RESUMO
BACKGROUND: The heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in their biological features, invasiveness, and response to chemotherapy, but the transcriptional regulators causing their differences remain nebulous. METHODS: In this study, we compared high-grade serous ovarian cancers (HGSOCs) to low malignant potential or serous borderline tumors (SBTs). Our aim was to discover new regulatory factors causing distinct biological properties of HGSOCs and SBTs. RESULTS: In a discovery dataset, we identified 11 differentially expressed genes (DEGs) between SBTs and HGSOCs. Their expression correctly classified 95% of 267 validation samples. Two of the DEGs, TMEM30B and TSPAN1, were significantly associated with worse overall survival in patients with HGSOC. We also identified 17 DEGs that distinguished stage II vs. III HGSOC. In these two DEG promoter sets, we identified significant enrichment of predicted transcription factor binding sites, including those of RARA, FOXF1, BHLHE41, and PITX1. Using published ChIP-seq data acquired from multiple non-ovarian cell types, we showed additional regulatory factors, including AP2-gamma/TFAP2C, FOXA1, and BHLHE40, bound at the majority of DEG promoters. Several of the factors are known to cooperate with and predict the presence of nuclear hormone receptor estrogen receptor alpha (ER-alpha). We experimentally confirmed ER-alpha and PITX1 presence at the DEGs by performing ChIP-seq analysis using the ovarian cancer cell line PEO4. Finally, RNA-seq analysis identified recurrent gene fusion events in our EOC tumor set. Some of these fusions were significantly associated with survival in HGSOC patients; however, the fusion genes are not regulated by the transcription factors identified for the DEGs. CONCLUSIONS: These data implicate an estrogen-responsive regulatory network in the differential gene expression between ovarian cancer subtypes and stages, which includes PITX1. Importantly, the transcription factors associated with our DEG promoters are known to form the MegaTrans complex in breast cancer. This is the first study to implicate the MegaTrans complex in contributing to the distinct biological trajectories of malignant and indolent ovarian cancer subtypes.
Assuntos
Carcinoma Epitelial do Ovário/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Fatores de Transcrição Box Pareados/metabolismo , Carcinoma Epitelial do Ovário/patologia , Feminino , HumanosRESUMO
OBJECTIVE: Our objective was to determine the rate of BRCA1/2 deficiency in platinum-sensitive and platinum-resistant tumors from a cohort of unselect patients with advanced epithelial ovarian cancer (EOH). METHODS: BRCA1/2 mutation analysis was performed in 29 patients with platinum-sensitive EOC and 24 patients with platinum-resistant disease. Germline DNA was analyzed in mutation carriers when normal tissue was available. BRCA expression was ascertained by quantitative rt-PCR. Associations between BRCA mutation status and expression levels and parameters of platinum response were analyzed. RESULTS: Fifteen of 53 (28.3%) EOC tumors had BRCA1/2 mutations. Twelve mutations were in BRCA1, while 3 involved BRCA2. Of the 12 mutation-carriers with normal tissue available for DNA analyses, 33.3% of the mutations were found to be somatic. Three mutations were novel. The majority of BRCA mutations (73%) were identified in patients with platinum-sensitive disease. In total, 38% of platinum-sensitive tumors were found to have a BRCA mutation, compared to 17% of the platinum-resistant patients. A statistical trend toward platinum-sensitive disease was seen in BRCA mutation carriers (p=0.079). Nineteen (36%) study patients had some form of BRCA deficiency, and these patients were less likely to have platinum-resistant tumors (OR=0.29; p value=0.048). CONCLUSIONS: BRCA mutations occurred more frequently in platinum-sensitive EOC than platinum-resistant disease. The high overall frequency of BRCA deficiency in EOC underscores the importance of tumor profiling as therapies targeting the DNA repair pathway are being investigated.
Assuntos
Genes BRCA1 , Genes BRCA2 , Mutação em Linhagem Germinativa , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Compostos Organoplatínicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Adulto , Idoso , Proteína BRCA1/biossíntese , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/biossíntese , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
OBJECTIVE: Approximately 20% of patients receiving platinum-based chemotherapy for epithelial ovarian cancer (EOC) are refractory or develop early recurrence. Identifying these patients early could reduce treatment-associated morbidity and allow quicker transfer to more effective therapies. Much attention has focused on ERCC1 as a potential predictor of response to therapy because of its essential role in the repair of platinum-induced DNA damage. The purpose of this study was to accurately measure protein levels of ERCC1 and its essential binding partner XPF from patients with EOC treated with platinum-based therapy and determine if protein levels correlate with mRNA levels, patient genotypes or clinical outcomes. METHODS: ERCC1 and XPF mRNA and protein levels were measured in frozen EOC specimens from 41 patients receiving intraperitoneal platinum-based chemotherapy using reverse transcription polymerase chain reaction and western blots. Genotypes of common nucleotide polymorphisms were also analyzed. Patient outcomes included progression free (PFS) and overall survival (OS). RESULTS: Expression of ERCC1 and XPF were tightly correlated with one another at both the mRNA and protein level. However, the mRNA and protein levels of ERCC1 were not positively correlated. Likewise, none of the SNPs analyzed correlated with ERCC1 or XPF protein levels. There was an inverse correlation between mRNA levels and patient outcomes. CONCLUSION: Neither genotype nor mRNA levels are predictive of protein expression. Despite this, low ERCC1 mRNA significantly correlated with improved PFS and OS.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Antineoplásicos/uso terapêutico , Carcinoma Epitelial do Ovário , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Feminino , Genótipo , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Estatísticas não ParamétricasRESUMO
PURPOSE: Efflux transporters of the ATP-binding cassette (ABC) family are major determinants of chemoresistance in tumor cells. This study examined associations between functional variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes in patients with epithelial ovarian/primary peritoneal cancer (EOC/PPC) following platinum and taxane-based chemotherapy. METHODS: Sequenom iPLEXTMGOLD Assay and MALDI-TOF platform were used to genotype the non-synonymous G2677T/A (rs2032582; encoding Ala893Ser/Thr) and synonymous C3435T (rs1045642; encoding Ile1145Ile) variants in ABCB1, the non-synonymous G1249A variant in ABCC2 (rs2273697; encoding Val417Ile), and the non-synonymous C421A variant in ABCG2 (rs2231142; encoding Q141K, Gln141Lys) in normal DNA from up to 511 women in Gynecologic Oncology Group (GOG) phase III trials, GOG-172 or GOG-182. Progression-free survival (PFS) and overall survival (OS) were analyzed in relation to genetic polymorphisms using Kaplan-Meier and Cox proportional hazards model. RESULTS: The C421A variant (CA+AA versus CC) in ABCG2 was associated with a 6-month longer median PFS (22.7 versus 16.8 months, p=0.041). In multivariate analysis, patients with variant genotypes were at a reduced risk of disease progression (hazard ratio [HR]=0.75, 95% confidence interval [CI]=0.59-0.96, p=0.022). The association between C421A and OS was not statistically significant (HR=0.88, 95% CI=0.67-1.15, p=0.356). None of the other variants measured in either ABCB1 or ABCC2 was associated with PFS or OS. CONCLUSION: The C421A variant in ABCG2, previously shown to be associated with enhanced protein degradation and drug sensitivity, was associated with longer PFS in advanced stage EOC/PPC patents treated with platinum+taxane-based chemotherapy. This finding requires further validation.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adulto , Idoso , Idoso de 80 Anos ou mais , Carboplatina/administração & dosagem , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Doxorrubicina/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Paclitaxel/administração & dosagem , Polimorfismo Genético , Topotecan/administração & dosagem , Adulto Jovem , GencitabinaRESUMO
