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1.
Anticancer Res ; 40(12): 6677-6684, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33288561

RESUMO

AIM: To analyze the biological role of the long non-coding RNA LINK-A. MATERIALS AND METHODS: An 850-bp segment from the second exon of LINK-A was removed using the CRISPR/Cas9 system in OVCA433 ovarian serous carcinoma cells. Spheroid formation, migration, invasion, proliferation, matrix metalloproteinase (MMP) activity and expression of cell-signaling proteins were assessed in vitro. RESULTS: OVCA433 cells with LINK-A deletion were more invasive (p=0.0008) but had reduced migration and MMP9 secretion compared to controls (p=0.003 and p=0.005, respectively). LINK-A deletion did not affect proliferation but induced phosphorylation of extracellular signal-regulated kinase (10-fold; p=0.005). LINK-A knock out additionally reduced spheroid formation. CONCLUSION: Added to our previous data from analysis of clinical specimens, LINK-A is likely to be a tumor suppressor.


Assuntos
Neoplasias Ovarianas/genética , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Conformação de Ácido Nucleico , Neoplasias Ovarianas/enzimologia , Fosforilação , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
2.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32718068

RESUMO

The objective of the present study was to analyze the biological and clinical role of the long non-coding RNA LOC642852 in ovarian carcinoma (OC). LOC642852 expression was analyzed in seven OC cell lines (OVCAR-3, OVCAR-8, OVCA 433, OVCA 429, OC 238, DOV13, ES-2) and 139 high-grade serous carcinoma (HGSC) specimens (85 effusions, 54 surgical specimens). Following LOC642852 knockout (KO) using the CRISPR/Cas9 system, OVCAR-8 HGSC cells were analyzed for spheroid formation, migration, invasion, proliferation, matrix metalloproteinase (MMP) activity, and expression of cell signaling proteins. OVCAR-8 cells with LOC642852 KO were significantly less motile and less invasive compared to controls, with no differences in spheroid formation, proliferation, or matrix metalloproteinase (MMP) activity. Total Akt and Erk levels were comparable in controls and KO cells, but their phosphorylation was significantly increased in the latter. In clinical specimens, LOC642852 was overexpressed in ovarian tumors and omental/peritoneal metastases compared to effusion specimens (p = 0.013). A non-significant trend for shorter overall (p = 0.109) and progression-free (p = 0.056) survival was observed in patients with HGSC effusions with high LOC642852 levels. Bioinformatics analysis showed potential roles for LOC642852 as part of the TLE3/miR-221-3p ceRNA network and in relation to the FGFR3 protein. In conclusion, LOC642852 inactivation via CRISPR/Cas9 affects cell signaling, motility, and invasion in HGSC cells. LOC642852 is differentially expressed in HGSC cells at different anatomical sites. Its potential role in regulating the TLE3/miR-221-3p ceRNA network and FGFR3 merits further research.


Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Ovarianas , RNA Longo não Codificante , Linhagem Celular , Colagenases/biossíntese , Colagenases/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
3.
J Biol Eng ; 14: 18, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32617119

RESUMO

Background: Implantation failure remains an unsolved obstacle in reproductive medicine. Previous studies have indicated that estrogen responsiveness, specifically by estrogen receptor alpha (ERα), is crucial for proper implantation. There is an utmost need for a reliable in vitro model that mimics the events in the uterine wall during the implantation process for studying the regulatory mechanisms governing the process. The current two-dimensional and hydrogel-based in vitro models provide only short-term endometrial cell culture with partial functionality. Results: Endometrial biopsies showed an increase in E-cadherin expression on the typical window of implantation of fertile women, compared to negligible expression in recurrent implantation failure (RIF) patients. These clinical results indicated E-cadherin as a marker for receptivity. Three-dimensional (3D) macroporous alginate scaffolds were the base for epithelial endometrial cell-seeding and long-term culture under hormone treatment that mimicked a typical menstrual cycle. The RL95-2 epithelial cell culture in macroporous scaffolds was viable for 3 weeks and showed increased E-cadherin levels in response to estrogen. Human choriocarcinoma (JAR) spheroids were used as embryo models, seeded onto cell constructs and successfully adhered to the RL95-2 cell culture. Moreover, a second model of HEC-1A with low ERα levels, showed lower E-cadherin expression and no JAR attachment. E-cadherin expression and JAR attachment were recovered in HEC-1A cells that were transfected with ERα plasmid. Conclusions: We present a novel model that enables culturing endometrial cells on a 3D matrix for 3 weeks under hormonal treatment. It confirmed the importance of ERα function and E-cadherin for proper implantation. This platform may serve to elucidate the regulatory mechanisms controlling the implantation process, and for screening and evaluating potential novel therapeutic strategies for RIF.

