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1.
Cancer Res ; 79(13): 3503-3513, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31097475

RESUMO

Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. SIGNIFICANCE: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Separação Celular/métodos , Cistadenocarcinoma Seroso/patologia , Exossomos/metabolismo , Microfluídica/métodos , Neoplasias Ovarianas/patologia , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/metabolismo , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-6/metabolismo , Neoplasias Ovarianas/metabolismo , Proteoma/análise , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
2.
J Natl Cancer Inst ; 110(5): 517-526, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29155953

RESUMO

Background: Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. Methods: MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Results: Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. Conclusion: These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma Endometrioide/genética , Códon sem Sentido , Neoplasias do Endométrio/genética , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos/genética , Animais , Animais não Endogâmicos , Arginina/genética , Carcinoma Endometrioide/epidemiologia , Carcinoma Endometrioide/patologia , Células Cultivadas , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/patologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Histidina/genética , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia
3.
PLoS One ; 12(1): e0170318, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28114390

RESUMO

OBJECTIVES: STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin. METHODS: In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin. RESULTS: Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins. CONCLUSIONS: These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.


Assuntos
Neoplasias do Endométrio/terapia , Glucose/administração & dosagem , Metformina/farmacologia , Fator de Transcrição STAT3/metabolismo , Animais , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Xenoenxertos , Humanos , Camundongos
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