RESUMO
BACKGROUND: Primary localized cutaneous amyloidosis (PLCA) is a chronic skin disease characterized by aberrant keratinocyte differentiation, epidermal hyperproliferation, and amyloid deposits. Previously, we demonstrated OSMR loss-function mutants enhanced basal keratinocyte differentiation through the OSMR/STAT5/KLF7 signaling in PLCA patients. OBJECTIVE: To investigate the underlying mechanisms involved in basal keratinocyte proliferation in PLCA patients that remain unclear. METHODS: Patients with pathologically confirmed PLCA visiting the dermatologic outpatient clinic were involved in the study. Laser capture microdissection and mass spectrometry analysis, gene-edited mice, 3D human epidermis culture, flow cytometry, western blot, qRT-PCR and RNA sequencing were used to explore the underlying molecular mechanisms. RESULTS: In this study, we found that AHNAK peptide fragments were enriched in the lesions of PLCA patients, as detected by laser capture microdissection and mass spectrometry analysis. The upregulated expression of AHNAK was further confirmed using immunohistochemical staining. qRT-PCR and flow cytometry revealed that pre-treatment with OSM can inhibit AHNAK expression in HaCaT cells, NHEKs, and 3D human skin models, but OSMR knockout or OSMR mutations abolished this down-regulation trend. Similar results were obtained in wild-type and OSMR knockout mice. More importantly, EdU incorporation and FACS assays demonstrated the knockdown of AHNAK could induce G1 phase cell cycle arrest and inhibit keratinocyte proliferation. Furthermore, RNA sequencing revealed that AHNAK knockdown regulated keratinocyte differentiation. CONCLUSION: Taken together, these data indicated that the elevated expression of AHNAK by OSMR mutations led to hyperproliferation and overdifferentiation of keratinocytes, and the discovered mechanism might provide insights into potential therapeutic targets for PLCA.
Assuntos
Amiloidose Familiar , Dermatopatias Genéticas , Humanos , Animais , Camundongos , Dermatopatias Genéticas/patologia , Pele/patologia , Amiloidose Familiar/genética , Queratinócitos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/genéticaRESUMO
Mutations in the tumor suppressor M receptor (OSMR) gene are associated with primary localized cutaneous amyloidosis (PLCA). Recently, we confirmed that OSMR loss-of-function mutations enhance epidermal keratinocyte differentiation via inactivation of the STAT5/KLF7 signaling. However, no disease model was available for PLCA. Accordingly, we generated an OSMR c.1538G > A mutant human embryonic stem cell line (SMUDHe010-A-82) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human skin organoid, which may provide a disease model for PLCA.
Assuntos
Células-Tronco Embrionárias Humanas , Sistemas CRISPR-Cas/genética , Linhagem Celular , Recombinação Homóloga , Homozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Mutação/genética , Subunidade beta de Receptor de Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/metabolismoRESUMO
Oncostatin M (OSM) is an IL-6 family member which exerts neuroprotective and remyelination-promoting effects after damage to the central nervous system (CNS). However, the role of OSM in neuro-inflammation is poorly understood. Here, we investigated OSM's role in pathological events important for the neuro-inflammatory disorder multiple sclerosis (MS). We show that OSM receptor (OSMRß) expression is increased on circulating lymphocytes of MS patients, indicating their elevated responsiveness to OSM signalling. In addition, OSM production by activated myeloid cells and astrocytes is increased in MS brain lesions. In experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, OSMRß-deficient mice exhibit milder clinical symptoms, accompanied by diminished T helper 17 (Th17) cell infiltration into the CNS and reduced BBB leakage. In vitro, OSM reduces BBB integrity by downregulating the junctional molecules claudin-5 and VE-cadherin, while promoting secretion of the Th17-attracting chemokine CCL20 by inflamed BBB-endothelial cells and reactive astrocytes. Using flow cytometric fluorescence resonance energy transfer (FRET) quantification, we found that OSM-induced endothelial CCL20 promotes activation of lymphocyte function-associated antigen 1 (LFA-1) on Th17 cells. Moreover, CCL20 enhances Th17 cell adhesion to OSM-treated inflamed endothelial cells, which is at least in part ICAM-1 mediated. Together, these data identify an OSM-CCL20 axis, in which OSM contributes significantly to BBB impairment during neuro-inflammation by inducing permeability while recruiting Th17 cells via enhanced endothelial CCL20 secretion and integrin activation. Therefore, care should be taken when considering OSM as a therapeutic agent for treatment of neuro-inflammatory diseases such as MS.