4.
Virchows Arch ; 477(2): 249-258, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31900634

RESUMO

The objective of this study was to analyze the expression and clinical role of Wnt pathway molecules in metastatic high-grade serous carcinoma (HGSC). mRNA expression by qPCR of 20 molecules related to Wnt signaling (WNT1, WNT2, WNT3, WNT4, WNT5A, WNT6, WNT7, WNT11, FZD1, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, LRP5, LRP6, DKK, CCND, RUNX2) was analyzed in 87 HGSC effusions. Thirty-nine surgical specimens (19 ovarian, 20 from other intra-abdominal sites) were analyzed for comparative purposes. Protein expression of YAP and LRP and their phosphorylated forms by western blotting were analyzed in 52 tumors. Significant differences in mRNA expression as a function of the anatomic site were observed for WNT3 (p = 0.005), WNT5A (p = 0.008), WNT7 (p < 0.001), FRZ5 (p = 0.04), and FRZ6 (p < 0.001). YAP and LRP and their phosphorylated forms were detected in HGSC specimens. FZD10 was overexpressed in effusions from patients who had complete response to chemotherapy compared with those with less favorable response (p = 0.037). WNT4 (p = 0.005), WNT7 (p = 0.047), RUNX2 (p = 0.038), LRP5 (p = 0.022), LRP6 (p = 0.011), FZD6 (p = 0.036), FZD7 (p = 0.004), and FZD10 (p = 0.015) levels were inversely related to primary chemoresistance. High FZD5 levels in pre-chemotherapy effusions tapped at diagnosis and high WNT2 levels in post-chemotherapy disease recurrence effusions were related to shorter overall survival (p = 0.018 and p = 0.011, respectively), whereas high RUNX2 (p = 0.031) and FZD1 (p = 0.029) in post-chemotherapy effusions were associated with longer overall survival. In multivariate analysis of post-chemotherapy cases, WNT2 (p = 0.002), RUNX2 (p = 0.017), FZD1 (p = 0.036), and FZD4 (p = 0.013) were independent prognosticators. In conclusion, expression of Wnt pathway molecules is anatomic site dependent. In HGSC effusions, it is informative of chemoresponse and survival.


Assuntos
Cistadenocarcinoma Seroso/patologia , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/patologia , Via de Sinalização Wnt/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores/métodos , RNA Mensageiro/genética , Proteínas Wnt/metabolismo
5.
J Oncol ; 2019: 7547469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31636669

RESUMO

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression, and resistance. It is produced mainly by autotaxin and acts on six G-protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors may involve any novel, thus far uncharacterized, signaling pathway(s). Here we show that three LPA receptors are involved in tumor progression by activation of both the AKT and ERK signaling pathways. CRISPR-edited LPAR2 and LPAR3 knockouts have opposing effects on ERK activation, whereas LPAR6 is involved in the activation of AKT, affecting cell migration and invasion. Our study identifies specific molecular machinery triggered by LPA and its receptors that modulates tumor cells and can serve as therapeutic target in this malignancy.

6.
Gynecol Oncol ; 153(3): 651-660, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30904337

RESUMO

OBJECTIVE: The aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC). METHODS: mRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells. RESULTS: OCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity. CONCLUSIONS: SOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.


