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1.
Artigo em Inglês | MEDLINE | ID: mdl-31965189

RESUMO

BACKGROUND: A test that helps predict the time to the final menstrual period (FMP) has been sought for many years. OBJECTIVE: To assess the ability of Anti-Mullerian Hormone (AMH) measurements to predictions the time to FMP. DESIGN: Prospective longitudinal cohort study. SETTING: The Study of Women's Health Across the Nation. PARTICIPANTS AND MEASUREMENTS: AMH and FSH were measured in 1537 pre- or early perimenopausal women, mean age 47.5 ± 2.6 years at baseline, then serially until 12 months of amenorrhea occurred. AMH was measured using a two-site ELISA with a detection limit of 1.85 pg/ml. MAIN OUTCOME MEASURE: Areas under the Receiver Operating Curves (AUC) for AMH-based and FSH-based predictions of time to FMP, stratified by age. Probabilities that women would undergo their FMP in the next 12, 24, or 36 months across a range of AMH values were assessed. RESULTS: AUCs for predicting that the FMP will occur within the next 24 months were significantly greater for AMH-based than FSH-based models. The probability that a woman with an AMH <10 pg/ml would undergo her FMP within the next 12 months ranged from 51% at <48 years old to 79% at ≥ 51 years old. The probability that a woman with an AMH >100 pg/mL would not undergo her FMP within the next 12 months ranged from 97% in women <48 years old to 90% in women ≥ 51 years old. CONCLUSIONS: AMH measurement helps estimate when a woman will undergo her FMP, and, in general, does so better than FSH.

2.
J Endocr Soc ; 3(11): 2123-2134, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31687639

RESUMO

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

3.
Elife ; 82019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232694

RESUMO

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.

4.
PLoS Genet ; 14(12): e1007822, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30532227

RESUMO

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.


Assuntos
Variação Genética , Hérnias Diafragmáticas Congênitas/genética , Proteínas de Membrana/genética , Mutação , Fatores de Transcrição/genética , Pré-Escolar , Variações do Número de Cópias de DNA , Deficiências do Desenvolvimento/genética , Feminino , Cardiopatias Congênitas/genética , Hérnias Diafragmáticas Congênitas/metabolismo , Humanos , Recém-Nascido , Estudos Longitudinais , Masculino , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Fenótipo , Análise de Sequência de RNA , Síndrome , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
5.
Curr Opin Endocrinol Diabetes Obes ; 25(6): 399-405, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30320617

RESUMO

PURPOSE OF REVIEW: The nascent field of oncofertility is quickly gaining traction as novel experimental treatments are being developed, driving a renewed interest in Müllerian inhibiting substance (MIS) as an ovarian fertoprotectant. RECENT FINDINGS: MIS is unique in its mechanisms of ovarian protection by virtue of acting directly on granulosa cells of primordial follicles and for being a benign reproductive hormone, with few side effects. We will explore in this review how it may be utilized to protect the ovary from chemotherapy, or to enhance ovarian tissue cryopreservation therapy. We will also examine potential mechanisms of action of MIS across multiple cell types, as well as current limitations in our understanding of the pharmacology of recombinant MIS. SUMMARY: The usefulness of MIS as a fertoprotectant may be dependent on the mechanisms of gonadotoxicity of each chemotherapeutic. Further investigation is needed to determine how to best deliver and combine MIS treatment to existing fertility management strategies.


Assuntos
Hormônio Antimülleriano/uso terapêutico , Fármacos para a Fertilidade Feminina/uso terapêutico , Preservação da Fertilidade/métodos , Folículo Ovariano/efeitos dos fármacos , Hormônio Antimülleriano/fisiologia , Criopreservação/métodos , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Humanos , Folículo Ovariano/fisiologia , Ovário
6.
Reprod Biomed Online ; 37(5): 631-640, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30241771

RESUMO

RESEARCH QUESTION: Is formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays? DESIGN: The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1-13.0 ng/ml. RESULTS: Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90-22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay. CONCLUSIONS: A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.


Assuntos
Hormônio Antimülleriano/sangue , Imunoensaio/normas , Calibragem , Feminino , Humanos
7.
J Clin Endocrinol Metab ; 103(11): 4187-4196, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30239805