Assuntos
Barreira Hematoencefálica , Encefalomielite Autoimune Experimental , Oncostatina M , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Oncostatina M/metabolismo , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/biossíntese , Subunidade beta de Receptor de Oncostatina M/genética , Células Th17/metabolismo , Células Th17/patologiaRESUMO
Chronic itch is a major symptom of many inflammatory skin diseases. This type of pruritus is thought to be facilitated by cytokines that activate cutaneous nerve fibers; however, the molecular components and mechanisms involved are poorly understood. We found that the cytokine oncostatin M (OSM) is highly up-regulated in psoriasis, atopic dermatitis, and cutaneous T cell lymphoma, diseases associated with chronic itch. OSM receptor (OSMR) is expressed by itch-selective natriuretic polypeptide B (Nppb) neurons, and single-cell sequencing showed that OSM is mainly produced by dermal T cells and monocytes. Unlike canonical pruritogens, OSM does not activate sensory neurons. Instead, it sensitizes neurons by potentiating neural responses to pruritogens and by enhancing neural excitability. Knockout of OSMR in sensory neurons attenuated OSM-sensitized itch and inflammatory itch in mice, and pharmacological antagonism of the OSMR complex effectively alleviated pruritus in experimental inflammatory dermatitis in a rodent model. Together, our results uncover OSM as an itch neuromodulator and reveal OSM signal transduction as a potential target for antipruritic therapy.
Assuntos
Prurido , Células Receptoras Sensoriais , Animais , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/genética , Células Receptoras Sensoriais/efeitos dos fármacos , Transdução de Sinais , PeleRESUMO
BACKGROUND: Oncostatin M (OSM), an interleukin-6 (IL-6) family proinflammatory cytokine, plays a critical role in inflammatory skin diseases, but its mechanism of action is not well understood. OBJECTIVE: To demonstrate the mechanism of OSM induced pyropotosis in normal human epidermal keratinocytes (NHEKs) and immortalized human keratinocytes (HaCaT cells). METHODS: NHEKs and HaCaT cells were treated with OSM. Knockout of OSM receptor (OSMR) with CRISPR/Cas9 system, knockdown of GSDME with small interfering RNA and primary keratinocytes from Osmr-/- and Gsdme-/- mice were used to study the effect of OSMR and GSDME. After treatment of OSM, NHEKs and HaCaT cells were irradiated with UVB. The mRNA was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and RNA sequencing, protein level was detected by Western Blotting, Elisa and immunofluorescence. Cell death was examined by lactate dehydrogenase (LDH) releasing. RESULTS: Here we found that OSM induced pyropotosis in NHEKs and HaCaT cells, but knockout of OSMR abolished pyropotosis. RNA sequencing revealed an upregulation of several key genes involved in NLRP3 inflammasome activation following OSM treatment, among which NLRP3, GSDME, and IL-1ß were confirmed by qRT-PCR and Western Blotting. Knockdown of GSDME alleviated OSM-induced pyropotosis. Pretreatment of OSM boosted UVB-induced pyroptosis and inflammation in NHEKs and HaCaT cells, and this priming function was lost in keratinocytes of Osmr-/- and Gsdme-/- mice. Similar results were obtained in a 3-dimensional culture of human epidermis. CONCLUSION: OSM functions as a priming cytokine to enhance UVB-induced inflammation in keratinocytes, providing insight into the pathogenesis of inflammatory skin diseases.