Assuntos
Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos , Exossomos , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pessoa de Meia-Idade , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Gradação de Tumores , Metástase Neoplásica , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/secundário , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteólise , RNA Mensageiro/metabolismo , Taxa de Sobrevida
7.
Virchows Arch ; 473(4): 463-470, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30032361

RESUMO

The aim of this study was to analyze the expression, biological role and clinical relevance of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA), and LPA receptors (LPAR) in high-grade serous carcinoma (HGSC). mRNA expression by qRT-PCR of LPAR1-6 was analyzed in 155 HGSC specimens (88 effusions, 67 solid lesions). ATX mRNA expression was analyzed in 97 specimens. ATX, ERK, and AKT protein expression was studied by Western blotting. LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein. Higher LPAR1 levels were significantly related to longer overall survival (OS) in pre-chemotherapy effusions (p = 0.027). Conversely, higher expression of LPAR1, LPAR2, and LPAR5 in post-chemotherapy effusions was significantly associated with shorter OS (p = 0.037, p = 0.025 and p = 0.021, respectively) and progression-free survival (PFS) (p < 0.001, p = 0.007 and p < 0.001, respectively) in univariate survival analysis. LPAR1 mRNA expression was an independent prognosticator of OS in patients with pre-chemotherapy effusions and PFS in patients with post-chemotherapy effusions (p = 0.013 both). In conclusion, LPAR mRNA and ATX protein levels are anatomic site-dependent in HGSC and the former are informative of disease outcome.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/enzimologia , Lisofosfolipídeos/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/enzimologia , Neoplasias Ovarianas/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma/genética , Carcinoma/secundário , Carcinoma/terapia , Intervalo Livre de Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Císticas, Mucinosas e Serosas/terapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Diester Fosfórico Hidrolases/genética , Fosforilação , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Receptores de Ácidos Lisofosfatídicos/genética , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Resultado do Tratamento
8.
Gynecol Oncol ; 148(3): 559-566, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29310950

RESUMO

OBJECTIVE: To profile long non-coding RNA (lncRNA) expression at the various anatomic sites of high-grades serous carcinoma (HGSC) and in effusion-derived exosomes. METHODS: LncRNA profiling was performed on 60 HGSC specimens, including 10 ovarian tumors, 10 solid metastases and 10 malignant effusions, as well as exosomes from 30 effusion supernatants. Anatomic site-related expression of ESRG, Link-A, GAS5, MEG3, GATS, PVT1 H19, Linc-RoR, HOTAIR and MALAT1 was validated by quantitative PCR and assessed for clinical relevance in a series of 77 HGSC effusions, 40 ovarian carcinomas, 21 solid metastases and 42 supernatant exosomes. RESULTS: Significantly different (p<0.05) expression of 241, 406 and 3634 lncRNAs was found in comparative analysis of the ovarian tumors to solid metastases, effusions and exosomes, respectively. Cut-off at two-fold change in lncRNA expression identified 54 lncRNAs present at the 3 anatomic sites and in exosomes. Validation analysis showed significantly different expression of 5 of 10 lncRNAs in the 4 specimen groups (ESRG, Link-A, MEG3, GATS and PVT1, all p<0.001). Higher ESRG levels in HGSC effusions were associated with longer overall survival in the entire effusion cohort (p=0.023) and in patients with pre-chemotherapy effusions tapped at diagnosis (p=0.048). Higher Link-A levels were associated with better overall (p=0.015) and progression-free (p=0.023) survival for patients with post-chemotherapy effusions. Link-A was an independent prognostic marker in Cox multivariate analysis in the latter group (p=0.045). CONCLUSIONS: We present the first evidence of differential LncRNA expression as function of anatomic site in HGSC. LncRNA levels in HGSC effusions are candidate prognostic markers.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Derrame Pleural Maligno/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida
9.
Virchows Arch ; 470(6): 665-678, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28432432

