RESUMO
Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through ανß3 integrin, while it decreases the stimulatory effect of vascular endothelial growth factor A (VEGFA) and inhibits cell migration in the absence of ανß3 through unknown mechanism(s). In the present work, by using a multitude of experimental approaches, we show that PTN binds to VEGF receptor type 2 (VEGFR2) with a KD of 11.6 nM. Molecular dynamics approach suggests that PTN binds to the same VEGFR2 region with VEGFA through its N-terminal domain. PTN inhibits phosphorylation of VEGFR2 at Tyr1175 and still stimulates endothelial cell migration in the presence of a selective VEGFR2 tyrosine kinase inhibitor. VEGFR2 downregulation by siRNA or an anti-VEGFR2 antibody that binds to the ligand-binding VEGFR2 domain also induce endothelial cell migration, which is abolished by a function-blocking antibody against ανß3 or the peptide PTN112-136 that binds ανß3 and inhibits PTN binding. In cells that do not express ανß3, PTN decreases both VEGFR2 Tyr1175 phosphorylation and cell migration in a VEGFR2-dependent manner. Collectively, our data identify VEGFR2 as a novel PTN receptor involved in the regulation of cell migration by PTN and contribute to the elucidation of the mechanism of activation of endothelial cell migration through the interplay between VEGFR2 and ανß3.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Citocinas/metabolismo , Integrina alfaVbeta3/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteínas de Transporte/química , Linhagem Celular Tumoral , Citocinas/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Neovascularização Fisiológica , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Domínios Proteicos , Ratos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis. DESIGN: DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo. RESULTS: We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer. KMT2D is transcriptionally repressed in human pancreatic tumours through DNA methylation. Clinically, lower levels of this methyltransferase associate with poor prognosis and significant weight alterations. RNAi-based genetic inactivation of KMT2D promotes tumour growth and results in loss of H3K4me3 mark. In addition, KMT2D inhibition increases aerobic glycolysis and alters the lipidomic profiles of pancreatic cancer cells. Further analysis of this phenomenon identified the glucose transporter SLC2A3 as a mediator of KMT2D-induced changes in cellular, metabolic and proliferative rates. CONCLUSION: Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.
Assuntos
Carcinoma/enzimologia , Carcinoma/patologia , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Animais , Estudos de Casos e Controles , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Camundongos , Transplante de NeoplasiasRESUMO
Inflammatory bowel diseases (IBD) are chronic inflammatory gastrointestinal diseases, primarily consisting of ulcerative colitis and Crohn's disease. The complex nature of the disease, as well as the limited therapeutic options characterized by low efficiency and major side effects, highlights the importance of developing novel strategies of therapeutic intervention in IBD. Susceptibility loci related to IBD are present only in a small percentage of IBD patients, implying that epigenetic modifications could influence the pathogenesis of the disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate multiple molecular pathways involved in IBD pathobiology. MiRNA inhibitors targeting the IBD-activated miRNAs could have therapeutic value for IBD patients. This review provides an overview of the recent advances in miRNA biology related to IBD pathogenesis and the pharmacological development of miRNA-based therapeutics.
Assuntos
Terapia Genética/métodos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/terapia , MicroRNAs/genética , MicroRNAs/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Loci Gênicos , Marcadores Genéticos , Predisposição Genética para Doença , Terapia Genética/efeitos adversos , Humanos , Doenças Inflamatórias Intestinais/metabolismo , MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Transdução de Sinais , TranscriptomaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates and limited therapeutic options. Thus elucidation of signaling pathways involved in PDAC pathogenesis is essential for identifying novel potential therapeutic gene targets. Here, we used a systems approach to elucidate those pathways by integrating gene and microRNA profiling analyses together with CRISPR/Cas9 technology to identify novel transcription factors involved in PDAC pathogenesis. FOXA2 transcription factor was found to be significantly downregulated in PDAC relative to control pancreatic tissues. Functional experiments revealed that FOXA2 has a tumor suppressor function through inhibition of pancreatic cancer cell growth, migration, invasion, and colony formation. In situ hybridization analysis revealed miR-199a to be significantly upregulated in pancreatic cancer. Bioinformatics and luciferase analyses showed that miR-199a negatively but directly regulates FOXA2 expression through binding in its 3'-untranslated region (UTR). Evaluation of the functional importance of miR-199a on pancreatic cancer revealed that miR-199a acts as an inhibitor of FOXA2 expression, inducing an increase in pancreatic cancer cell proliferation, migration, and invasion. Additionally, gene ontology and network analyses in PANC-1 cells treated with a small interfering RNA (siRNA) against FOXA2 revealed an enrichment for cell invasion mechanisms through PLAUR and ERK activation. FOXA2 deletion (FOXA2Δ) by using two CRISPR/Cas9 vectors in PANC-1 cells induced tumor growth in vivo resulting in upregulation of PLAUR and ERK pathways in FOXA2Δ xenograft tumors. We have identified FOXA2 as a novel tumor suppressor in pancreatic cancer and it is regulated directly by miR-199a, thereby enhancing our understanding of how microRNAs interplay with the transcription factors to affect pancreatic oncogenesis.