Assuntos
Queratinócitos/efeitos da radiação , Oncostatina M/farmacologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/efeitos dos fármacos , Raios Ultravioleta , Animais , Epiderme/metabolismo , Técnicas de Inativação de Genes , Células HaCaT , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Subunidade beta de Receptor de Oncostatina M/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptose/genética , Piroptose/efeitos da radiação , RNA Interferente Pequeno , Regulação para CimaRESUMO
The mesenchymal subtype of glioblastoma is thought to be determined by both cancer cell-intrinsic alterations and extrinsic cellular interactions, but remains poorly understood. Here, we dissect glioblastoma-to-microenvironment interactions by single-cell RNA sequencing analysis of human tumors and model systems, combined with functional experiments. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived oncostatin M (OSM) that interacts with its receptors (OSMR or LIFR) in complex with GP130 on glioblastoma cells and activates STAT3. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications.
Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Glioblastoma/imunologia , Glioblastoma/patologia , Linfócitos T/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Neoplasias Encefálicas/genética , Células Cultivadas , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citotoxicidade Imunológica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oncostatina M/metabolismo , Subunidade beta de Receptor de Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/patologiaRESUMO
OBJECTIVES: This study aimed to determine whether mRNA expression of oncostatin-M (OSM) and its receptor (OSMR) in initial, pre-treatment intestinal biopsies is predictive of response to tumor necrosis factor antagonists (anti-TNF) in a pediatric inflammatory bowel disease (IBD) cohort. Secondary outcomes correlated OSM and OSMR expression with demographic variables; IBD type, extent, phenotype, and severity; laboratory values; and endoscopic findings. METHODS: A retrospective chart review was conducted on 98 pediatric patients. Patients' clinical courses were stratified as follows: failed anti-TNF (nâ=â14), quiescent on anti-TNF (nâ=â36), anti-TNF naïve (nâ=â19), and age-matched non-IBD controls (nâ=â29). The mRNA from each patient's pre-treatment ileal or colonic biopsy was isolated, and expression of OSM and OSMR was analyzed. RESULTS: There was no difference in OSM or OSMR expression among the three IBD groups; however, expression was significantly higher in patients with IBD than non-IBD controls (Pâ<â0.001). OSM and OSMR were more highly expressed in patients with ulcerative colitis (UC) with a Mayo score of 3 (Pâ=â0.0092 and Pâ=â0.0313, respectively). High OSM expression correlated with severe disease activity indices at diagnosis (Pâ=â0.002), anemia at diagnosis (Pâ=â0.0236), and need for immunomodulators (Pâ=â0.0193) and steroids (Pâ=â0.0273) during patients' clinical courses. CONCLUSIONS: OSM and OSMR expression were not predictive of response to anti-TNF in our pediatric cohort. OSM expression did correlate with IBD compared with healthy controls as well as with several clinical indicators of severe IBD.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Subunidade beta de Receptor de Oncostatina M/genética , Oncostatina M/genética , Criança , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Estudos Retrospectivos , Inibidores do Fator de Necrose TumoralRESUMO
