RESUMO
Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.
Assuntos
Adenoma/patologia , Neoplasias Hipofisárias/patologia , RNA Longo não Codificante/fisiologia , Adenoma/genética , Adenoma/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Ensaios de Migração Celular , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Proteína Forkhead Box M1/análise , Proteína Forkhead Box M1/metabolismo , Humanos , Janus Quinase 1/análise , Janus Quinase 1/metabolismo , Luciferases , MicroRNAs/análise , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/análise , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/análise , Serina-Treonina Quinases TOR/metabolismo , TransfecçãoRESUMO
BACKGROUND: Lymphangioleiomyomatosis (LAM) is a low-grade neoplasm characterized by the pulmonary infiltration of smooth muscle-like cells (LAM cells) and cystic destruction. Patients usually present with airway obstruction in pulmonary function tests (PFTs). Previous studies have shown correlations among histological parameters, lung function abnormalities and prognosis in LAM. We investigated the lung tissue expression of proteins related to the mTOR pathway, angiogenesis and enzymatic activity and its correlation with functional parameters in LAM patients. METHODS: We analyzed morphological and functional parameters of thirty-three patients. Two groups of disease severity were identified according to FEV1 values. Lung tissue from open biopsies or lung transplants was immunostained for SMA, HMB-45, mTOR, VEGF-D, MMP-9 and D2-40. Density of cysts, density of nodules and protein expression were measured by image analysis and correlated with PFT parameters. RESULTS: There was no difference in the expression of D2-40 between the more severe and the less severe groups. All other immunohistological parameters showed significantly higher values in the more severe group (p ≤ 0.002). The expression of VEGF-D, MMP-9 and mTOR in LAM cells was associated with the density of both cysts and nodules. The density of cysts and nodules as well as the expression of MMP-9 and VEGF-D were associated with the impairment of PFT parameters. CONCLUSIONS: Severe LAM represents an active phase of the disease with high expression of VEGF-D, mTOR, and MMP-9, as well as LAM cell infiltration. Our findings suggest that the tissue expression levels of VEGF-D and MMP-9 are important parameters associated with the loss of pulmonary function and could be considered as potential severity markers in open lung biopsies of LAM patients.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Serina-Treonina Quinases TOR/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/patologia , Metaloproteinase 9 da Matriz/análise , Pessoa de Meia-Idade , Estudos Retrospectivos , Serina-Treonina Quinases TOR/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Isocitrato Desidrogenase/análise , Proteínas Quinases S6 Ribossômicas/análise , Serina-Treonina Quinases TOR/análise , Regulação para Cima , Adulto , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/epidemiologia , Criança , Feminino , Glioblastoma/diagnóstico , Glioblastoma/epidemiologia , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Taxa de SobrevidaRESUMO
BACKGROUND: TNF-α is a key cytokine involved in prostate carcinogenesis and is mediated by the TNF-α receptor type 1 (TNFR-1). This receptor triggers two opposite pathways: cell death or cell survival and presents a protective or stimulator role in cancer. Thus, the purpose of this study was to evaluate the role of TNF signaling in chemically induced prostate carcinogenesis in mice. METHODS: C57bl/6 wild type (WT) and p55 TNFR-1 knockout mice (KO) were treated with mineral oil (control) or N-methyl N-nitrosurea (MNU) in association with testosterone (MNU+T, single injection of 40 mg/kg and weekly injection 2 mg/kg, respectively) over the course of 6 months. After this induction period, prostate samples were processed for histological and biochemical analysis. RESULTS: MNU+T treatment led to the development of prostate intraepithelial neoplasia (PIN) and adenocarcinoma (PCa) in both WT and KO animals; however, the incidence of PCa was lower in KO group than in WT. Cell proliferation analysis showed that PCNA levels were significantly lower in the KO group, even after carcinogenesis induction. Furthermore, the prostate of KO animals had lower levels of p65 and p-mTOR after treatment with MNU+T than WT. There was also a decrease in prostate androgen receptor levels after induction of carcinogenesis in both KO and WT mice. Regarding the extracellular matrix in the prostate, KO mice had higher levels of fibronectin and lower levels of matrix metalloproteinase 2 (MMP2) after carcinogenesis. Finally, there was a similar increase in apoptosis in both groups after carcinogenesis, indicating that the TNAFr1 pathway in prostate carcinogenesis presented proliferative, and not apoptotic, stimuli. CONCLUSIONS: TNF-α, through its receptor TNFR-1, promoted cell proliferation and cell survival in prostate by activation of the AKT/mTOR and NFKB pathway, which stimulated prostate carcinogenesis in chemically induced mice. Prostate 76: 917-926, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Carcinogênese , Neoplasias da Próstata , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Adenocarcinoma/patologia , Animais , Apoptose , Carcinogênese/patologia , Proliferação de Células , Sobrevivência Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/análise , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/química , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/análise , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Serina-Treonina Quinases TOR/análise , Serina-Treonina Quinases TOR/metabolismo , Fator de Transcrição RelA/análiseRESUMO
Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.