Assuntos
Fator 3-beta Nuclear de Hepatócito/genética , Neoplasias Pancreáticas/genética , Transcriptoma , Proteínas Supressoras de Tumor/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Colite Ulcerativa/prevenção & controle , Colo/metabolismo , Neoplasias do Colo/prevenção & controle , MicroRNAs/metabolismo , Terapêutica com RNAi , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Azoximetano , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Linhagem Celular , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Proteínas com Domínio LIM/metabolismo , Camundongos , MicroRNAs/genética , NF-kappa B/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Pancreatic cancer is a devastating malignancy that ranks as the fourth leading cause of cancer-related deaths worldwide. Dismal prognosis is mainly attributable to limited knowledge of the molecular pathogenesis of the disease. miRNAs have been found to be deregulated in pancreatic cancer, affecting several steps of initiation and aggressiveness of the disease by regulating important signaling pathways, such as the KRAS and Notch pathways. Moreover, the effect of miRNAs on regulating cell cycle events and expression of transcription factors has gained a lot of attention. Recent studies have highlighted the application of miRNAs as biomarkers and therapeutic tools. The current review focuses on latest advances with respect to the roles of miRNAs in pancreatic ductal adenocarcinoma associated signaling pathways and miRNA-based therapeutics.
Assuntos
MicroRNAs/genética , Neoplasias Pancreáticas/genética , Transdução de Sinais/genética , Animais , Biomarcadores Tumorais/genética , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , HumanosRESUMO
BACKGROUND: Receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPß/ζ interacts with ανß3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, ß3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated ß3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανß3 integrin association and subsequent signaling. In the present work, we studied whether RPTPß/ζ mediates angiogenic actions of VEGF. METHODS: Human umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different ß3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPß/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 µm pores. RESULTS: RPTPß/ζ mediates VEGF165-induced c-Src-dependent ß3 Tyr773 phosphorylation, which is required for VEGFR2-ανß3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPß/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPß/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect. CONCLUSIONS: These data identify RPTPß/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies.
Assuntos
Ligação Proteica/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células CHO , Linhagem Celular , Movimento Celular/genética , Cricetulus , Regulação para Baixo/genética , Glioma , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrinas/genética , Integrinas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Mapas de Interação de Proteínas/genética , RNA Interferente Pequeno/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α(v)ß(3) integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α(v)ß(3) by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α(v)ß(3) and depended on the phosphorylation of ß(3) at Tyr(773) through receptor protein-tyrosine phosphatase ß/ζ (RPTPß/ζ) and c-Src activation. Downstream of α(v)ß(3,) PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α(v)ß(3) and RPTPß/ζ. Positive correlation of cell surface NCL and α(v)ß(3) expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α(v)ß(3). Collectively, these data suggest that both expression and ß(3) integrin phosphorylation at Tyr(773) determine the cell surface localization of NCL downstream of the RPTPß/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.
Assuntos
Movimento Celular , Células Endoteliais/citologia , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Células CHO , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Cricetinae , Citocinas/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Microscopia de Fluorescência/métodos , Neovascularização Patológica , Fosforilação , Ratos , Transdução de Sinais , NucleolinaRESUMO
Background and Aims: Decoding pancreatic ductal adenocarcinoma heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in pancreatic ductal adenocarcinoma progression. Methods: Global DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss-of-function and gain-of function studies and in vivo xenograft and the KPC (LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in 3 different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. Results: We identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. Conclusion: HNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.