BACKGROUND: Invasive ductal carcinoma (IDC) is a serious problem for patients as it metastasizes, decreasing 5-year patient survival from > 95 to ~ 27%. The breast tumor microenvironment (TME) is often saturated with proinflammatory cytokines, such as oncostatin M (OSM), which promote epithelial-to-mesenchymal transitions (EMT) in IDC and increased metastasis. The extracellular matrix (ECM) also plays an important role in promoting invasive and metastatic potential of IDC. Specifically, the reorganization and alignment of collagen fibers in stromal ECM leads to directed tumor cell motility, which promotes metastasis. Lysyl oxidase like-2 (LOXL2) catalyzes ECM remodeling by crosslinking of collagen I in the ECM. We propose a novel mechanism whereby OSM induces LOXL2 expression, mediating stromal ECM remodeling of the breast TME. METHODS: Bioinformatics was utilized to determine survival and gene correlation in patients. IDC cell lines were treated with OSM (also IL-6, LIF, and IL-1ß) and analyzed for LOXL2 expression by qRT-PCR and immunolabelling techniques. Collagen I contraction assays, 3D invasion assays, and confocal microscopy were performed with and without LOXL2 inhibition to determine the impact of OSM-induced LOXL2 on the ECM. RESULTS: Our studies demonstrate that IDC patients with high LOXL2 and OSM co-expression had worse rates of metastasis-free survival than those with high levels of either, individually, and LOXL2 expression is positively correlated to OSM/OSM receptor (OSMR) expression in IDC patients. Furthermore, human IDC cells treated with OSM resulted in a significant increase in LOXL2 mRNA, which led to upregulated protein expression of secreted, glycosylated, and enzymatically active LOXL2. The expression of LOXL2 in IDC cells did not affect OSM-promoted EMT, and LOXL2 was localized to the cytoplasm and/or secreted. OSM-induced LOXL2 promoted an increase in ECM collagen I fiber crosslinking, which led to significant fiber alignment between cells and increased IDC cell invasion. CONCLUSIONS: Aligned collagen fibers in the ECM provide pathways for tumor cells to migrate more easily through the stroma to nearby vasculature and tissue. These results provide a new paradigm through which proinflammatory cytokine OSM promotes tumor progression. Understanding the nuances in IDC metastasis will lead to better potential therapeutics to combat against the possibility.
Assuntos
Aminoácido Oxirredutases/metabolismo , Neoplasias da Mama/metabolismo , Matriz Extracelular/metabolismo , Oncostatina M/metabolismo , Aminoácido Oxirredutases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Glicosilação , Humanos , Inflamação , Metástase Neoplásica , Oncostatina M/genética , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/metabolismo , Prognóstico , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima/genéticaRESUMO
Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.
Assuntos
Suscetibilidade a Doenças , Enterocolite/etiologia , Enterocolite/metabolismo , Doença de Hirschsprung/complicações , Doença de Hirschsprung/genética , Subunidade beta de Receptor de Oncostatina M/genética , Transdução de Sinais , Alelos , Enterocolite/patologia , Expressão Gênica , Frequência do Gene , Variação Genética , Genótipo , Doença de Hirschsprung/diagnóstico , Humanos , Modelos Moleculares , Subunidade beta de Receptor de Oncostatina M/química , Subunidade beta de Receptor de Oncostatina M/metabolismo , Conformação Proteica , Proteômica/métodos , Relação Estrutura-Atividade , Sequenciamento do Exoma , Sequenciamento Completo do GenomaAssuntos
Amiloidose Familiar/genética , Queratinócitos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Subunidade beta de Receptor de Oncostatina M/genética , Oncostatina M/genética , Fator de Transcrição STAT5/genética , Dermatopatias Genéticas/genética , Amiloidose Familiar/metabolismo , Amiloidose Familiar/patologia , Animais , Sequência de Bases , Diferenciação Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Queratinócitos/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anotação de Sequência Molecular , Mutação , Oncostatina M/metabolismo , Subunidade beta de Receptor de Oncostatina M/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Dermatopatias Genéticas/metabolismo , Dermatopatias Genéticas/patologiaRESUMO