RESUMO

Context: There is increasing evidence for Müllerian-inhibiting substance (MIS)/anti-Müllerian hormone (AMH) physiologic activity in the human uterus, so it is relevant to study how MIS/AMH levels impact pregnancy. Objective: To investigate the association of MIS/AMH levels with the risk of adverse obstetric outcomes. Design: Retrospective cohort study. Setting: Academic fertility center. Patients: Women who became pregnant through in vitro fertilization between January 2012 and October 2016. Exclusion criteria were: oocyte donation, gestational carrier, multiple gestations, miscarriage before 20 weeks, or medically indicated preterm deliveries. Interventions: None. Main Outcome Measures: There were two primary outcomes, preterm birth and cesarean delivery for arrest of labor. Because MIS/AMH level is highly skewed by certain infertility diagnoses, the preterm birth analysis was stratified by polycystic ovary syndrome (PCOS) diagnosis, and the cesarean delivery for arrest of labor analysis was stratified by diminished ovarian reserve diagnosis. χ2, Mann-Whitney, and t tests were used as appropriate. A P value of <0.05 was considered statistically significant. Results: Among women with PCOS, those who delivered prematurely had substantially higher MIS/AMH levels (18 vs 6.4 ng/mL, P = 0.003) than did those who delivered at term. At the highest MIS/AMH values, preterm deliveries predominated; above the 90th percentile in women with PCOS, all deliveries were premature. No effect of MIS/AMH level was observed in women without PCOS. We found no association between MIS/AMH values and cesarean delivery for labor arrest. Conclusion: In women with PCOS, substantially elevated MIS/AMH levels are significantly associated with preterm birth, suggesting closer follow-up and further studies to elucidate the underlying mechanisms.


Assuntos
Hormônio Antimülleriano/sangue , Cesárea/estatística & dados numéricos , Distocia/diagnóstico , Síndrome do Ovário Policístico/sangue , Nascimento Prematuro/diagnóstico , Adulto , Distocia/sangue , Distocia/etiologia , Distocia/cirurgia , Feminino , Humanos , Recém-Nascido , Síndrome do Ovário Policístico/complicações , Gravidez , Nascimento Prematuro/etiologia , Prognóstico , Estudos Retrospectivos , Inércia Uterina
8.
Proc Natl Acad Sci U S A ; 115(20): 5247-5252, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712845

RESUMO

Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX, LHX1, and HNF1B We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects.


Assuntos
Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Marcadores Genéticos , Hérnias Diafragmáticas Congênitas/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Humanos , Prognóstico
9.
Hum Genet ; 136(6): 679-691, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28303347

RESUMO

Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.


Assuntos
Estudo de Associação Genômica Ampla , Hérnias Diafragmáticas Congênitas/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligação Proteica
10.
Proc Natl Acad Sci U S A ; 114(9): E1688-E1697, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28137855

RESUMO

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.


Assuntos
Hormônio Antimülleriano/farmacologia , Antineoplásicos/efeitos adversos , Anticoncepcionais/farmacologia , Folículo Ovariano/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Animais , Anticoncepção/métodos , Dependovirus/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Ovariana Primária/prevenção & controle , Reprodução/efeitos dos fármacos
11.
Int J Oncol ; 50(3): 1022-1028, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28197641

RESUMO

Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) has been suggested as a biotherapeutic agent in gynecological cancers that highly express the MIS/AMH type II receptors (MISRII/AMHRII) but the anticancer mechanisms by which MIS/AMH acts are not fully understood. Our experiments show that MIS/AMH inhibits ovarian cancer by deregulating the Wnt signal pathway via the ß-catenin interacting protein (ICAT). MIS/AMH inhibition of ICAT by small interfering RNAs (siRNA) decreased ICAT driven ovarian cancer cell viability as measured by the methylthiazoltetrazolium assay, reversed cell cycle arrest and annexin V expression and diminished migration by scratch wound assay. Changes in expression of regulatory proteins were shown by western blotting. We determined that MIS/AMH upregulated ICAT in ovarian cancer cell line which caused decreased cell viability, cell cycle arrest and apoptosis. This effect, however, was blocked when ICAT was downregulated by siRNA. The present study demonstrates a role for ICAT in MIS/AMH mediated inhibition of the Wnt signaling pathway in ovarian cancer.


Assuntos
Hormônio Antimülleriano/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Ovarianas/patologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética
13.
Am J Pathol ; 186(10): 2532-43, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27565037

RESUMO

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models.


Assuntos
Aberrações Cromossômicas , Hérnias Diafragmáticas Congênitas/genética , Hérnias Diafragmáticas Congênitas/patologia , Animais , Diafragma/embriologia , Diafragma/patologia , Modelos Animais de Doenças , Feminino , Hérnias Diafragmáticas Congênitas/terapia , Humanos , Lactente , Pulmão/embriologia , Pulmão/patologia , Camundongos , Camundongos Knockout
14.
BMC Biol ; 14: 59, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27412481

RESUMO

BACKGROUND: Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown. RESULTS: We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture. CONCLUSIONS: Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.