RESUMO
Pleiotrophin (PTN) is a growth factor that appears to play an important role in prostate cancer growth and angiogenesis. We have previously shown that decreased PTN expression in human prostate cancer PC3 cells leads to decreased adhesion of prostate cancer cells to osteoblasts, suggesting that PTN mediates this interaction. In the current work, using peptides that correspond to different regions of the PTN protein, we identified that a domain responsible for the adhesion of prostate cancer cells to osteoblasts corresponds to amino acids 16-24 of the mature PTN protein. Given that a synthetic PTN16-24 peptide which disturbs the interaction of PTN with nucleolin (NCL) was found to inhibit prostate cancer cells' adhesion to osteoblasts, it seems that NCL mediates the cellular interactions involved in the adhesion process. Two pseudopeptides that bind to cell surface NCL and an anti-NCL antibody also decrease prostate cancer cell adhesion to osteoblasts to the same degree as PTN16-24, further supporting the involvement of cell surface NCL in this interaction. Collectively, our data suggest that NCL on the cell surface of osteoblasts may mediate adhesion of prostate cancer cells through PTN and identify peptides that could be exploited therapeutically to target this component of prostate cancer bone metastases.
Assuntos
Citocinas , Neoplasias da Próstata , Proteínas de Transporte , Adesão Celular , Citocinas/metabolismo , Humanos , Masculino , Osteoblastos/metabolismo , Fosfoproteínas , Neoplasias da Próstata/patologia , Proteínas de Ligação a RNA , NucleolinaRESUMO
The secreted growth factor pleiotrophin (PTN) is expressed in all species and is evolutionarily highly conserved, suggesting that it plays a significant role in the regulation of important processes. The observation that it is highly expressed at early stages during development and in embryonic progenitor cells highlights a potentially important contribution to development. There is ample evidence of the role of PTN in the development of the nervous system and hematopoiesis, some, albeit inconclusive, evidence of its role in the skeletomuscular system, and limited evidence of its role in the development of other organs. Studies on its role in the cardiovascular system and angiogenesis suggest that PTN has a significant regulatory effect by acting on endothelial cells, while its role in the functions of smooth or cardiac muscle cells has not been studied. This review highlights what is known to date regarding the role of PTN in the development of various organs and in angiogenesis. Wherever possible, evidence on the crosstalk between the receptors that mediate PTN's functions is also quoted, highlighting the complex regulatory pathways that affect development and angiogenesis.
Assuntos
Proteínas de Transporte , Células Endoteliais , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Humanos , Neovascularização PatológicaRESUMO
Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) ß(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) ß(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of ß(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) ß(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPß/ζ in endothelial cells and induced ß(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPß/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPß/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) ß(3) integrin.
Assuntos
Proteínas de Transporte/química , Citocinas/química , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Western Blotting , Proteínas de Transporte/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Integrina alfaVbeta3/metabolismo , Espectroscopia de Ressonância Magnética , Peptídeos/síntese química , Peptídeos/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismoRESUMO
We have previously shown that the angiogenic growth factor pleiotrophin (PTN) induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). In this study, we show that a monoclonal antibody against alpha(nu)beta(3) but not alpha(5)beta(1) integrin abolished PTN-induced human endothelial cell migration in a concentration-dependent manner. Integrin alpha(nu)beta(3) was found to directly interact with PTN in an RGD-independent manner, whereas a synthetic peptide corresponding to the specificity loop of the beta(3) integrin extracellular domain ((177)CYDMKTTC(184)) inhibited PTN-alpha(nu)beta(3) interaction and totally abolished PTN-induced endothelial cell migration. Interestingly, alpha(nu)beta(3) was also found to directly interact with RPTPbeta/zeta, and PTN-induced Y773 phosphorylation of beta(3) integrin was dependent on both RPTPbeta/zeta and the downstream c-src kinase activation. Midkine was found to interact with RPTPbeta/zeta, but not with alpha(nu)beta(3), and caused a small but statistically significant decrease in cell migration. In the same line, PTN decreased migration of different glioma cell lines that express RPTPbeta/zeta but do not express alpha(nu)beta(3), while it stimulated migration of U87MG cells that express alpha(nu)beta(3) on their cell membrane. Overexpression or down-regulation of beta(3) stimulated or abolished, respectively, the effect of PTN on cell migration. Collectively, these data suggest that alpha(nu)beta(3) is a key molecule that determines the stimulatory or inhibitory effect of PTN on cell migration.