RESUMO

The present study analyzed the expression and clinical role of the transforming growth factor-ß (TGFß) pathway in high-grade serous carcinoma (HGSC), with focus on malignant effusions. TGFß1-3 and TGFßRI-III mRNA expression by qRT-PCR was analyzed in 70 HGSC effusions and 55 solid specimens (28 ovarian, 27 abdominal metastases). Protein expression of Smad2 and Smad3 and their phosphorylated forms by Western blotting was analyzed in 73 specimens (42 effusions, 13 ovarian carcinomas, 18 solid metastases). Expression was analyzed for association with anatomic site and clinical parameters, including survival. TGFßRI and TGFßRII mRNA was overexpressed in effusions and solid metastases, particularly the former, compared to that in the ovarian tumors (p < 0.001 to p = 0.05), with anatomic site-dependent expression of splice variants. Conversely, Smad2, p-Smad2, and p-Smad3 were overexpressed in solid specimens (ovarian and peritoneal) compared to those in effusions (p < 0.001 for all). In univariate survival analysis, higher TGFßRI variant 1 and TGFßRIII mRNA levels were associated with a trend for shorter overall survival in patients with post-chemotherapy effusions (p = 0.066 and p = 0.087, respectively), and the latter was an independent prognostic marker in Cox multivariate analysis (p = 0.041). Smad3 protein expression was associated with a trend for shorter overall survival in univariate survival analysis (p = 0.052). TGFß receptor splice variant expression is anatomic site-dependent in HGSC. Elevated levels of TGFß signaling pathway mRNAs are seen in metastatic HGSC, but are not accompanied by increased Smad expression and activation in HGSC effusions, evidence of failure to activate canonical TGFß signaling. Assessment of the prognostic role of this pathway in HGSC effusions merits further research.


Assuntos
Cistadenocarcinoma Seroso/patologia , Neoplasias Ovarianas/patologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Western Blotting , Cistadenocarcinoma Seroso/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Reação em Cadeia da Polimerase , Isoformas de Proteínas/metabolismo
10.
Hum Pathol ; 62: 115-121, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28087476

RESUMO

The objective of this study was to analyze the expression and clinical role of phospholipase D (PLD) in high-grade serous carcinoma (HGSC). PLD1 and PLD2 isoform expression was studied in 125 HGSC specimens (73 effusions, 28 ovarian tumors, 24 solid metastases) using quantitative real-time reverse-transcription polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters, including chemoresponse, and survival. PLD1 and PLD2 isoforms were found in most specimens at all anatomic sites, and their levels were strongly positively related (P<.001 for effusions and solid lesions). PLD2 messenger RNA (mRNA) expression was significantly higher in effusions compared with both carcinomas in the ovary and solid metastases (P<.001). Higher levels of both isoforms were associated with higher CA 125 levels at diagnosis (P<.001), and higher PLD2 mRNA levels in effusions were associated with unfavorable response to chemotherapy (P=.021). Expression levels of the studied isoforms were unrelated to the levels of previously studied mRNAs that form part of the phospholipase A2 pathway or to survival. The present study provides the first evidence of PLD expression in HGSC and suggests a role in mediating progression to effusions and chemoresistance in this cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Fosfolipase D/genética , RNA Mensageiro/genética , Adulto , Idoso , Biomarcadores Tumorais/análise , Biópsia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/enzimologia , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Císticas, Mucinosas e Serosas/terapia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do Tratamento
11.
Hum Pathol ; 60: 180-187, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27825812

RESUMO

The objective of this study was to analyze the expression and clinical role of molecules involved in exosome synthesis and secretion in high-grade serous carcinoma, with focus on malignant effusions. The mRNA expression levels of ARF6, nSMase2, TSAP6, Rab27a and Rab27b by quantitative real-time reverse-transcription polymerase chain reaction were analyzed in 103 HGSC effusions and 65 solid specimens (35 ovarian, 30 abdominal metastases). Protein expression of ARF6, nSMase2, TSAP6 and Rab27a by Western blotting was analyzed in 150 specimens (94 effusions, 29 ovarian carcinomas, 27 solid metastases). Secreted ARF6, nSMase2 and Rab27a protein levels in exosomes were analyzed in supernatants from 75 effusions. Expression levels were analyzed for association with anatomic site and clinical parameters, including survival. nSMase2 and TSAP6 mRNA was overexpressed in effusions compared to solid lesions (P<.001 and P=.003, respectively), whereas ARF6, nSMase2, TSAP6 and Rab27a protein was overexpressed in solid specimens (ovarian and peritoneal) compared to effusions (P<.001 for all). Secreted ARF6, nSMase2 and Rab27a levels were found in all effusion supernatants. In univariate survival analysis, higher TSAP6 protein levels in effusions were associated with shorter overall survival (P=.01), a finding which was reproduced in analysis of specimens from patients with pre-chemotherapy effusions tapped at diagnosis (P=.026). Higher levels of exosomal Rab27a protein were significantly related to longer overall survival (P=.025). Molecules which are part of the exosome secretion machinery are differentially expressed in HGSC effusions and solid lesions and are present in effusion supernatant-derived exosomes. TSAP6 and Rab27a may be novel prognostic markers in metastatic HGSC.


Assuntos
Biomarcadores Tumorais/análise , Exossomos/química , Neoplasias Císticas, Mucinosas e Serosas/química , Proteínas Oncogênicas/análise , Neoplasias Ovarianas/química , Neoplasias Peritoneais/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Western Blotting , Proteínas de Ciclo Celular , Intervalo Livre de Doença , Exossomos/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Císticas, Mucinosas e Serosas/terapia , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Oxirredutases , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Resultado do Tratamento , Proteínas rab de Ligação ao GTP/análise , Proteínas rab27 de Ligação ao GTP
12.
PLoS One ; 11(9): e0162502, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27622508

RESUMO

OBJECTIVE: Ezrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC). METHODS: OC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression. RESULTS: Higher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome. CONCLUSIONS: The 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido Ascítico/metabolismo , Líquido Ascítico/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/antagonistas & inibidores , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/metabolismo , Derrame Pleural Maligno/patologia , RNA Interferente Pequeno/genética , Esferoides Celulares
13.
Hum Pathol ; 54: 74-81, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27063471

RESUMO

The objective of this study was to analyze the clinical role of 9 microRNAs (miRs) previously found to be overexpressed in ovarian carcinoma effusions compared with primary ovarian carcinomas. High-grade serous carcinoma effusions (n=148) were analyzed for expression of miR-29a, miR-31, miR-99b, miR-182, miR-210, miR-221, miR-222, miR-224, and miR-342 using quantitative polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters and survival. miR-29a and miR-31 levels were further assessed for association with protein expression of their targets Stathmin and DNA methyltransferase-3A (DNMT3A) by immunohistochemistry and Western blotting, respectively. miRNA levels were unrelated to clinicopathological parameters. However, higher miR-29a levels were significantly related to longer overall survival in univariate (P=.007) and Cox multivariate survival analysis (P=.045). miR-29a levels were inversely related to those of its target DNMT3A (P=.048), and higher DNMT3A expression was significantly related to poor overall survival in univariate (P=.03) and Cox multivariate (P=.016) survival analysis. In contrast, miR-31 levels were directly related to cytoplasmic phospho-Stathmin expression (P=.029) and unrelated to Stathmin and nuclear phospho-Stathmin, and both Stathmin and phospho-Stathmin expressions were unrelated to survival. miR-29a and its target DNMT3A are novel candidate biomarkers of longer and shorter survival, respectively, in metastatic high-grade serous carcinoma.


Assuntos
Biomarcadores Tumorais/genética , MicroRNAs/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , DNA (Citosina-5-)-Metiltransferases/análise , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Ovarianas/química , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Fenótipo , Fosforilação , Modelos de Riscos Proporcionais , Fatores de Risco , Estatmina/análise , Fatores de Tempo , Regulação para Cima
14.
Gynecol Oncol ; 140(3): 512-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26768834

RESUMO

OBJECTIVE: To compare the microRNA (miRNA) profiles of uterine endometrial stromal sarcoma (ESS) and leiomyosarcoma (LMS), and to compare the miRNA signatures of primary and metastatic uterine LMS. METHODS: Eight primary LMS, 9 primary ESS and 8 metastatic LMS were analyzed for miRNA profiles using TaqMan Human miRNA Array Cards. Findings for 20 differentially expressed miRNAs were validated in a series of 44 uterine sarcomas (9 primary uterine ESS, 17 primary uterine LMS, 18 metastatic LMS) using qPCR. Frizzled-6 protein expression was analyzed in 30 LMS (15 primary, 15 metastases). Frizzled-6 was silenced in SK-LMS-1 uterine LMS cells using siRNA and the effect on invasion, wound healing and matrix metalloproteinase-2 (MMP2) activity was assessed. RESULTS: Ninety-four miRNAs were significantly differentially expressed in ESS and LMS, of which 76 were overexpressed in ESS and 18 overexpressed in LMS. Forty-nine miRNAs were differentially expressed in primary and metastatic LMS, of which 45 were overexpressed in primary LMS and 4 in metastases. Differential expression was confirmed for 10/20 miRNA analyzed using qPCR. Frizzled-6 silencing in SK-LMS-1 cells significantly inhibited cellular invasion, wound healing and MMP-2 activity. CONCLUSIONS: Differential miRNA signatures of ESS and LMS provide novel data regarding transcriptional regulation in these cancers, based on which new potential diagnostic markers, prognostic biomarkers and therapeutic targets may be explored. Differences in miRNA profiles of primary and metastatic LMS may improve our understanding of disease progression in this aggressive malignancy.


