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BACKGROUND: Elevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM's role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC. RESULTS: Despite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells. CONCLUSION: This pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.
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Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Carcinoma Epitelial do Ovário , Progressão da Doença , Proteínas da Matriz Extracelular/urina , Feminino , Humanos , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologiaRESUMO
Epithelial ovarian cancer is a leading cause of death in gynecological cancers. While several systematic studies have revealed the mutation landscape of serous epithelial ovarian cancer, other non-serous subtypes of the disease have not been explored as extensively. Here we conduct exome sequencing of nine non-serous epithelial ovarian tumors (six endometrioid and three mucinous) and their corresponding normal DNA as well as a tumor-only granulosa cell sample. We integrated the exome data with targeted gene sequencing for 1,321 genes selected for their involvement in cancer from additional 28 non-serous ovarian tumors and compared our results to TCGA ovarian serous cystadenocarcinoma and uterine corpus endometrial carcinomas. Prevalence of TP53 mutations in non-serous was much lower than in serous epithelial OC, whereas the prevalence of PIK3CA, PIK3R1, PTEN, CTNNB1, ARID1A, and KRAS was higher. We confirmed the high prevalence of FOXL2 and KRAS mutations in granulosa cell tumors and in mucinous tumors, respectively. We also identified POLE proofreading domain mutations in three endometrioid ovarian tumors. These results highlight mutational differences between serous and non-serous ovarian cancers, and further distinguish different non-serous subtypes.
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Biomarcadores Tumorais , Cistadenocarcinoma Seroso/genética , Heterogeneidade Genética , Mutação , Neoplasias Ovarianas/genética , Adulto , Idoso , Cistadenocarcinoma Seroso/patologia , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , DNA Polimerase II/genética , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Sequenciamento do ExomaRESUMO
Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25D3) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D3 suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D3, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D3 as well as a 1,25D3-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages. This article is part of a Special Issue entitled "Vitamin D Workshop".
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Calcitriol/farmacologia , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Omento/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Calcitriol/metabolismo , Vitaminas/farmacologia , Animais , Carcinoma Epitelial do Ovário , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Omento/metabolismo , Omento/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
BACKGROUND: Early detection of epithelial ovarian cancer (OC) is necessary to overcome the high mortality rate of late stage diagnosis; and, examining the molecular changes that occur at early disease onset may provide new strategies for OC detection. Since the deregulation of inflammatory mediators can contribute to OC development, the purpose of this pilot study was to determine whether elevated urinary levels of Interleukin-1beta (IL-1 beta) are associated with OC and associated clinical parameters. METHODS: Urinary and serum levels of IL-1 beta were analyzed by ELISA from a patient cohort consisting of healthy women (N = 10), women with ovarian benign disease (N = 23), women with OC (N = 32), women with other benign gynecological conditions (N = 22), and women with other gynecological cancers (N = 6). RESULTS: Average urinary IL-1 beta levels tended to be elevated in ovarian benign (1.26 pg/ml) and OC (1.57 pg/ml) patient samples compared to healthy individuals (0.36 pg/ml). Among patients with benign disease, urinary IL-1ß levels were statistically higher in patients with benign inflammatory gynecologic disease compared to patients with non-inflammatory benign disease. Interestingly, urinary IL-1 beta levels tended to be 3-6x greater in patients with benign ovarian disease or OC as well as with a concomitant family history of ovarian and/or breast cancer compared to similar patients without a family history of ovarian and/or breast cancer. Lastly, there was a pattern of increased urinary IL-1 beta with increasing body mass index (BMI); patients with a normal BMI averaged urinary IL-1 beta levels of 0.92 pg/ml, overweight BMI averaged urinary IL-1 beta levels of 1.72 pg/ml, and obese BMI averaged urinary IL-1 beta levels of 5.26 pg/ml. CONCLUSIONS: This pilot study revealed that urinary levels of IL-1 beta are elevated in patients with epithelial OC supporting the thought that inflammation might be associated with cancer progression. Consequently, further studies of urinary IL-1 beta and the identification of an inflammatory profile specific to OC development may be beneficial to reduce the mortality associated with this disease.