OBJECTIVE: This study evaluated common polymorphisms in excision repair cross-complementation group 1 (ERCC1) involved in repair of platinum-induced DNA damage in advanced-stage, epithelial ovarian/peritoneal/tubal cancer (EOC/PPC/FTC) patients treated with intravenous carboplatin- and paclitaxel-based chemotherapy. METHODS: Pyrosequencing was performed to examine single nucleotide polymorphisms (SNPs) in codon 118 and C8092A in ERCC1 in leukocyte DNA from the Gynecologic Oncology Group phase III protocol, GOG-182. Kaplan-Meier method and adjusted Cox regression modeling were used to examine associations between ERCC1 polymorphisms and progression-free survival (PFS) and overall survival (OS). RESULTS: The genotype distribution at codon 118 (n=278) in ERCC1 for CC, CT, and TT was 23%, 45% and 32%, and the median OS was 32, 47 and 43 months, respectively. Patients with the CT+TT versus CC genotype in codon 118 in ERCC1 were at a reduced risk of death (hazard ratio [HR]=0.68, 95% confidence interval [CI]=0.49-0.95, p=0.025). The genotype distribution for C8092A in ERCC1 (N=280) was 50%, 42% and 8%, and the median OS was 45, 40 or 30 months for CC, CA and AA, respectively. Women with the CA+AA versus CC genotype in C8092A in ERCC1 had a trend suggesting an increased risk of death (HR=1.29, 95% CI=0.97-1.72, p=0.077). CONCLUSIONS: The polymorphism in codon 118 in the DNA repair gene ERCC1 was an independent predictor for better survival in EOC/PPC/FTC patients treated with intravenous carboplatin- and paclitaxel-based chemotherapy. The relationship between the C8092A polymorphisms in ERCC1 and survival was modest with an effect size that was not always statistically significant.
Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Progressão da Doença , Doxorrubicina/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/patologia , Polimorfismo de Nucleotídeo Único , Topotecan/administração & dosagem , GencitabinaRESUMO
Smoking is associated with multiple adverse pregnancy outcomes, including fetal growth restriction. The objective of this study was to determine whether cigarette smoke exposure during pregnancy in a mouse model affects the functional properties of maternal uterine, mesenteric, and renal arteries as a possible mechanism for growth restriction. C57Bl/CJ mice were exposed to whole body sidestream smoke for 4 h/day. Smoke particle exposure was increased from day 4 of gestation until late pregnancy (day 16-19), with mean total suspended particle levels of 63 mg/m(3), representative of moderate-to-heavy smoking in humans. Uterine, mesenteric, and renal arteries from late-pregnant and virgin mice were isolated and studied in a pressure-arteriograph system (n = 23). Plasma cotinine was measured by ELISA. Fetal weights were significantly reduced in smoke-exposed compared with control fetuses (0.88 +/- 0.1 vs. 1.0 +/- 0.08 g, P < 0.02), while litter sizes were not different. Endothelium-mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. This difference was not apparent in isolated renal arteries from pregnant mice exposed to cigarette smoke; however, relaxation was significantly reduced in renal arteries from smoke-exposed virgin mice. In conclusion, we found that passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in pregnant mice. Functional maternal vascular perturbations during pregnancy, specifically impaired peripheral and uterine vasodilation, may contribute to a mechanism by which smoking results in fetal growth restriction.
Assuntos
Retardo do Crescimento Fetal/etiologia , Complicações Cardiovasculares na Gravidez/etiologia , Fumar/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Pressão Sanguínea/fisiologia , Peso Corporal/fisiologia , Cotinina/sangue , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/fisiologia , Gravidez , Complicações Cardiovasculares na Gravidez/fisiopatologia , Artéria Renal/fisiologia , Artéria Uterina/fisiologia , Vasodilatação/fisiologiaRESUMO
OBJECTIVE: Current firstline chemotherapy for ovarian cancer consists of carboplatin combined with either paclitaxel or docetaxel. Disposition of carboplatin is determined by renal clearance, while the taxanes are metabolized by cytochrome P450 (CYP450) enzymes. Although the majority of taxane metabolism occurs in the liver, recent data have shown that some solid tumors express CYP450 enzymes in the tumors themselves. The objective of this study was to determine whether ovarian tumors express genes regulating cellular efflux and subsequent metabolism, and whether any clinico-pathologic features correlated with expression. METHODS: Gene expression of CYP2C8, CYP3A4/A5 and the ABC transporter ABCB1 was determined in 56 primary epithelial ovarian tumors. Cells were grown from seven different tumors and exposed ex vivo to paclitaxel (PAC) and docetaxel (DOC) for up to 24 h. PAC and DOC concentrations were measured in the media by an LC-MS assay. RESULTS: Results from this analysis demonstrate that ovarian cancer cells do express functional taxane-metabolizing enzymes. Such expression appeared to enhance the ability of cancer cells to metabolize DOC. Specifically, the PK of DOC was correlated with the ratio of CYP4A5 to ABCB1 gene expression, thus representing a novel mechanism of chemotherapy resistance. There was no relationship between PAC PK parameters and gene expression. CONCLUSIONS: Knowledge of inter-individual variation in CYP450 enzyme and ABC transporter tumor expression and activity may influence the individualization of chemotherapy, by avoiding agents that are rapidly metabolized and selecting agents that are not.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias Ovarianas/enzimologia , Paclitaxel/farmacocinética , Taxoides/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Docetaxel , Feminino , Expressão Gênica , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Células Tumorais CultivadasRESUMO
OBJECTIVE: The objectives of this study were to determine whether there are distinct gene expression profiles for endometrial and ovarian endometrioid carcinomas and to create a statistical model using these profiles to predict their organ of origin. METHODS: Expression profiles of seven stage I, grades 1 and 2, endometrioid endometrial carcinomas and seven stage I ovarian endometrioid carcinomas were analyzed as the training set. The test set included seven advanced endometrial carcinomas and nine dual primary endometrial and ovarian primary tumors of endometrioid histology. Unsupervised hierarchical clustering, multidimensional scaling (MDS) and predictive analysis of microarrays (PAM) were used to analyze the data. RESULTS: We identified 163 differentially expressed (DE) genes between endometrial and ovarian tumors. Both unsupervised hierarchical clustering and multidimensional scaling showed clear separation of the two groups. Pathway analysis of the 163 DE genes revealed significant biological differences between the two groups, which included metabolic and biosynthetic pathways. Further classification analysis on the training data using predictive analysis of microarray generated a 119-gene predictive model with 100% cross-validation accuracy. When the 119-gene model was applied to the test set of 16 samples, we found concordance in 11/16 samples and discordance in 5/16 samples with the pathologic classification. CONCLUSION: We conclude that, although similar in histologic appearance, endometrioid carcinomas of the ovary and endometrium have distinct gene expression patterns. A larger test set is needed to prospectively evaluate this prediction model further.
Assuntos
Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adulto , Idoso , Carcinoma Endometrioide/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Neoplasias do Endométrio/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/metabolismo , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Ovarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood. METHODS: To determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts. RESULTS: We identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells. CONCLUSION: These findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.
Assuntos
Colesterol/biossíntese , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/genética , Progesterona/farmacologia , Adulto , Idoso , Linhagem Celular , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/citologia , Ovário/metabolismo , Ovário/patologia , Progesterona/metabolismo , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The native form of human chorionic gonadotropin (hCG) is a heterodimer protein with two asparagine (Asn)-linked carbohydrate chains on each subunit. Removal of the Asn-linked carbohydrate chains from hCG has resulted in hCG variants with consistent antagonistic properties on isolated murine cells. Specific and direct enzymatic removal of these carbohydrate chains from native hCG with resultant antagonistic properties has not been reported. An antagonist to the hCG/luteinising hormone (LH) receptor could be used as an anticancer therapy, emergency contraceptive or for therapeutic resolution of ectopic pregnancies. Therefore, our aim was to use enzymes to specifically remove Asn-linked carbohydrate chains from hCG in the heterodimer form and analyse the resultant bioactivity. Native hCG was treated with endoglycosidases, carbohydrate removal was analysed with electrophoresis and the hCG variants were tested for altered bioactivity with human and murine cells. Endoglycosidases were able to cleave most of the Asn-linked carbohydrate chains from the native hCG. The deglycosylated hCG demonstrated a 75% reduction in bioactivity on a murine Leydig cell line and a 65% reduction in bioactivity on human granulosa cells. These results exemplify a simple and efficient method for creating deglycosylated hCG and provide the most direct evidence for the importance of Asn-linked carbohydrate chains in maintaining hCG bioactivity.
Assuntos
Gonadotropina Coriônica/química , Glicosídeo Hidrolases/química , Animais , Asparagina/química , Asparagina/metabolismo , Gonadotropina Coriônica/metabolismo , Gonadotropina Coriônica/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosídeo Hidrolases/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Progesterona/metabolismo , Subunidades Proteicas , Especificidade por SubstratoRESUMO
Despite the poor prognosis of ovarian cancer and the importance of early diagnosis, there are no reliable noninvasive biomarkers for detection in the early stages of disease. Therefore, to identify novel ovarian cancer markers with potential utility in early-stage screening protocols, we have undertaken an unbiased and comprehensive analysis of gene expression in primary ovarian tumors and normal human ovarian surface epithelium (HOSE) using Serial Analysis of Gene Expression (SAGE). Specifically, we have generated SAGE libraries from three serous adenocarcinomas of the ovary and, using novel statistical tools, have compared these to SAGE data derived from two pools of normal HOSE. Significantly, in contrast to previous SAGE-based studies, our normal SAGE libraries are not derived from cultured cell lines. We have also compared our data with publicly available SAGE data obtained from primary tumors and "normal" HOSE-derived cell lines. We have thus identified several known and novel genes whose expressions are elevated in ovarian cancer. These include but are not limited to CLDN3, WFDC2, FOLR1, COL18A1, CCND1, and FLJ12988. Furthermore, we found marked differences in gene expression patterns in primary HOSE tissue compared with cultured HOSE. The use of HOSE tissue as a control for these experiments, along with hierarchical clustering analysis, identified several potentially novel biomarkers of ovarian cancer, including TACC3, CD9, GNAI2, AHCY, CCT3, and HMGA1. In summary, these data identify several genes whose elevated expressions have not been observed previously in ovarian cancer, confirm the validity of several existing markers, and provide a foundation for future studies in the understanding and management of this disease.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Biologia Molecular , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Both hereditary and sporadic ovarian tumors frequently have decreased BRCA1 expression. One mechanism of downregulating BRCA1 expression is hypermethylation of the BRCA1 promoter. Studies have shown that the BRCA1 promoter is aberrantly hypermethylated in a subset of ovarian tumors, although the proportion varies widely between reports. High-resolution analysis of the BRCA1 promoter in ovarian cancer may provide information regarding the extent and heterogeneity of methylation and guide future studies using methylation-specific polymerase chain reaction (MS-PCR). We screened 50 primary epithelial ovarian tumors for BRCA1 promoter hypermethylation using MS-PCR. The BRCA1 promoter was hypermethylated in 16% (8 of 50) of the tumors, including two stage IA tumors. Sequence analysis of the promoter revealed that methylation of the CpG island is both extensive and mosaic in the methylated samples. Two CpG dinucleotides in the BRCA1 promoter, within and adjacent to a Myb consensus binding site, were most frequently methylated in ovarian tumors. BRCA1 expression was significantly lower in methylated than in unmethylated samples. Our analysis of the BRCA1 promoter revealed preferential methylation of specific CpG sites in ovarian tumors. This finding could be exploited in the design of highly sensitive MS-PCR assays for direct assessment of tumor DNA and potentially for early detection of ovarian cancer in body fluids.
Assuntos
Metilação de DNA , Genes BRCA1 , Neoplasias Ovarianas/genética , Sequência de Bases , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Fatores de TempoRESUMO
Pregnancy is characterized by the presence of generalized leukocyte activation. We used flow cytometry to investigate changes in phenotype and intracellular cytokines of circulating granulocytes, monocytes, and T lymphocytes of pregnant women during gestation. We report that peripheral circulation of pregnancy is characterized by an increased percentage of granulocytes and a decrease in lymphocytes. The proportion of monocytes remains stable throughout gestation; however, a progressive up-regulation of surface markers CD11a, CD54, and CD64 was detected. Monocytes also showed higher production of interleukin (IL)-12 and IL-1beta compared with the nonpregnant state, and granulocytes had greater potential to synthesize IL-8. All these changes were particularly marked in late gestation. T lymphocytes did not have any characteristics of the activated state and showed a decreased IL-6 production. These findings demonstrate that activation of maternal monocytes and granulocytes increases during pregnancy and support the idea that pregnancy results in an elevation of the innate immune system and suppression of the adaptive immune system.
Assuntos
Monócitos/imunologia , Gravidez/imunologia , Adulto , Especificidade de Anticorpos , Moléculas de Adesão Celular/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Citometria de Fluxo , Granulócitos/imunologia , Humanos , Imunofenotipagem , Cinética , Leucócitos/classificação , Gravidez/sangue , Estudos Prospectivos , Linfócitos T/imunologiaRESUMO
OBJECTIVE: To determine the role of estrogen in leukocyte recruitment to the human endometrium. DESIGN: Prospective, controlled in vivo study. SETTING: Academic research laboratory. PATIENT(S): Ten patients presenting for donor oocytes. INTERVENTION(S): Endometrial biopsies for the evaluation of leukocyte populations were collected from perimenopausal women in two consecutive regulated cycles who were given two different regimens of estrogen with identical progesterone treatment. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of endometrial leukocyte populations and relative levels of expression of three chemokine genes. RESULT(S): The total uterine leukocyte population increased significantly when the women received oral estrogen, which resulted in higher serum estrogen levels. This rise in leukocytes was due to a significant increase in both the uterine natural killer cells and the macrophage populations. T-cell numbers did not change relative to circulating estrogen levels. The relative abundance of mRNA from three chemokines was also determined. No changes were found in the expression of M-CSF or MCP-1. Interleukin 8 decreased in glands relative to estrogen levels. CONCLUSION(S): These data demonstrate that changes in circulating levels of estrogen can regulate the recruitment of bone marrow-derived cells to the uterine endometrium; however, the mechanism whereby that occurs remains elusive.
Assuntos
Endométrio/fisiologia , Estrogênios/farmacologia , Leucócitos/fisiologia , Adulto , Biópsia , Quimiocinas/biossíntese , Quimiocinas/sangue , Quimiocinas/genética , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Estrogênios/sangue , Feminino , Humanos , Imuno-Histoquímica , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: BRCA1/BRCA2 germline mutations appear to enhance the platinum-sensitivity, but little is known about the prognostic relevance of polymorphisms in BRCA1/BRCA2 in epithelial ovarian cancer (EOC). This study evaluated whether common variants of BRCA1/BRCA2 are associated with progression-free survival (PFS) and overall survival (OS) in patients with advanced stage sporadic EOC. EXPERIMENTAL DESIGN: The allelic frequency of BRCA1 (2612C > T, P871L-rs799917) and BRCA2 (114A > C, N372H-rs144848) were determined in normal blood DNA from women in Gynecologic Oncology Group protocol #172 phase III trial with optimally resected stage III EOC treated with intraperitoneal or intravenous cisplatin and paclitaxel (C + P). Associations between polymorphisms and PFS or OS were assessed. RESULTS: Two hundred and thirty-two women were included for analyses. African Americans (AA) had different distributions for the two polymorphisms from Caucasians and others. For non-AA patients, the genotype for BRCA1 P871L was distributed as 38% for CC, 49% for CT, and 13% for TT. Median PFS was estimated to be 31, 21, and 21 months, respectively. After adjusting for cell type, residual disease, and chemotherapy regimen, CT/TT genotypes were associated with a 1.40-fold increased risk of disease progression [95% confidence interval (CI) = 1.00-1.95, p = 0.049]. After removing seven patients with known BRCA1 germline mutations, the hazard ratio (HR) was 1.36 (95% CI = 0.97-1.91, p = 0.073). The association between BRCA1 P871L and OS was not significant (HR = 1.25, 95% CI = 0.88-1.76, p = 0.212). Genotype distribution of BRCA2 N372H among non-AA patients was 50, 44, and 6% for AA, AC, and CC, respectively and there is no evidence that this BRCA2 polymorphism was related to PFS or OS. CONCLUSION: Polymorphisms in BRCA1 P871L or in BRCA2 N372H were not associated with either PFS or OS in women with optimally resected, stage III EOC treated with cisplatin and paclitaxel.
RESUMO
The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.
Assuntos
Carcinoma Endometrioide/genética , Caspase 8/genética , Metilação de DNA , Neoplasias do Endométrio/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma Endometrioide/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Neoplasias do Endométrio/metabolismo , Epigênese Genética , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas , Dedos de ZincoRESUMO
Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.
Assuntos
Biologia Computacional/métodos , Metilação de DNA/genética , Neoplasias do Endométrio/genética , Neoplasias Ovarianas/genética , Fenótipo , Análise por Conglomerados , Epigênese Genética/genética , Feminino , Humanos , Laboratórios , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Positron emission tomography (PET)/computed tomography (CT) imaging of suspected new and recurrent ovarian carcinoma was performed to assess the relationship between [(18)F] 3'deoxy-3'fluorothymidine ((18)FLT) uptake and histopathological tissue markers of cellular proliferation (Ki67) and thymidine kinase-1 (TK-1) expression. METHODS: Six subjects were included in this pilot study. Subjects were injected with 5 mCi of (18)FLT prior to a planned surgery and then scanned on a GE Discovery-ST PET/CT scanner within an hour of injection. Regions of interest in tumor and control tissue were identified on the diagnostic CT scans and marked for later surgical biopsy. Surgery was performed within 2 days after the scan. At the time of surgery, the regions of interest identified on PET/CT were available to guide the surgeon to the tumor biopsy sites. Tissue from normal ovarian tissue control regions was also sampled. (18)FLT uptake in tumor and control tissue regions was calculated by measuring the maximum standardized uptake values (SUV(max)). The excised tumor and normal ovarian tissue control tissues were analyzed by immunohistochemical staining for Ki67 and CD34. TK-1 messenger RNA expression was measured by real-time polymerase chain reaction. RESULTS: (18)FLT uptake (SUV(max)) was higher in malignant (mean 4.85/range 1.7-8.8) compared to benign (1.65/range 1.4-1.9) and normal ovarian control tissue (1.12/range 0.6-1.5). Mitotic index, as determined by Ki67 staining, was higher in malignant (18.89/range 11.97-27.19) compared to benign (0.59/range 0.23-0.95) and control tissue (0.45/range 0.06-1.20). TK-1 expression was also higher in malignant (35.52/range 5.21-106.62) compared to benign (8.71/range 4.74-12.67) and control tissue (9.79/range 0.85-39.46). An increasing trend between (18)FLT uptake and Ki67 mitotic index is seen in malignant tissue CD 34 staining between malignant, benign and control tissues was not qualitatively different. CONCLUSION: An increasing trend between (18)FLT uptake and Ki67 mitotic index is seen in malignant tissue. Additional studies will determine whether (18)FLT PET/CT is specific enough to distinguish between cancerous and noncancerous cells and to assess its role in ovarian carcinoma patient management.
