RESUMEN
5-Fluorouracil (5-FU) resistance has been long considered as an obstacle to the efficacy of chemotherapy in colorectal cancer (CRC). In this study, we demonstrated the role of miR-20b-5p-regulated syndecan-2 (SDC2) in 5-FU resistance of CRC cells. 5-FU-resistant SW480 CRC cells were established by treatment of SW480 cells with stepwise increase of 5-FU concentration. The results showed that SDC2 was expressed significantly higher in SW480/5-FU cells than in SW480/WT cells as revealed by quantitative real-time polymerase chain reaction and western blot analysis. MTT assay and BrdU assay showed that SDC2 overexpression led to increased cell survival rate, while SDC2 knockdown reversed the drug resistance of SW480/5-FU cells. Wound healing and transwell invasion assays revealed that knockdown of SDC2 inhibited the migratory and invasive ability of SW480/5-FU cells. Moreover, animal experiments indicated that si-SDC2 plays a suppressive role in tumor growth in vivo. We also confirmed that miR-20b-5p interacted with SDC2, which reversed the effect of SDC2 in SW480/5-FU cells via the c-Jun N-terminal kinase (JNK)/extracellular regulated protein kinases (ERK) signaling pathway. These findings showed that JNK/ERK signaling pathway is involved in miR-20b-5p/SDC2 axis-mediated 5-FU resistance in SW480/5-FU cells, indicating that the miR-20b-5p/SDC2 axis is a potential target for reversing 5-FU resistance in CRC.
Asunto(s)
Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Fluorouracilo/farmacología , MicroARNs/genética , Sindecano-2/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Emparejamiento Base , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional/métodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Survivin/genética , Survivin/metabolismo , Sindecano-2/antagonistas & inhibidores , Sindecano-2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumour stromal cells support tumourigenesis. We report that Syndecan-2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan-2 modulated TGFß signalling (SMAD7, PAI-1), migration and immunosuppression of patient-derived tumour-associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan-2 in TASCs significantly enhanced TGFß signalling (SMAD7, PAI-1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFß signalling (SMAD7, PAI-1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan-2-peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFß-regulated immunosuppressive genes, such as CXCR4 and PD-L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan-2-peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan-2 within the TME inhibits tumour growth and metastasis due to decreased TGFß signalling and increased immune control.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Evasión Inmune , Sindecano-2/antagonistas & inhibidores , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Sindecano-2/fisiología , Factor de Crecimiento Transformador beta/fisiología , Microambiente TumoralRESUMEN
The syndecan family of heparan sulfate proteoglycans contributes to cell adhesion and communication by serving as co-receptors for cell signaling and extracellular matrix molecules. Syndecan-2 is located at the cell surface, and we previously reported that it induces matrix metalloproteinase-7 (MMP-7) expression in colon cancer cells. However, the underlying regulatory mechanisms are unknown. Here, we report that overexpression of syndecan-2 in HT-29 colon cancer cells increases the phosphorylation of focal adhesion kinase (FAK) and ERK in parallel with up-regulated MMP-7 expression, but a syndecan-2 mutant lacking the cytoplasmic domain showed significant reductions in these effects. Consistent with this observation, FAK inhibition via FAK-related non-kinase expression or inhibition of ERK with the ERK1/2 inhibitor SCH772984 diminished the syndecan-2-mediated up-regulation of MMP-7. Activation of PKC enhanced syndecan-2-mediated MMP-7 expression, whereas inhibition of PKC had the opposite effect. Of note, the exogenous expression of syndecan-2 triggered localization of PKCγ to the membrane. Expression of syndecan-2 harboring a phosphomimetic (S198E) mutation of the variable region of the cytoplasmic domain enhanced MMP-7 expression and FAK phosphorylation. Finally, experimental suppression of shedding of the syndecan-2 extracellular domain did not significantly affect the syndecan-2-mediated up-regulation of MMP-7 in the early period after syndecan-2 overexpression. Taken together, these findings suggest that syndecan-2's cytoplasmic domain up-regulates MMP-7 expression in colon cancer cells via PKCγ-mediated activation of FAK/ERK signaling.
Asunto(s)
Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Sindecano-2/metabolismo , Sustitución de Aminoácidos , Animales , Carcinoma/tratamiento farmacológico , Carcinoma/enzimología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Inducción Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal/química , Quinasa 1 de Adhesión Focal/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sindecano-2/antagonistas & inhibidores , Sindecano-2/química , Sindecano-2/genéticaRESUMEN
Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CßII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases.
Asunto(s)
Melaninas/biosíntesis , Melanocitos/metabolismo , Melanoma/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteína Quinasa C beta/fisiología , Sindecano-2/fisiología , Animales , Línea Celular Tumoral , Membrana Celular/enzimología , Activación Enzimática , Células Epidérmicas , Epidermis/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Melanocitos/efectos de la radiación , Melanoma/patología , Melanosomas/enzimología , Ratones , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Sindecano-2/antagonistas & inhibidores , Sindecano-2/genética , Rayos Ultravioleta , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Herpes simplex virus type 1 (HSV-1) is an important human pathogen and a leading cause of infectious blindness in the developed world. HSV-1 exploits heparan sulfate proteoglycans (HSPG) for attachment to cells. While the significance of heparan sulphate (HS) moieties in HSV-1 infection is well established, the role of specific proteoglycan core proteins in the infection process remains poorly understood. The objective of this study was to assess the roles of syndecan-1 and syndecan-2 core proteins in HSV-1 infection, both of which are expressed by many HSV-1 target cell types. Our results demonstrate that syndecan-1 and syndecan-2 gene silencing by RNA interference reduces HSV-1 entry, plaque formation and facilitates cell survival. Furthermore, HSV-1 infection increases syndecan-1 and syndecan-2 protein synthesis and a resultant increase in cell surface expression of HS. Our observations suggest that changes in syndecan-1 and syndecan-2 expression levels may be related to active viral infection. Taken together, our findings provide new insights into HSPG functions during HSV-1 entry and spread.
Asunto(s)
Herpesvirus Humano 1/patogenicidad , Interacciones Huésped-Patógeno , Sindecano-1/metabolismo , Sindecano-2/metabolismo , Internalización del Virus , Animales , Línea Celular , Chlorocebus aethiops , Silenciador del Gen , Humanos , Interferencia de ARN , Sindecano-1/antagonistas & inhibidores , Sindecano-2/antagonistas & inhibidoresRESUMEN
We developed laminin-derived AG73 peptide labeled liposomes for cancer specific gene therapy. AG73 peptide is well known as a ligand for syndecan-2 which is highly expressed in various cancer cells. Liposomes labeled with AG73 showed high efficient transfection efficiency in syndecan-2 overexpressing cells, and found that AG73 could be a superior molecule in the development of non-viral vector using liposomes for the gene delivery to syndecan-2 overexpressing cancer cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Laminina/química , Liposomas/química , Péptidos/administración & dosificación , Secuencia de Aminoácidos , Antineoplásicos/química , Línea Celular , Humanos , Datos de Secuencia Molecular , Neoplasias/terapia , Péptidos/química , Sindecano-2/antagonistas & inhibidores , Sindecano-2/metabolismo , TransfecciónRESUMEN
The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in "in vitro" tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.