Assuntos
Colágeno Tipo IV/metabolismo , Células Endoteliais/citologia , Células Epiteliais/citologia , Morfogênese , Fragmentos de Peptídeos/metabolismo , Células A549 , Animais , Membrana Basal/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Colágeno Tipo IV/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Pulmão/citologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Mutação , Miofibroblastos/citologia , Fragmentos de Peptídeos/genética , Regulação para Cima
15.
Am J Med Genet A ; 170(9): 2457-61, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27363585

RESUMO

COUP-TFII (NR2F2) is mapped to the 15q26 deletion hotspot associated with the common and highly morbid congenital diaphragmatic hernia (CDH). Conditional homozygous deletions of COUP-TFII in mice result in diaphragmatic defects analogous to the human Bochdalek-type hernia phenotype. Despite evidence from animal models however, mutations in the coding sequence of COUP-TFII have not been reported in patients, prompting the speculation that additional coding or non-coding sequences in the 15q26 locus are necessary for diaphragmatic hernias to develop. In this report, we describe a case of a patient with a heterozygous de novo COUP-TFII frameshift mutation, presenting with CDH and an atrial septal defect. The p.Pro33AlafsTer77 mutation specifically disrupts protein isoform 1 which contains the DNA binding domain. In addition, we review other COUP-TFII sequence variations and deletions that have been described in cases of CDH. We conclude that COUP-TFII mutations can cause diaphragmatic hernias, and should be included in the differential diagnosis of CDH patients, particularly those with comorbid congenital heart defects. © 2016 Wiley Periodicals, Inc.


Assuntos
Fator II de Transcrição COUP/genética , Mutação da Fase de Leitura , Estudos de Associação Genética , Hérnias Diafragmáticas Congênitas/diagnóstico , Hérnias Diafragmáticas Congênitas/genética , Fenótipo , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Recém-Nascido , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único
16.
Cell Rep ; 16(3): 657-71, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27396341

RESUMO

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-ß)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-ß/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-ß/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.


Assuntos
Hormônio Antimülleriano/metabolismo , Plasticidade Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo
17.
PLoS One ; 11(6): e0156595, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27253518

RESUMO

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.


Assuntos
Biomarcadores Tumorais/genética , Receptores de Hialuronatos/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Isoformas de Proteínas/genética , Adulto , Idoso , Processamento Alternativo/genética , Carcinoma Epitelial do Ovário , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Prognóstico , Análise Serial de Tecidos
18.
PLoS One ; 11(2): e0149425, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26891231

RESUMO

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.


Assuntos
Pulmão/embriologia , Pulmão/metabolismo , MicroRNAs/genética , Organogênese/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Proliferação de Células , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Pulmão/patologia , Camundongos , MicroRNAs/química , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Interferência de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Transcrição Genética
19.
Proc Natl Acad Sci U S A ; 112(32): E4418-27, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216943

RESUMO

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.


Assuntos
Hormônio Antimülleriano/genética , Hormônio Antimülleriano/uso terapêutico , Dependovirus/metabolismo , Terapia Genética , Neoplasias Ovarianas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ascite/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Músculos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise Serial de Tecidos , Transgenes , Tropismo , Carga Tumoral
20.
Int J Oncol ; 46(5): 2039-46, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25760378

RESUMO

Müllerian inhibiting substance (MIS) has been shown to inhibit growth of a number of tumors in vitro and/or in vivo, but the downstream pathways which it regulates are not fully understood. In the present study we show that MIS type II receptor was highly expressed in AN3CA cells, a cell line derived from human endometrial cancer cell in which MIS-treatment caused a reduction of cell viability, and induced cellular apoptosis and genes involved cell cycle arrest. To understand the genome-wide effects of MIS on gene regulation, we performed serial gene expression analyses from 0 to 96 h at 24 h intervals after treating AN3CA cells with MIS. Transcriptomic analysis of molecular changes induced by MIS identified 2,688 differentially expressed genes that were significantly up- or down-regulated during the 96 h study period. When the 2,688 differentially expressed genes were mapped to known biological processes, Wnt-, cancer-, proteolysis-, cytoskeleton-, cell cycle-, apoptosis-, and MAPK-signaling pathways emerged as the functions most significantly changed by MIS in AN3CA cells. Furthermore, western blot analysis validated that protein expression of cell cycle inhibitory genes, apoptotic protease activating factor-1 (APAF-1), ß-catenin-interacting protein (ICAT), Rb related protein 130 (p130), and inhibitor of disheveled Dvl and Axin complex (IDAX), were gradually increased over the time of the study, whereas downstream cell cycle activating genes, cyclin-dependent kinase 2 (CDK2) and phospho-c-Jun were downregulated in MIS-treated AN3CA cells. These transcriptome analyses support previous observations that MIS functions as a tumor suppressor, potentially by regulating signaling pathways that could contribute to endometrial carcinogenesis, and indicating that MIS should be considered as a potential treatment for endometrial cancer.


Assuntos
Neoplasias do Endométrio/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Receptores de Peptídeos/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/fisiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo
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