Assuntos
Didesoxinucleosídeos , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Idoso , Transporte Biológico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Didesoxinucleosídeos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Projetos Piloto , Recidiva , Timidina Quinase/genéticaRESUMO
PURPOSE: The objectives of this study were to determine whether the midazolam clearance predicted docetaxel pharmacokinetics, CA-125 change, and response and to assess the impact of cytochrome P450 (CYP) 3A5 and ATP-binding cassette, subfamily B, member 1 (ABCB1) genotypes on docetaxel pharmacokinetics and pharmacodynamics in ovarian or primary peritoneal cancer patients. METHODS: Thirty-four patients with advanced ovarian and primary peritoneal cancer were administered docetaxel at 75 mg/m(2) as a 1-h infusion in combination with carboplatin IV over 30 min at a target AUC of 5 mg/ml min. Cycles were repeated every 21 days for 6 cycles. Midazolam was administered at 2 mg as a 30-min IV infusion the day prior to cycle one of docetaxel administration. Pharmacokinetic studies of docetaxel and CYP3A5 and ABCB1 genotype studies were performed. RESULTS: There was an inverse relationship between midazolam clearance (CL) and CA-125 level after cycle 6 where a higher midazolam CL was associated with a CA-125 <10 U/ml (P = 0.007) and CA-125 <15 U/ml (P = 0.048). The CA-125 categories were associated with response achieved (complete response/partial response) (CR/PR), stable disease (SD), and progressive disease (PD) at the end of therapy (P = 0.0173). Docetaxel CL was not related to midazolam CL or genotype. Docetaxel exposure and genotypes were not related to toxicity or response (P > 0.05). CONCLUSIONS: The midazolam CL predicted CA-125 levels and response that was independent of other factors including docetaxel pharmacokinetics. Future studies need to evaluate the mechanism for the relationship between midazolam CL and response in patients with ovarian cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Peritoneais/tratamento farmacológico , Taxoides/uso terapêutico , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Docetaxel , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Farmacogenética , Reação em Cadeia da Polimerase , Espectrometria de Massas em Tandem , Taxoides/efeitos adversos , Taxoides/farmacocinéticaRESUMO
AIM: Identifying the factors responsible for reducing the proliferation, syncytialization, and invasiveness of trophoblast tissues, as seen with preeclampsia, intrauterine growth restriction, and spontaneous miscarriage, is a current challenge in reproductive biology. These factors, transforming growth factor (TGF)-beta as an example, can work by altering trophoblast differentiation or proliferation. We therefore investigated and compared specific markers of trophoblast proliferation and differentiation in three commonly used trophoblast tissue cell models, and also investigated the influence of TGF-beta on these markers. METHODS: In this study, we isolated human trophoblasts from first trimester and term placentas, and additionally used human choriocarcinoma cells (JEG-3). Baseline values of human chorionic gonadotropin (hCG) secretion and relative mRNA levels of cell cycle regulators (cyclin E, p21, p27, and p57) were investigated for each cell type. We also investigated the influence of TGF-beta on these parameters. RESULTS: Quantitative and longitudinal production of hCG differed between the three cell types. Significantly different amounts of cyclin E, p21, p27, and p57 mRNA were demonstrated within each cell type, as well as between all the cell types, throughout the culture time period. Each trophoblast type demonstrated a reduction of hCG secretion in response to TGF-beta. TGF-beta did not show a consistent effect on the cell cycle mRNA of any of the cell types. CONCLUSION: We were able to characterize and compare the differential production of hCG, as well as the differential expression of cell cycle-associated mRNA of early trophoblasts, term trophoblasts, and choriocarcinoma cells. The production of hCG was altered by TGF-beta, although mRNA levels were not markedly altered by TGF-beta.