Assuntos
Neoplasias do Endométrio/genética , Leiomiossarcoma/genética , Leiomiossarcoma/secundário , MicroRNAs/análise , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/secundário , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Endométrio/patologia , Feminino , Receptores Frizzled/análise , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
15.
Hum Pathol ; 48: 95-101, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26640230

RESUMO

The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.


Assuntos
Biomarcadores Tumorais/genética , Cistadenocarcinoma Seroso/genética , Proteína Semelhante a ELAV 1/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Intervalo Livre de Doença , Proteína Semelhante a ELAV 1/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/análise , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Adulto Jovem
16.
Hum Pathol ; 46(9): 1245-56, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26216350

RESUMO

Ovarian cancer, consisting predominantly of ovarian carcinoma, is the eighth most common cancer in women and the most lethal gynecologic malignancy. Efforts focus on identifying biomarkers which may aid in early diagnosis and reduce mortality, as well as on characterizing therapeutic targets with the aim of circumventing chemoresistance and prolonging survival at advanced-stage disease. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression, and have been found to play an important role in ovarian carcinoma. Recent research has identified multiple miRNAs involved in the biology and progression of the disease, and supports a role for miRNAs as potential biomarkers, predictive markers and prognostic factors. Many of the studies published to date nevertheless suffer from critical weaknesses which affect data quality and reproducibility, including the comparison of normal ovaries to tumor tissue without compensation for the highly discrepant target cell fraction in these two specimen types and the inclusion of carcinomas of different histotypes, non-epithelial tumors or tumors of non-specified histology. These shortcomings highlight the critical role of pathologists as part of the team in the setting of such research. This review summarizes current knowledge in this area and discusses the potential clinical relevance of miRNAs in ovarian carcinoma, with focus on studies of clinical specimens in which tissue selection has been deemed adequate.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Técnicas de Diagnóstico Molecular , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Medicina de Precisão , Valor Preditivo dos Testes , Prognóstico
17.
J Enzyme Inhib Med Chem ; 30(5): 767-72, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25669348

RESUMO

Autotaxin is an extracellular, two zinc-centered enzyme that hydrolyzes lysophosphatidyl choline to lysophosphatidic acid, involved in various cancerous processes, e.g. migration, proliferation and tumor progression. We examined the autotaxin inhibitory properties of extended structure carbamoylphosphonates (CPOs) PhOC(6)H(4)SO(2)NH(CH(2))nNHCOPO(3)H(2), with increasing lengths of methylene chains, (CH(2))(n), n = 4-8. Carbamoylphosphonates having n = 6, 7, 8 inhibited autotaxin in vitro with IC(50) ≈ 1.5 µM. Using an imaging probe we demonstrated that compound n = 6 inhibits recombinant autotaxin activity in vitro and in vivo, following oral CPO administration. Additionally, daily oral administration of compound n = 7 inhibited over 90% of lung metastases in a murine melanoma metastasis model. Both the carbamoylphosphonates and the enzymes reside and interact in the extracellular space expecting minimal toxic side effects, and presenting a novel approach for inhibiting tumor proliferation and metastasis dissemination.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Organofosfonatos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Sulfonamidas/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Organofosfonatos/administração & dosagem , Organofosfonatos/química , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/química
18.
Carcinogenesis ; 35(9): 2113-20, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24925027