Assuntos
Proteínas de Transporte/fisiologia , Movimento Celular/fisiologia , Citocinas/fisiologia , Integrina alfaVbeta3/fisiologia , Células Cultivadas , Humanos , Midkina , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Quinases da Família src/metabolismoRESUMO
Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and ανß3 integrin. Screening for proteins that interact with RPTPß/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPß/ζ interaction and revealed the molecular association of CDK5 and RPTPß/ζ. In endothelial cells, PTN activates CDK5 in an RPTPß/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανß3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-α]carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPß/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.
Assuntos
Proteínas de Transporte/farmacologia , Movimento Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/genética , Citocinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Carbazóis/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Guanina/análogos & derivados , Guanina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Roscovitina/farmacologia , Transdução de SinaisRESUMO
Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21-induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A-ERK network in mice. Mol Cancer Ther; 17(7); 1430-40. ©2018 AACR.
Assuntos
MicroRNAs/genética , Oligonucleotídeos Antissenso/administração & dosagem , Proteína Fosfatase 2/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , MicroRNAs/antagonistas & inibidores , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
International experts gathered at the Mayo Clinic (Rochester MN, USA) on February 27th-28th, 2017 for a meeting entitled 'Basic and Translational Facets of the Epigenetics of GI Diseases'. This workshop summarized recent advances on the role of epigenetics in the pathobiology of gastrointestinal (GI) diseases. Highlights of the meeting included recent advances on the involvement of different epigenetic mechanisms in malignant and nonmalignant GI disorders and the epigenetic heterogeneity exhibited in these diseases. The translational value of epigenetic drugs, as well as the current and future use of epigenetic changes (i.e., DNA methylation patterns) as biomarkers for early detection tools or disease stratification were also important topics of discussion.
Assuntos
Epigênese Genética , Gastroenteropatias/genética , Metilação de DNA , Heterogeneidade Genética , Marcadores Genéticos , Humanos , Pesquisa Translacional BiomédicaRESUMO
OBJECTIVE: Chronic HIV-1 infection leads to widespread inflammation and immune dysregulation. The gastrointestinal mucosa, a primary site for HIV-1 replication, is thought to play a significant role in this response. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression, including immune activation and inflammation. Here we investigate miR expression and function in the colonic mucosa during HIV-1 infection. DESIGN AND METHODS: Using miR profiling, we examined miR expression in the colonic mucosa of HIV-infected patients. These miRs were further parsed to identify those that most likely function in HIV-related inflammation. Using bioinformatics tools, we identified potential target genes which were confirmed using in-vitro functional testing. RESULTS: We identified 12 miRs that were differentially expressed in the colonic mucosa of HIV-infected patients with high versus undetectable plasma viral concentrations. Of these, both miR-26a and miR-29a were downregulated in untreated HIV-1 infection, yet not in the colonic mucosa from inflammatory bowel disease. This downregulation occurs within the first hours after infection. These miRs were further shown to directly target IL-6 and STAT3, respectively, with similar changes confirmed in an ex-vivo explant infection model. CONCLUSION: miR-26a and miR-29a levels are decreased in the colonic mucosa during chronic HIV-1 infection, and this change may be initiated during acute infection. Both miRs de-repress the IL-6/STAT3 signaling pathway, which could contribute to increased inflammation during infection. These miRs may represent novel therapeutic targets for HIV-1-associated inflammation in the colonic mucosa.
Assuntos
Colo/patologia , Perfilação da Expressão Gênica , Infecções por HIV/patologia , Mucosa Intestinal/patologia , MicroRNAs/análise , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-IdadeRESUMO
Vascular endothelial growth factor A (VEGF-A) is a key molecule in angiogenesis acting through VEGF receptors (VEGFRs), ανß3 integrin, receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and cell surface nucleolin (NCL). Pleiotrophin (PTN) stimulates endothelial cell migration and limits the angiogenic effects of VEGF-A165 to the levels of its own effect, possibly acting as a VEGF-A165 modifier. Since PTN and VEGF-A165 share receptors and actions on endothelial cells, in the present work we studied whether and how VEGF-A165 affects PTN expression or secretion. VEGF-A165 decreased PTN mRNA and protein levels acting at the transcriptional level. Bevacizumab, a selective VEGFR2 tyrosine kinase inhibitor and down-regulation of VEGFR2 expression by siRNA did not affect this decrease, suggesting that it is VEGFR-independent. VEGF-A121 also decreased PTN mRNA and protein levels, suggesting that heparin binding of VEGF-A165 is not involved. Blockage of cell surface NCL, lack of expression or mutation of ß3 integrin and down-regulation of RPTPß/ζ abolished the inhibitory effect of VEGF-A165 on PTN expression and secretion. Down-regulation of endogenous PTN in endothelial cells enhanced VEGF-A165-induced increase in migration and tube formation on matrigel. Collectively, these data suggest that VEGF-A down-regulates PTN expression and secretion through the RPTPß/ζ-ανß3-NCL axis to enhance its own effect on cell migration and further highlight the role of RPTPß/ζ in VEGF-A actions.
