmacologia , Ratos , Transfecção , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Ovarianas/genética , Fator 1 de Elongação de Peptídeos/genética , Lesões Pré-Cancerosas/genética , Transativadores/genética , Apoptose/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Dosagem de Genes , Expressão Gênica , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fator 1 de Elongação de Peptídeos/biossíntese , Fator 1 de Elongação de Peptídeos/sangue , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , RNA Interferente Pequeno/genética , Transativadores/biossínteseRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Ovário/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Feminino , Fator 9 de Diferenciação de Crescimento/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Proteína Meis1 , Proteínas de Neoplasias/genética , Ovário/imunologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Neoplásica/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adenoviridae/genética , Adulto , Caderinas/genética , Movimento Celular/genética , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação para Baixo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes do Retinoblastoma , Genes p53 , Vetores Genéticos/genética , Humanos , Mesoderma/patologia , Pessoa de Meia-Idade , Peptídeo Hidrolases/metabolismo , Proteína Fosfatase 2/genética , Transfecção , Células Tumorais CultivadasRESUMO
Despite evidence that gonadotropins may facilitate peritoneal metastasis of ovarian cancer by increasing cell adhesion, the action and molecular mechanism of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in ovarian cancer invasion is not well characterized. In the present study, we investigated the effects of FSH and LH on the invasive activity and the expression of metastasis-related proteinases in human epithelial ovarian cancer by Western blot, zymography, reverse transcription-PCR (RT-PCR), ELISA, and Boyden chamber assay. Treatment with FSH or LH (10, 100, or 1,000 ng/mL) significantly increased the invasion of ovarian cancer cell lines, including BG-1, CaOV-3, and SKOV-3 cells but not OVCAR-3 cells. In addition, treatment of SKOV-3 cells with FSH or LH (100 or 1,000 ng/mL) enhanced the expression and activation of matrix metalloproteinases (MMP-2 and MMP-9) as shown by RT-PCR, gelatin zymography, and ELISA. Pretreatment with [(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (10 micromol/L), a total MMP inhibitor, and 3-(4-phenoxyphenylsulfonyl)-propylthiirane (20 micromol/L), a specific gelatinase inhibitor, neutralized the proinvasive effect of gonadotropins in SKOV-3 cells. In addition, the secretion of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and plasminogen activator inhibitor-1 was significantly decreased by FSH and LH (100 or 1,000 ng/mL). We further showed that gonadotropins induced an increase in SKOV-3 invasiveness via the activation of protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Taken together, these results suggest that gonadotropins may contribute to ovarian cancer metastasis via activation of proteolysis and increase in invasion through the PKA and PI3K pathways.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Neoplasias Ovarianas/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Feminino , Humanos , Isoquinolinas/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Morfolinas/farmacologia , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologia , Sulfonamidas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Transforming growth factor-beta superfamily has been implicated in tumorigenesis. We have recently shown that Nodal, a member of transforming growth factor-beta superfamily, and its receptor, activin receptor-like kinase 7 (ALK7), inhibit proliferation and induce apoptosis in human epithelial ovarian cancer cell lines. In this study, we further investigated the cellular mechanisms underlying the apoptotic action of ALK7 using an immortalized ovarian surface epithelial cell line, IOSE397, and an epithelial ovarian cancer cell line, OV2008. Infection of these cells with an adenoviral construct carrying constitutively active ALK7 (Ad-ALK7-ca) potently induced cell death; all cells died after 3 and 5 days of Ad-ALK7-ca infection in IOSE397 and OV2008 cells, respectively. ALK7-ca induced the expression of proapoptotic factor Bax but suppressed the expression of antiapoptotic factors Bcl-2, Bcl-XL, and Xiap. Silencing of Bax by small interfering RNA in IOSE397 cells significantly reduced ALK7-ca-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay but partially blocked ALK7-ca-induced caspase-3 activation and did not affect the down-regulation of Xiap by ALK7-ca. Dominant-negative Smad2, Smad3, and Smad4 blocked ALK7-ca-regulated Xiap and Bax expression and caspase-3 activation. Thus, ALK7-induced apoptosis is at least in part through two Smad-dependent pathways, Bax/Bcl-2 and Xiap.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Apoptose , Carcinoma/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/metabolismo , Receptores de Ativinas Tipo I/genética , Carcinoma/genética , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Neoplasias Ovarianas/genética , Ovário/citologia , Ovário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Transfecção , Regulação para Cima , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genéticaRESUMO
The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.