BACKGROUND: Feiyangchangweiyan capsule (FYC) is a traditional Chinese medicine formulation used in the clinical treatment of acute and chronic gastroenteritis and bacterial dysentery. However, the effect of FYC on ulcerative colitis (UC) and the mechanism thereof remains unknown. PURPOSE: To investigate the protective effect of FYC on UC mice induced by dextran sulfate sodium and illustrate the potential mechanism of this effect. METHODS: Here, we established a model of UC mice by dextran sulfate sodium and administered with FYC. The disease activity index (DAI), colon length, myeloperoxidase (MPO) content in serum, pathological structure and ultrastructural changes, and inflammatory cell infiltration of colon tissue were evaluated. Transcriptome and 16S rDNA sequencing were employed to illuminate the mechanism of FYC in the protection of UC mice. RESULTS: FYC significantly alleviates the pathological damage and the infiltration of inflammatory cells in colon tissue of dextran sulfate sodium induced UC mice, rescues shortened colon length, reduces DAI score, MPO content in serum, and pro-inflammatory factors including IL-1ß, IL-6, CCL11, MCP-1 and MIP-2, and increases anti-inflammatory factors such as IL-10. Transcriptomics revealed that Oncostatin M (OSM) and its receptor (OSMR) are the critical pathway for UC treatment by FYC. OSM and OSMR increased in UC mice compared to control mice, and decreased with FYC, which was verified via measurement of OSM and OSMR mRNA and protein levels. Furthermore, we observed that FYC modulates intestinal microbiome composition (e.g., the proportion of Barnesiella/Proteobacteria) by affecting the inflammatory factors. CONCLUSION: FYC exerts an effect on UC by inhibiting the OSM/OSMR pathway and regulating inflammatory factors to improve the intestinal flora.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Subunidade beta de Receptor de Oncostatina M/metabolismo , Oncostatina M/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Cápsulas , Quimiocinas/sangue , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/microbiologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Colo/ultraestrutura , Citocinas/sangue , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Microbioma Gastrointestinal/genética , Masculino , Camundongos Endogâmicos C57BL , Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/genética , Substâncias Protetoras/farmacologiaRESUMO
BACKGROUND: Scleroderma (SSc) is a rare autoimmune disease characterized by vascular impairment and progressive fibrosis of the skin and other organs. Oncostatin M, a member of the IL-6 family, is elevated in SSc serum and was recognized as a significant player in various stages of fibrosis. The goal of this study was to assess the contribution of the OSM/OSMRß pathway to endothelial cell (EC) injury and activation in SSc. METHODS: IHC and IF were used to assess the distribution of OSM and OSMRß in SSc (n = 14) and healthy control (n = 7) skin biopsies. Cell culture experiments were performed in human dermal microvascular endothelial cells (HDMECs) and included mRNA and protein analysis, and cell migration and proliferation assays. Ex vivo skin organoid culture was used to evaluate the effect of OSM on perivascular fibrosis. RESULTS: OSMRß protein was elevated in dermal ECs and in fibroblasts of SSc patients. Treatments of HDMECs with OSM or IL-6+sIL-6R have demonstrated that both cytokines similarly stimulated proinflammatory genes and genes related to endothelial to mesenchymal transition (EndMT). OSM was more effective than IL-6+sIL-6R in inducing cell migration, while both treatments similarly induced cell proliferation. The effects of OSM were mediated via OSMRß and STAT3, while the LIFR did not contribute to these responses. Both OSM and IL-6+sIL-6R induced profibrotic gene expression in HDMECs, as well as expansion of the perivascular PDGFRß+ cells in the ex vivo human skin culture system. Additional studies in HDMECs showed that siRNA-mediated downregulation of FLI1 and its close homolog ERG resulted in increased expression of OSMRß in HDMECs. CONCLUSIONS: This work provides new insights into the role of the OSM/OSMRß axis in activation/injury of dermal ECs and supports the involvement of this pathway in SSc vascular disease.