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Biomarcadores Tumorais/urina , Interleucina-1beta/urina , Neoplasias Epiteliais e Glandulares/urina , Neoplasias Ovarianas/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Ovarianas/diagnóstico , Projetos Piloto , Adulto JovemRESUMO
MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.
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Histona Desacetilases/fisiologia , Proteína 2 Homóloga a MutS/metabolismo , Acetilação , Animais , Células Cultivadas , Células HEK293 , Células HeLa , Desacetilase 6 de Histona , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Camundongos , Estabilidade Proteica , UbiquitinaçãoRESUMO
OBJECTIVE: In epithelial ovarian cancer, concordance between results of microsatellite instability (MSI) and immunohistochemical (IHC) testing has not been demonstrated. This study evaluated the association of MSI-high (MSI-H) status with loss of expression (LoE) of mismatch repair (MMR) proteins on IHC and assessed for potential factors affecting the strength of the association. METHODS: Tumor specimens from three population-based studies of epithelial ovarian cancer were stained for MMR proteins through manual or automated methods, and results were interpreted by one of two pathologists. Tumor and germline DNA was extracted and MSI testing performed. Multivariable logistic regression models were fitted to predict loss of IHC expression based on MSI status after adjusting for staining method and reading pathologist. RESULTS: Of 834 cases, 564 (67.6%) were concordant; 41 were classified as MSI-H with LoE and 523 as microsatellite stable (MSS) with no LoE. Of the 270 discordant cases, 83 were MSI-H with no LoE and 187 were MSS with LoE. Both IHC staining method and reading pathologist were strongly associated with discordant results. CONCLUSIONS: Lack of concordance in the current study may be related to inconsistencies in IHC testing methods and interpretation. Results support the need for validation studies before routine screening of ovarian tumors is implemented in clinical practice for the purpose of identifying Lynch syndrome.
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Pareamento Incorreto de Bases , Reparo do DNA , Instabilidade de Microssatélites , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Idoso , Carcinoma Epitelial do Ovário , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Fallopian tube intraepithelial cancer is a postulated precursor of epithelial ovarian carcinomas. As research continues on epithelial ovarian carcinomas' developmental pathways, representative tubal tissue must be procured for diagnostic, biological, and molecular studies without compromising pathological diagnosis. MATERIALS AND METHODS: Fallopian tube fimbrial epithelia were harvested from postmenopausal women undergoing surgery for non-neoplastic gynecologic lesions (n = 16) and epithelial ovarian carcinomas (n = 6). Cytological imprints and washings were obtained from each fimbria and stained by Diff-Quik and rapid Papanicolaou for general cytomorphology; by Trypan blue for cell viability; and by rapid immunohistochemistry for evaluation of low molecular weight cytokeratin, MIB-1, p53, and high-mobility group A (HMGA2) expression. RESULTS: Benign and malignant tubal imprints harvests yielded means of 3.5 × 105 and 1.2 × 106 cells/fimbria, respectively, with viabilities higher than 85%. A mean of 2.5 × 105 cells/fimbria was obtained from fimbrial washings. The mean DNA, RNA, and protein contents of benign imprints were 2.4, 1.5, and 67 µg/fimbria, respectively. Benign cell populations contained nearly 97% cytokeratin-positive and p53/HMGA2-negative cells, which were dispersed within a watery to proteinaceous material and rare microcalcifications. Fimbrial imprints from serous carcinomas involving the fimbriae exhibited abnormal p53 and HMGA2 expression, high proliferation, and diagnostic criteria of malignancy, including prominent nucleoli and cell crowding. CONCLUSIONS: Ex vivo harvest from operative specimens allows for collection of cell populations representative of native fimbrial epithelium and free of significant contaminants. Tubal harvest facilitates triaging of cellular material for basic, clinical, and translational studies on cancer pathobiology and also represents a potential diagnostic adjunct to emerging in vivo high-resolution optical technologies.