Assuntos
Proteínas de Transporte , Movimento Celular , Citocinas , Expressão Gênica/efeitos dos fármacos , Fragmentos de Peptídeos/fisiologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Bevacizumab/farmacologia , Western Blotting , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Cross-Talk/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The purpose of this paper is to describe a novel minimally invasive endovascular model of progressive myocardial hypertrophy in rabbits as an experimental protocol of hypertrophic cardiomyopathy. Nine New Zealand White rabbits underwent transauricular aortic catheterization. Under fluoroscopy a bare metal stent was partially deployed in the descending thoracic aorta (balloon length/stent length=1/2) so as to produce a funnel-shaped thoracic stent. Another nine animals underwent a sham procedure without stent placement (control). Follow-up computed tomography imaging was performed to exclude aortic occlusion. Subjects were killed after 3 months and their hearts were harvested and weighed. Cardiac hypertrophy was assessed with the heart weight-to-body weight (HW/BW) ratio and post-mortem histology was performed. We also used immunohistochemical staining for myogenin to compare the thickness of the wall between the two groups. The stents were polymer embedded for histomorphometry. Expressions of vascular endothelial growth factor (VEGF) and pleiotrophin (PTN) were analyzed by western blot analysis of total protein heart extracts. Computerized image analysis of CD34 and VEGF immunoreactivity was used to quantify myocardial angiogenesis. After 3 months, cross-sectional microscopic analysis of the harvested aortas showed total stent occlusion of the distal underdeployed area and some evidence of thrombus formation at the transitional zone toward the fully deployed stent in all cases. There was a nearly +10% increase of the adjusted HW/BW ratio compared with controls (absolute ratio difference was 0.02±0.01%; P=0.02). VEGF and CD34 expression was significantly suppressed, but expression of PTN was significantly increased in case of myocardial hypertrophy (stent group). Cardiac hypertrophy was evidenced using immunohistochemical staining for myogenin by significantly increased cardiomyocyte cross-sectional area (+38.4%; P<0.0001) compared with the control animals. In conclusion, this minimally invasive novel technique of transauricular funnel-shaped stent insertion in the descending thoracic aorta may achieve progressive myocardial hypertrophy in rabbits.
Assuntos
Cardiomegalia/etiologia , Modelos Animais de Doenças , Miocárdio/metabolismo , Animais , Aorta Torácica/diagnóstico por imagem , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/metabolismo , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Imuno-Histoquímica , Coelhos , Tomografia Computadorizada por Raios X , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A large number of mostly recent reports show enhanced expression of the multi-functional protein nucleolin (NCL) on the surface of activated lymphocytes, angiogenic endothelial and many different types of cancer cells. Translocation of NCL at the external side of the plasma membrane occurs via a secretory pathway independent of the endoplasmic reticulum-Golgi complex, requires intracellular intact actin cytoskeleton, and seems to be mediated by a variety of factors. Cell surface NCL serves as a binding partner of several molecules implicated in cell differentiation, adhesion, and leukocyte trafficking, inflammation, angiogenesis and tumor development, mediating their biological activities and in some cases, leading to their internalization. Accumulating evidence validates cell surface NCL as a strategic target for treatment of cancer, while its property of tumor-specific uptake of targeted ligands seems to be useful for the development of non-invasive imaging tools for the diagnosis of cancer and for the targeted release of chemotherapeutic drugs. The observation that cell surface NCL exists in complexes with several other proteins implicated in tumorigenesis and angiogenesis suggests that targeting cell surface NCL might trigger multi-inhibitory effects, depending on the cell type. This review summarizes papers and patents related to the redistribution and the biological functions of cell surface NCL, with emphasis on the potential importance and advantages of developing efficient anti-cell surface NCL strategies.