Assuntos
Subunidade beta de Receptor de Oncostatina M , Escleroderma Sistêmico , Células Endoteliais , Fibrose , Humanos , Oncostatina M , Subunidade beta de Receptor de Oncostatina M/genéticaRESUMO
BACKGROUND & AIMS: Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. METHODS: Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. RESULTS: Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966-0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. CONCLUSIONS: LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR-extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Proteínas do Citoesqueleto/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Subunidade beta de Receptor de Oncostatina M/genética , Neoplasias Pancreáticas/genética , Fator de Transcrição Sp1/genética , Transcriptoma , Adulto Jovem , Gencitabina , Neoplasias PancreáticasRESUMO
Glioblastoma (GBM) is characterized by extensive tumor cell invasion, angiogenesis, and proliferation. We previously established subclones of GBM cells with distinct invasive phenotypes and identified annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we further explored the role of ANXA2 in regulating phenotypic transition in GBM. We identified oncostatin M receptor (OSMR) as a key ANXA2 target gene in GBM utilizing microarray analysis and hierarchical clustering analysis of the Ivy Glioblastoma Atlas Project and The Cancer Genome Atlas datasets. Overexpression of ANXA2 in GBM cells increased the expression of OSMR and phosphorylated signal transducer and activator of transcription 3 (STAT3) and enhanced cell invasion, angiogenesis, proliferation, and mesenchymal transition. Silencing of OSMR reversed the ANXA2-induced phenotype, and STAT3 knockdown reduced OSMR protein expression. Exposure of GBM cells to hypoxic conditions activated the ANXA2-STAT3-OSMR signaling axis. Mice bearing ANXA2-overexpressing GBM exhibited shorter survival times compared with control tumor-bearing mice, whereas OSMR knockdown increased the survival time and diminished ANXA2-mediated tumor invasion, angiogenesis, and growth. Further, we uncovered a significant relationship between ANXA2 and OSMR expression in clinical GBM specimens, and demonstrated their correlation with tumor histopathology and patient prognosis. Our results indicate that the ANXA2-STAT3-OSMR axis regulates malignant phenotypic changes and mesenchymal transition in GBM, suggesting that this axis is a promising therapeutic target to treat GBM aggressiveness.
Assuntos
Anexina A2/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Subunidade beta de Receptor de Oncostatina M/genética , Fator de Transcrição STAT3/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anexina A2/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células/genética , Criança , Cães , Transição Epitelial-Mesenquimal/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Subunidade beta de Receptor de Oncostatina M/metabolismo , Fenótipo , Receptores de Oncostatina M/genética , Receptores de Oncostatina M/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Hipóxia Tumoral/genéticaRESUMO
AIMS: Metaplastic breast carcinoma (MBC) is a rare type of triple-negative breast cancer that shows vast histological and genetic heterogeneity. Osseous differentiation can be found in different subtypes of MBC. Whether MBCs with osseous differentiation are underpinned by specific genetic alterations has yet to be defined. The aim of this study was to investigate the repertoire of somatic mutations and copy number alterations (CNAs) in three MBCs with extensive osseous differentiation. METHODS AND RESULTS: Tumour and normal DNA samples from three MBCs with extensive osseous differentiation were subjected to whole-exome sequencing. Somatic mutations, CNAs and mutational signatures were determined by use of a validated bioinformatics pipeline. Our analyses revealed clonal TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele coupled with mutations affecting genes related to the Wnt and/or the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathways in all cases analysed. All cases showed a dominant mutational signature 1, with two cases showing a secondary signature 3 in addition to other features of homologous recombination DNA repair defects. The oncostatin M receptor gene, which plays a role in mesenchymal differentiation and bone formation, was found to be mutated in two MBCs with extensive osseous differentiation and in none of 35 previously published 35 MBCs. CONCLUSION: Our findings suggest that MBCs with osseous differentiation have somatic mutations similar to those of other forms of MBC.