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Over the last few decades, study of cancer in mouse models has gained popularity. Sophisticated genetic manipulation technologies and commercialization of these murine systems have made it possible to generate mice to study human disease. Given the large socio-economic burden of cancer, both on academic research and the health care industry, there is a need for in vivo animal cancer models that can provide a rationale that is translatable to the clinic. Such a bench-to-bedside transition will facilitate a long term robust strategy that is economically feasible and clinically effective to manage cancer. The major hurdles in considering mouse models as a translational platform are the lack of tumor heterogeneity and genetic diversity, which are a hallmark of human cancers. The present review, while critical of these pitfalls, discusses two newly emerging concepts of personalized mouse models called "Mouse Avatars" and Co-clinical Trials. Development of "Mouse Avatars" entails implantation of patient tumor samples in mice for subsequent use in drug efficacy studies. These avatars allow for each patient to have their own tumor growing in an in vivo system, thereby allowing the identification of a personalized therapeutic regimen, eliminating the cost and toxicity associated with non-targeted chemotherapeutic measures. In Co-clinical Trials, genetically engineered mouse models (GEMMs) are used to guide therapy in an ongoing human patient trial. Murine and patient trials are conducted concurrently, and information obtained from the murine system is applied towards future clinical management of the patient's tumor. The concurrent trials allow for a real-time integration of the murine and human tumor data. In combination with several molecular profiling techniques, the "Mouse Avatar" and Co-clinical Trial concepts have the potential to revolutionize the drug development and health care process. The present review outlines the current status, challenges and the future potential of these two new in vivo approaches in the field of personalized oncology.
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Modelos Animais de Doenças , Oncologia/tendências , Neoplasias/terapia , Medicina de Precisão/tendências , Animais , Humanos , Oncologia/métodos , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Medicina de Precisão/métodosRESUMO
Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as α-tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of α-tubulin. We have identified two novel ERK-mediated phosphorylation sites: threonine 1031 and serine 1035 in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas Ser-1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration compared with those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. These data indicate that ERK/HDAC6-mediated cell motility is through deacetylation of α-tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling.
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Movimento Celular/fisiologia , Histona Desacetilases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Células CHO , Cricetinae , Cricetulus , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Mutantes , Proteína Quinase 3 Ativada por Mitógeno/genética , Tubulina (Proteína)/genéticaRESUMO
BACKGROUND: Utility of immunohistochemistry (IHC) for mismatch repair (MMR) protein expression has been demonstrated in colorectal cancer but remains incompletely defined in ovarian cancer. We evaluated MMR protein expression in three population-based samples of epithelial ovarian cancers. MATERIALS AND METHODS: IHC staining was performed on full-section (FS) or tissue microarray (TMA) slides for MLH1, MSH2, and MSH6 expression. RESULTS: Out of 487 cases, 147 and 340 were performed through FS and TMA, respectively. Overall, Loss of Expression (LoE) of at least one MMR protein was observed in 12.7% based on an expression score of ≤3 (on a scale of 9). Notably, LoE was significantly higher in TMAs (17.9%) compared to FS cases (0.7%) (p<0.001). CONCLUSION: A substantial proportion of epithelial ovarian cancers have a loss of MMR protein expression. Protein expression results vary significantly by the tissue sampling methodology utilized, raising concerns about the clinical utility of this test for ovarian tumors.
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Biomarcadores Tumorais/análise , Imuno-Histoquímica/métodos , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Análise Serial de Tecidos/métodos , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Carcinoma Epitelial do Ovário , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Feminino , Humanos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/análise , Proteína 2 Homóloga a MutS/biossíntese , Proteínas Nucleares/análise , Proteínas Nucleares/biossínteseRESUMO
BACKGROUND: More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant. METHODOLOGY/PRINCIPAL FINDINGS: We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates. RESULTS: The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%. CONCLUSIONS/SIGNIFICANCE: Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical presentation, to distinguish between women with ovarian cancer and those with benign conditions with shared symptoms and features.
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Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Fosfolipídeos/sangue , Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário , Feminino , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Neoplasias Epiteliais e Glandulares/classificação , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/patologia , Sensibilidade e Especificidade , Máquina de Vetores de SuporteRESUMO
Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).