Assuntos
Técnicas Citológicas/métodos , Células Tumorais Cultivadas/citologia , Contagem de Células , Quimiotaxia , Filtros MicroporosRESUMO
Elevated levels of the bioactive lipid lysophosphatidic acid (LPA) are detectable in the majority of patients with both early- and late-stage ovarian cancer, suggesting that LPA promotes early events in ovarian carcinoma dissemination. LPA contributes to the development, progression, and metastasis of ovarian cancer in part by inducing the expression of genes that contribute to proliferation, survival, or invasion, including cyclooxgenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2). We have previously shown that LPA promotes proMMP-2 activation and MMP-2-dependent migration and invasion in ovarian cancer cells. The purpose of the current study was to determine whether the effect of LPA on acquisition of the metastatic phenotype in ovarian cancer cells is mediated via a COX-2-dependent mechanism. Immunohistochemical analysis of 173 ovarian tumors showed strong COX-2 immunoreactivity in 63% of tumor specimens, including 50% of borderline tumors. LPA increased COX-2 protein expression in a time- and concentration-dependent manner in two of three immortalized borderline ovarian epithelial cells as well as in four of six ovarian cancer cell lines. This was accomplished by both activation of the Edg/LPA receptor and LPA-mediated transactivation of the epidermal growth factor receptor, which increased COX-2 expression via the Ras/mitogen-activated protein kinase pathway. COX-2 also played a role in LPA-induced invasion and migration, as treatment with the COX-2 specific inhibitor NS-398 reduced LPA-induced proMMP-2 protein expression and activation and blocked MMP-dependent motility and invasive activity. These data show that COX-2 functions as a downstream mediator of LPA to potentiate aggressive cellular behavior.
Assuntos
Lisofosfolipídeos/farmacologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Movimento Celular/genética , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Indução Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Receptores ErbB/biossíntese , Receptores ErbB/genética , Feminino , Gelatinases/metabolismo , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana , Metaloendopeptidases/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Receptores de Lisoesfingolipídeo/fisiologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Caspase-1 plays a key role in the processing of cytokines and in the apoptosis of neurons and macrophages. Whether it also causes apoptosis of cancer cells has been unclear. In this study, we screened an array of apoptosis-related proteins in ovarian carcinoma cell lines and their tissue of origin, ovarian surface epithelium (OSE). Caspase-1alpha protein was abundant in OSE and in nontumorigenic OSE with extended but limited life spans (immortalized OSE), but was reduced in the cancer lines A2780 and OVCAR10. By Western blot and immunofluorescence, caspase-1alpha levels were greatly reduced in six of eight ovarian carcinoma lines compared with OSE. By real-time reverse transcription-PCR, steady-state transcripts of the CASP1 gene were proportional to protein levels. Caspase-1alpha overexpression caused significant apoptosis, but overexpression of a caspase-1alpha mutant without catalytic activity did not, confirming that the effect was caspase-1alpha-specific. Immunofluorescence of caspase-1alpha and terminal nucleotidyl transferase-mediated dUTP-X nick end labeling colocalization clearly established a link between apoptosis and caspase-1alpha expression. Caspase-9 and caspase-3 were activated in caspase-1alpha overexpressing A2780 cells, suggesting involvement of an intrinsic apoptotic pathway. Caspase-1alpha overexpression did not change the apoptotic effect of cisplatin in A2780 and OVCAR10 cells, suggesting that this agent activates a different pathway. Immunohistochemically, caspase-1 was lower in ovarian serous carcinomas than in OSE. Our study indicates, for the first time, that caspase-1alpha is proapoptotic in ovarian cancer cells, and raises the possibility that its down-regulation is one of the mechanisms which increase resistance to apoptosis in cancer cells.