Assuntos
Sequenciamento do Exoma , Fosfatidilinositol 3-Quinases/genética , Neoplasias de Mama Triplo Negativas , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteína Oncogênica v-akt/genética , Subunidade beta de Receptor de Oncostatina M/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Hepatic myofibroblasts (MFs) can originate from hepatic stellate cells, portal fibroblasts, or bone marrow-derived mesenchymal stem cells and can migrate towards the site of injury by aligning with nascent and established fibrotic septa in response to several mediators. Oncostatin M (OSM) is known to orchestrate hypoxia-modulated hepatic processes involving the hypoxia-inducible factor 1 (HIF-1). METHODS: In vivo and in vitro experiments were performed to analyze the expression of OSM and OSM-receptor (OSMR) in three murine models of non-alcoholic-fatty liver disease (NAFLD) and -steatohepatitis (NASH) and in human NASH patients as well as the action of OSM on phenotypic responses of human MFs. RESULTS: Hepatic OSM and OSMR levels were overexpressed in three murine NASH models and in NASH patients. OSM stimulates migration in human MFs by involving early intracellular ROS generation and activation of Ras/Erk, JNK1/2, PI3K/Akt as well as STAT1/STAT3 pathways and HIF-1α. OSM-dependent migration relies on a biphasic mechanism requiring early intracellular generation of reactive oxygen species (ROS) and late HIF1-dependent expression and release of VEGF. CONCLUSION: OSM is overexpressed in experimental and human progressive NAFLD and can act as a profibrogenic factor by directly stimulating migration of hepatic MFs.
Assuntos
Miofibroblastos/citologia , Hepatopatia Gordurosa não Alcoólica/genética , Subunidade beta de Receptor de Oncostatina M/genética , Oncostatina M/genética , Regulação para Cima , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Miofibroblastos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Oncostatina M/metabolismo , Subunidade beta de Receptor de Oncostatina M/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied. APPROACH AND RESULTS: Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457). CONCLUSIONS: Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.
Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/patologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Oncostatina M/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/genética , Biomarcadores/metabolismo , Doença das Coronárias/sangue , Doença das Coronárias/genética , Doença das Coronárias/mortalidade , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/patologia , Interleucina-6/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Camundongos Transgênicos , Monócitos/patologia , Oncostatina M/sangue , Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/metabolismo , Fenótipo , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Probabilidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Molécula 1 de Adesão de Célula Vascular/metabolismoAssuntos
Amiloidose Familiar/epidemiologia , Disparidades nos Níveis de Saúde , Subunidade beta de Receptor de Oncostatina M/genética , Dermatopatias Genéticas/epidemiologia , Adulto , Idade de Início , Amiloidose Familiar/diagnóstico , Amiloidose Familiar/genética , China/epidemiologia , Análise Mutacional de DNA , Éxons/genética , Feminino , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Prevalência , Distribuição por Sexo , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/genéticaRESUMO
BACKGROUND: Oncostatin M receptor (OSMR) is a member of the interleukin 6 (IL-6) receptor family that transduces signaling events of Oncostatin M (OSM). OSM-OSMR signaling plays a key role in inflammation and cancer progression. However, the role of OSM-OSMR in gastric cancer (GC) is still unknown. METHODS: OSMR expression in GC was determined by real-time PCR (RT-PCR), immunohistochemistry (IHC) and Western blot. The effects of OSM-OSMR on GC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and metastasis in vivo were examined. The pathways underlying OSM-OSMR signaling were explored by Western blot. Regulatory mechanism between SP1 and OSMR was explored in vitro. RESULTS: OSMR was highly expressed in GC tissues and its expression level was closely associated with age, T stage, Lauren classification, lymph node metastasis, TNM stage and worse prognosis of patients with GC. Knockdown of OSMR expression in GC cells significantly inhibited cell proliferation, migration, invasion, and EMT in vitro, as well as tumorigenesis and peritoneal metastasis in vivo induced by OSM. These effects mediated by OSM-OSMR were dependent on the activation of STAT3/FAK/Src signaling. SP1 could bind to the promoter region of human OSMR gene from - 255 to - 246 bp, and transcriptionally regulated OSMR overexpression in GC cells. CONCLUSIONS: OSM-OSMR contributes to GC progression through activating STAT3/FAK/Src signaling, and OSMR is transcriptionally activated by SP1.