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Calcitriol/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Neoplasias/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/biossíntese , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Feminino , Genoma , Humanos , Camundongos , Camundongos Nus , MicroRNAs/fisiologia , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , RNA não Traduzido/metabolismoRESUMO
Histone deacetylase inhibitors (HDACi) are promising therapeutic agents which are currently used in combination with chemotherapeutic agents in clinical trials for cancer treatment including non-small cell lung cancer (NSCLC). However, the mechanisms underlying their anti-tumor activities remain elusive. Previous studies showed that inhibition of HDAC6 induces DNA damage and sensitizes transformed cells to anti-tumor agents such as etoposide and doxorubicin. Here, we showed that depletion of HDAC6 in two NSCLC cell lines, H292 and A549, sensitized cells to cisplatin, one of the first-line chemotherapeutic agents used to treat NSCLC. We suggested that depletion of HDAC6 increased cisplatin-induced cytotoxicity was due to the enhancement of apoptosis via activating ATR/Chk1 pathway. Furthermore, we showed that HDAC6 protein levels were positively correlated with cisplatin IC(50) in 15 NSCLC cell lines. Lastly, depletion of HDAC6 in H292 xenografts rendered decreased tumor weight and volume and exhibited increased basal apoptosis compared with the controls in a xenograft mouse model. In summary, our findings suggest that HDAC6 is positively associated with cisplatin resistance in NSCLC and reveal HDAC6 as a potential novel therapeutic target for platinum refractory NSCLC.
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Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Dano ao DNA , Histona Desacetilases/genética , Histona Desacetilases/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Ciclo Celular , Linhagem Celular Tumoral , Ensaio Cometa , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Feminino , Desacetilase 6 de Histona , Humanos , Immunoblotting , Imuno-Histoquímica/métodos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.
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Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , RNA Neoplásico/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , MicroRNAs/genética , Proteína Homeobox Nanog , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone.
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Calcitriol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Ovarianas/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclinas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/genética , Feminino , Genes Reporter , Humanos , Íntrons , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Transdução de Sinais , Elemento de Resposta à Vitamina DRESUMO
Separating adenocarcinoma of the lung from non-pulmonary adenocarcinoma or malignant mesothelioma is difficult, especially in cytology specimens. Consequently, it is important to identify markers that may facilitate this distinction. Thyroid transcription factor-1 (TTF-1) is a homeodomain containing transcription factor expressed selectively in the thyroid, lung, and diencephalon. TTF-1 is also expressed in adenocarcinomas of the lung and is widely used as a pulmonary adenocarcinoma marker in surgical specimens. However, the utility of TTF-1 has rarely been investigated in cytology. In this study, we evaluated the expression of TTF-1 in malignant pleural effusions. The primary tumors included 26 pulmonary adenocarcinomas, 26 non-pulmonary adenocarcinomas (13 breast, 5 ovarian, 2 gastric, 2 prostatic, 1 esophageal, 1 colonic, 1 pancreatic and 1 renal) and 4 malignant mesotheliomas. Immunocytochemistry was performed on sections of cell blocks, using a mouse monoclonal TTF-1 antibody (clone 8G7G3/1) and a biotin-streptavidin detection system. Nuclear immunoreactivity for TTF-1 was detected in 19 pulmonary adenocarcinomas. All non-pulmonary adenocarcinomas and malignant mesotheliomas were negative. These data indicate that TTF-1 maintains its sensitivity (73%) and specificity (100%) in cell block preparations and is useful in separating adenocarcinoma of the lung from non-pulmonary adenocarcinoma and malignant mesothelioma in cytology specimens.
Assuntos
Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/análise , Neoplasias Pulmonares/diagnóstico , Proteínas Nucleares/biossíntese , Derrame Pleural Maligno/metabolismo , Fatores de Transcrição/biossíntese , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mesotelioma/diagnóstico , Derrame Pleural Maligno/etiologia , Sensibilidade e Especificidade , Fator Nuclear 1 de TireoideRESUMO
OBJECTIVE: The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC. METHODS: VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. RESULTS: AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements. CONCLUSIONS: Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted.