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Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology.
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Autofagia , Carcinoma Hepatocelular , Hepatocitos , Neoplasias Hepáticas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Hepatocitos/metabolismo , Hepatocitos/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Sistema de Señalización de MAP Quinasas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mitofagia/genética , Proteínas RepresorasRESUMEN
Angiogenesis is considered essential for tumor progression; however, whether histological counting of blood vessel numbers, expressed as microvessel density (MVD), can be a prognostic factor in breast cancer remains controversial. It has been suggested that the specific morphology of blood vessels such as glomeruloid microvascular proliferation (GMP) is associated with clinical parameters. Here, we aimed to clarify the significance of MVD with revised immunohistochemistry and to identify new blood vessel shapes that predict prognosis in breast cancer. Four hundred and eleven primary breast cancer specimens were collected, and the sections were immunohistochemically stained with CD31 (single staining) and CD31 and Collagen IV (double staining). The prognosis of patients was examined based on the MVD value, and the presence of GMP and other blood vessels with other specific shapes. As a result, high MVD value and the presence of GMP were not associated with worse prognosis. By contrast, patients with deep-curved capillaries surrounding tumor cell nests (C-shaped) or excessively branched capillaries near tumor cell nests showed a significantly poor prognosis. The presence of these capillaries was also correlated with clinicopathological parameters such as Ki-67 index. Thus, the morphology of capillaries rather than MVD can be a better indicator of tumor aggressiveness.
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Neoplasias de la Mama , Capilares , Densidad Microvascular , Neovascularización Patológica , Humanos , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/irrigación sanguínea , Pronóstico , Persona de Mediana Edad , Neovascularización Patológica/patología , Anciano , Adulto , Capilares/patología , Inmunohistoquímica , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismoRESUMEN
BACKGROUND: Formyl peptide receptor 2 (Fpr2) plays a crucial role in colon homeostasis and microbiota balance. Commensal E. coli is known to promote the regeneration of damaged colon epithelial cells. The aim of the study was to investigate the connection between E. coli and Fpr2 in the recovery of colon epithelial cells. RESULTS: The deficiency of Fpr2 was associated with impaired integrity of the colon mucosa and an imbalance of microbiota, characterized by the enrichment of Proteobacteria in the colon. Two serotypes of E. coli, O22:H8 and O91:H21, were identified in the mouse colon through complete genome sequencing. E. coli O22:H8 was found to be prevalent in the gut of mice and exhibited lower virulence compared to O91:H21. Germ-free (GF) mice that were pre-orally inoculated with E. coli O22:H8 showed reduced susceptibility to chemically induced colitis, increased proliferation of epithelial cells, and improved mouse survival. Following infection with E. coli O22:H8, the expression of Fpr2 in colon epithelial cells was upregulated, and the products derived from E. coli O22:H8 induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency increased susceptibility to chemically induced colitis, delayed the repair of damaged colon epithelial cells, and heightened inflammatory responses. Additionally, the population of E. coli was observed to increase in the colons of Fpr2-/- mice with colitis. CONCLUSION: Commensal E. coli O22:H8 stimulated the upregulation of Fpr2 expression in colon epithelial cells, and the products from E. coli induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency led to an increased E. coli population in the colon and delayed recovery of damaged colon epithelial cells in mice with colitis. Therefore, Fpr2 is essential for the effects of commensal E. coli on colon epithelial cell recovery.
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Breast cancer is the most prevalent cancer worldwide, and metastasis is the leading cause of death in cancer patients. Human monocyte chemoattractant protein-1 (MCP-1/CCL2) was isolated from the culture supernatants of not only mitogen-activated peripheral blood mononuclear leukocytes but also malignant glioma cells based on its in vitro chemotactic activity toward human monocytes. MCP-1 was subsequently found to be identical to a previously described tumor cell-derived chemotactic factor thought to be responsible for the accumulation of tumor-associated macrophages (TAMs), and it became a candidate target of clinical intervention; however, the role of TAMs in cancer development was still controversial at the time of the discovery of MCP-1. The in vivo role of MCP-1 in cancer progression was first evaluated by examining human cancer tissues, including breast cancers. Positive correlations between the level of MCP-1 production in tumors and the degree of TAM infiltration and cancer progression were established. The contribution of MCP-1 to the growth of primary tumors and metastasis to the lung, bone, and brain was examined in mouse breast cancer models. The results of these studies strongly suggested that MCP-1 is a promoter of breast cancer metastasis to the lung and brain but not bone. Potential mechanisms of MCP-1 production in the breast cancer microenvironment have also been reported. In the present manuscript, we review studies in which the role of MCP-1 in breast cancer development and progression and the mechanisms of its production were examined and attempt to draw a consensus and discuss the potential use of MCP-1 as a biomarker for diagnosis.
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Neoplasias de la Mama , Quimiocina CCL2 , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Leucocitos Mononucleares , Monocitos , Microambiente Tumoral , Melanoma Cutáneo MalignoRESUMEN
Over the past thirty years, the importance of chemokines and their seven-transmembrane G protein-coupled receptors (GPCRs) has been increasingly recognized. Chemokine interactions with receptors trigger signaling pathway activity to form a network fundamental to diverse immune processes, including host homeostasis and responses to disease. Genetic and nongenetic regulation of both the expression and structure of chemokines and receptors conveys chemokine functional heterogeneity. Imbalances and defects in the system contribute to the pathogenesis of a variety of diseases, including cancer, immune and inflammatory diseases, and metabolic and neurological disorders, which render the system a focus of studies aiming to discover therapies and important biomarkers. The integrated view of chemokine biology underpinning divergence and plasticity has provided insights into immune dysfunction in disease states, including, among others, coronavirus disease 2019 (COVID-19). In this review, by reporting the latest advances in chemokine biology and results from analyses of a plethora of sequencing-based datasets, we outline recent advances in the understanding of the genetic variations and nongenetic heterogeneity of chemokines and receptors and provide an updated view of their contribution to the pathophysiological network, focusing on chemokine-mediated inflammation and cancer. Clarification of the molecular basis of dynamic chemokine-receptor interactions will help advance the understanding of chemokine biology to achieve precision medicine application in the clinic.
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COVID-19 , Medicina de Precisión , Humanos , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , COVID-19/genética , Quimiocinas/genética , Quimiocinas/metabolismo , Epigénesis GenéticaRESUMEN
Formyl peptide receptor 2 (FPR2) and its mouse counterpart Fpr2 are the members of the G protein-coupled receptor (GPCR) family. FPR2 is the only member of the FPRs that interacts with ligands from different sources. FPR2 is expressed in myeloid cells as well as epithelial cells, endothelial cells, neurons, and hepatocytes. During the past years, some unusual properties of FPR2 have attracted intense attention because FPR2 appears to possess dual functions by activating or inhibiting intracellular signal pathways based on the nature, concentration of the ligands, and the temporal and spatial settings of the microenvironment in vivo, the cell types it interacts with. Therefore, FPR2 controls an abundant array of developmental and homeostatic signaling cascades, in addition to its "classical" capacity to mediate the migration of hematopoietic and non-hematopoietic cells including malignant cells. In this review, we summarize recent development in FPR2 research, particularly in its role in diseases, therefore helping to establish FPR2 as a potential target for therapeutic intervention.
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Células Endoteliales , Receptores de Formil Péptido , Ratones , Animales , Receptores de Formil Péptido/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Receptores de Lipoxina/metabolismoRESUMEN
The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Transición Epitelial-Mesenquimal/genética , Línea Celular Tumoral , Células Hep G2 , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Represoras/genéticaRESUMEN
Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.
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BACKGROUND: Patients with triple-negative breast cancer (TNBC) often have poorer prognosis than those with other subtypes because of its aggressive behaviors. Cancer cells are heterogeneous, and only a few highly metastatic subclones metastasize. Although the majority of subclones may not metastasize, they could contribute by releasing factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Here, we analyzed the interclonal communication in TNBC which leads to efficient cancer progression, particularly lung metastasis, using the polyclonal murine 4T1 BC model. METHODS: We isolated two 4T1 subclones, LM.4T1 and HM.4T1 cells with a low and a high metastatic potential, respectively, and examined the effects of LM.4T1 cells on the behaviors of HM.4T1 cells using the cell scratch assay, sphere-forming assay, sphere invasion assay, RT-qPCR, and western blotting in vitro. We also examined the contribution of LM.4T1 cells to the lung metastasis of HM.4T1 cells and TME in vivo. To identify a critical factor which may be responsible for the effects by LM.4T1 cells, we analyzed the data obtained from the GEO database. RESULTS: Co-injection of LM.4T1 cells significantly augmented lung metastases by HM.4T1 cells. LM.4T1-derived exosomes promoted the migration and invasion of HM.4T1 cells in vitro, and blocking the secretion of exosome abrogated their effects on HM.4T1 cells. Analyses of data obtained from the GEO database suggested that Wnt7a might be a critical factor responsible for the enhancing effects. In fact, a higher level of Wnt7a was detected in LM.4T1 cells, especially in exosomes, than in HM.4T1 cells, and deletion of Wnt7a in LM.4T1 cells significantly decreased the lung metastasis of HM.4T1 cells. Further, treatment with Wnt7a increased the spheroid formation by HM.4T1 cells via activation of the PI3K/Akt/mTOR signaling pathway. Finally, infiltration of αSMA-positive fibroblasts and angiogenesis was more prominent in tumors of LM.4T1 cells and deletion of Wnt7a in LM.4T1 cells markedly reduced angiogenesis. CONCLUSIONS: We demonstrated, for the first time, that a low metastatic subclone can enhance lung metastasis of highly metastatic subclone via exosomal Wnt7a and propose Wnt7a as a molecular target to treat TNBC patients.
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Neoplasias Pulmonares , Metástasis de la Neoplasia , Neoplasias de la Mama Triple Negativas , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Neovascularización Patológica , Fosfatidilinositol 3-Quinasas , Neoplasias de la Mama Triple Negativas/genética , Microambiente TumoralRESUMEN
Under normal conditions, fasting results in decreased protein disulfide isomerase (PDI) activity and accumulation of unfolded proteins, leading to the subsequent activation of the unfolded protein response (UPR)/autophagy signaling pathway to eliminate damaged mitochondria. Fasting also induces upregulation of thioredoxin-interacting protein (TXNIP) expression and mice deficient of this protein (TXNIP-KO mice) was shown to develop severe hypoglycemia, hyperlipidemia and liver steatosis (LS). In the present study, we aimed to determine the role of TXNIP in fasting-induced LS by using male TXNIP-KO mice that developed LS without severe hypoglycemia. In TXNIP-KO mice, fasting induced severe microvesicular LS. Examinations by transmission electron microscopy revealed mitochondria with smaller size and deformities and the presence of few autophagosomes. The expression of ß-oxidation-associated genes remained at the same level and the level of LC3-II was low. PDI activity level stayed at the original level and the levels of p-IRE1 and X-box binding protein 1 spliced form (sXBP1) were lower. Interestingly, treatment of TXNIP-KO mice with bacitracin, a PDI inhibitor, restored the level of LC3-II after fasting. These results suggest that TXNIP regulates PDI activity and subsequent activation of the UPR/autophagy pathway and plays a protective role in fasting-induced LS.
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Hígado Graso , Hipoglucemia , Animales , Ayuno , Hígado Graso/genética , Masculino , Ratones , Transducción de Señal , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Introduction: Mitogen-activated protein kinases (MAPKs) are involved in T cell-mediated liver damage. However, the inhibitory mechanism(s) that controls T cell-mediated liver damage remains unknown. Objectives: We investigated whether Spred2 (Sprouty-related, EVH1 domain-containing protein 2) that negatively regulates ERK-MAPK pathway has a biological impact on T cell-mediated liver damage by using a murine model. Methods: We induced hepatotoxicity in genetically engineered mice by intravenously injecting Concanavalin A (Con A) and analyzed the mechanisms using serum chemistry, histology, ELISA, qRT-PCR, Western blotting and flow cytometry. Results: Spred2-deficient mice (Spred2-/-) developed more sever liver damage than wild-type (WT) mice with increased interferon-γ (IFNγ) production. Hepatic ERK phosphorylation was enhanced in Spred2-/- mice, and pretreatment of Spred2-/- mice with the MAPK/ERK inhibitor U0126 markedly inhibited the liver damage and reduced IFNγ production. Neutralization of IFNγ abolished the damage with decreased hepatic Stat1 activation in Spred2-/- mice. IFNγ was mainly produced from CD4+ and CD8+ T cells, and their depletion decreased liver damage and IFNγ production. Transplantation of CD4+ and/or CD8+ T cells from Spred2-/- mice into RAG1-/- mice deficient in both T and B cells caused more severe liver damage than those from WT mice. Hepatic expression of T cell attractants, CXCL9 and CXCL10, was augmented in Spred2-/- mice as compared to WT mice. Conversely, liver damage, IFNγ production and the recruitment of CD4+ and CD8+ T cells in livers after Con A challenge were lower in Spred2 transgenic mice, and Spred2-overexpressing CD4+ and CD8+ T cells produced lower levels of IFNγ than WT cells upon stimulation with Con A in vitro. Conclusion: We demonstrated, for the first time, that Spred2 functions as an endogenous regulator of T cell IFNγ production and Spred2-mediated inhibition of ERK-MAPK pathway may be an effective remedy for T cell-dependent liver damage.
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Linfocitos T CD8-positivos , Interferón gamma , Animales , Linfocitos T CD4-Positivos , Concanavalina A/toxicidad , Interferón gamma/genética , Hígado , Ratones , Proteínas RepresorasRESUMEN
Human cathelicidin antimicrobial peptide LL-37 (LL-37) is an antimicrobial peptide derived from its precursor protein hCAP18, which is an only cathelicidin in human. LL-37 not only serves as a mediator of innate immune defense against invading microorganisms, but it also plays an essential role in tissue homeostasis, regenerative processes, regulation of proinflammatory responses, and inhibition of cancer progression. Therefore, LL-37 has been considered as a drug lead for diseases. However, high levels of LL-37 may reduce cell viability and promote apoptosis of osteoblasts, vascular smooth muscle cells, periodontal ligament cells, neutrophils, airway epithelial cells and T cells. Recent evidence reveals that LL-37-derived short peptides possess similar biological activities as the whole LL-37 with reduced cytotoxicity. Thus, such small molecules constitute a pool of potential therapeutic agents for diseases.
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Péptidos Antimicrobianos , Catelicidinas , Células Epiteliales , Humanos , NeutrófilosRESUMEN
Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.
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Carcinogénesis/genética , Carcinoma de Células Transicionales/genética , Proteínas Represoras/genética , Neoplasias de la Vejiga Urinaria/genética , Urotelio/metabolismo , Carcinogénesis/patología , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Bases de Datos Factuales , Humanos , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) is shown to promote the progression of breast cancer. We previously identified cancer cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential regulator of MCP-1 production in the murine 4T1 breast cancer, but it played a minimum role in overall MCP-1 production. Here, we evaluated the crosstalk between 4T1 cells and fibroblasts. When fibroblasts were co-cultured with 4T1 cells or stimulated with the culture supernatants of 4T1 cells (4T1-sup), MCP-1 production by fibroblasts markedly increased. 4T1 cells expressed mRNA for platelet-derived growth factor (PDGF)-a, b and c, and the PDGF receptor inhibitor crenolanib almost completely inhibited 4T1-sup-induced MCP-1 production by fibroblasts. However, PDGF receptor antagonists failed to reduce MCP-1 production in tumor-bearing mice. Histologically, 4T1 tumors contained a small number of αSMA-positive fibroblasts, and Mcp-1 mRNA was mainly associated with macrophages, especially those surrounding necrotic lesions on day 14, by in situ hybridization. Thus, although cancer cells have the capacity to crosstalk with fibroblasts via PDGFs, this crosstalk does not play a major role in MCP-1 production or cancer progression in this model. Unraveling complex crosstalk between cancer cells and stromal cells will help us identify new targets to help treat breast cancer patients.
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Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Comunicación Celular , Quimiocina CCL2/biosíntesis , Animales , Biomarcadores , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Ratones , Especificidad de Órganos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Microambiente Tumoral , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patologíaRESUMEN
BACKGROUNDS: Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-ß is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-ß-treated A549 human LC cells. METHODS AND RESULTS: By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-ß1 at the concentration range up to 10 µg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-ß1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-ß1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. CONCLUSION: PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.
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Adenocarcinoma Bronquioloalveolar/metabolismo , Movimiento Celular/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Poli I-C/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Células A549 , Adenocarcinoma Bronquioloalveolar/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Compuestos Organometálicos/farmacología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación/efectos de los fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Receptor Toll-Like 3/agonistasRESUMEN
Colorectal cancer (CRC) remains a leading cause of cancer mortality. Here, we define the colonic epithelial expression of cathelicidin (LL-37) in CRC. Cathelicidin exerts pleotropic effects including anti-microbial and immunoregulatory functions. Genetic knockout of cathelicidin led to increased size and number of colorectal tumours in the azoxymethane-induced murine model of CRC. We aimed to translate this to human disease. The expression of LL-37 in a large (n = 650) fully characterised cohort of treatment-naïve primary human colorectal tumours and 50 matched normal mucosa samples with associated clinical and pathological data (patient age, gender, tumour site, tumour stage [UICC], presence or absence of extra-mural vascular invasion, tumour differentiation, mismatch repair protein status, and survival to 18 years) was assessed by immunohistochemistry. The biological consequences of LL-37 expression on the epithelial barrier and immune cell phenotype were assessed using targeted quantitative PCR gene expression of epithelial permeability (CLDN2, CLDN4, OCLN, CDH1, and TJP1) and cytokine (IL-1ß, IL-18, IL-33, IL-10, IL-22, and IL-27) genes in a human colon organoid model, and CD3+ , CD4+ , and CD8+ lymphocyte phenotyping by immunohistochemistry, respectively. Our data reveal that loss of cathelicidin is associated with human CRC progression, with a switch in expression intensity an early feature of CRC. LL-37 expression intensity is associated with CD8+ T cell infiltrate, influenced by tumour characteristics including mismatch repair protein status. There was no effect on epithelial barrier gene expression. These data offer novel insights into the contribution of LL-37 to the pathogenesis of CRC and as a therapeutic molecule.
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Linfocitos T CD8-positivos/patología , Catelicidinas/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Inmunohistoquímica , Anciano , Animales , Estudios de Cohortes , Citocinas/genética , Femenino , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Organoides , PermeabilidadRESUMEN
Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/patología , Catarata/metabolismo , Receptores de Formil Péptido/metabolismo , Animales , Catarata/patología , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Citometría de Flujo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Receptores de Formil Péptido/genética , UltrasonografíaRESUMEN
Host-derived antimicrobial peptides play an important role in the defense against extracellular bacterial infections. However, the capacity of antimicrobial peptides derived from macrophages as potential antibacterial effectors against intracellular pathogens remains unknown. In this study, we report that normal (wild-type, WT) mouse macrophages increased their expression of cathelin-related antimicrobial peptide (CRAMP, encoded by Camp) after infection by viable E. coli or stimulation with inactivated E. coli and its product lipopolysaccharide (LPS), a process involving activation of NF-κB followed by protease-dependent conversion of CRAMP from an inactive precursor to an active form. The active CRAMP was required by WT macrophages for elimination of phagocytosed E. coli, with participation of autophagy-related proteins ATG5, LC3-II and LAMP-1, as well as for aggregation of the bacteria with p62 (also known as SQSTM1). This process was impaired in CRAMP-/- macrophages, resulting in retention of intracellular bacteria and fragmentation of macrophages. These results indicate that CRAMP is a critical component in autophagy-mediated clearance of intracellular E. coli by mouse macrophages.