RESUMO

The objective of this study was to analyze the expression, biological role and clinical relevance of exosomal microRNAs (miRNAs) from ovarian carcinoma (OC) effusion supernatants. Exosomal miRNA expression profiling was performed using miRNA Taqman arrays. Selected miRNAs were validated using quantitative PCR in 86 OC effusion supernatants. The role of exosomal miRNA in this cancer was further studied using in vitro and in vivo models. miRNA profiling identified 99 miRNAs with high expression levels in exosomes from OC effusion supernatants. Quantitative PCR validation of 11 miRNAs showed significant associations with effusion site (peritoneum versus pleura) and International Federation of Gynecology and Obstetrics stage. In univariate survival analysis, high levels of miRNAs 21, 23b and 29a were associated with poor progression-free survival (P = 0.01, P = 0.015 and P = 0.009, respectively), whereas high expression of miRNA 21 correlated with poor overall survival (P = 0.017). The latter association was retained in Cox multivariate analysis (P = 0.001). Exposure of LP9 mesothelial cells and ES2 OC cells to OC effusion-derived exosomes inhibited tumor spheroid expansion and reduced mesothelial clearance area. Treatment of severe combined immunodeficiency mice with exosomes from OC effusions prior to injection of tumor cells was associated with larger tumor load, more infiltrative tumors and shorter survival. Patient-derived OC effusion exosomes contain multiple miRNAs, of which some may have clinical relevance. In experimental models, OC exosomes affect both tumor cells and cells in the tumor microenvironment and induce more aggressive disease. Collectively, these data demonstrate the central role of miRNAs and their content in the biology of this cancer.


Assuntos
Exossomos/metabolismo , MicroRNAs/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Líquido Ascítico/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Intervalo Livre de Doença , Exossomos/genética , Feminino , Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos SCID , MicroRNAs/genética , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Derrame Pleural Maligno/metabolismo , Modelos de Riscos Proporcionais , Transcriptoma , Carga Tumoral
19.
Hum Pathol ; 45(7): 1520-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24814803

RESUMO

The involvement of VICKZ proteins has been implicated in a large number of cancers. The aim of the present study was to investigate the biological and clinical role of VICKZ proteins in ovarian carcinoma (OC). VICKZ1-3 protein expression was analyzed in 82 serous OC specimens (51 effusions, 14 primary carcinomas, 17 solid metastases) by immunoblotting. Protein localization was studied using immunohistochemistry in 101 tumors (40 effusions, 25 primary carcinomas, 36 solid metastases). The effect of VICKZ silencing using short hairpin RNA on collagenolytic activity and invasion was assessed in ES-2 OC cells. VICKZ2 was the most frequently expressed family member in serous carcinomas. VICKZ levels measured by pan-VICKZ antibody were significantly higher in primary carcinomas and solid metastases compared to effusions (P < .001). In contrast, VICKZ1 and VICKZ2 were overexpressed in effusions compared to primary carcinomas and solid metastases (P = .016 and P = .024, respectively), and higher VICKZ2 expression in effusions was associated with shorter overall survival in univariate analysis (P = .01). All 3 proteins were localized to OC cells by immunohistochemistry, with tumor-specific expression observed for VICKZ1 and VICKZ2. VICKZ silencing in ES-2 cells led to reduced matrix metalloproteinase 9 activity and reduced invasion. In conclusion, VICKZ2 is the most frequently expressed VICKZ family member in serous OCs. VICKZ1 and VICKZ2 are overexpressed in effusions compared to primary carcinomas and solid metastases, suggesting a biological role at this anatomical site, and appear to have a role in proteolysis and invasion. VICKZ2 may be a prognostic marker in ovarian serous carcinoma effusions.


Assuntos
Líquido Ascítico/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Líquido Ascítico/patologia , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Taxa de Sobrevida
20.
Front Oncol ; 4: 104, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24860785

RESUMO

The tumor microenvironment, consisting of stromal myofibroblasts, endothelial cells, and leukocytes, is growingly perceived to be a major contributor to the pathogenesis and disease progression in practically all cancer types. Stromal myofibroblasts produce angiogenic factors, proteases, growth factors, immune response-modulating proteins, anti-apoptotic proteins, and signaling molecules, and express surface receptors and respond to stimuli initiated in the tumor cells to establish a bi-directional communication network in the microenvironment to promote tumor cell invasion and metastasis. Many of these molecules are candidates for targeted therapy and the cancer stroma has been recently regarded as target for biological intervention. This review provides an overview of the biology and clinical role of the stroma in ovarian cancer.

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