RESUMEN
In the present study, we investigated the effect of simultaneous downregulation of uPAR and cathepsin B (pUC), alone or in combination with radiation, on JNK-MAPK signaling pathway in regulating the migration of non-GICs (glioma-initiating cells) and GICs. The increase in the expression of p-JNK with pUC treatment was mostly localized to nucleus whereas increase in the expression of p-JNK with radiation and overexpression of uPAR and cathepsin B was confined to cytoplasm of the cells. Depletion of cytosolic p-JNK with pUC treatment inhibited migration by downregulating the expression of the adapter proteins of the focal adhesion complex. We also observed that knockdown of uPAR and cathepsin B regulated the Ras-Pak-1 pathway to induce the translocation of p-JNK from cytosol to nucleus. In control cells, Pak-1 served as a functional inhibitor for MEKK-1, which inhibits the complex formation of MEKK-1 and p-JNK and thus inhibits the translocation of this complex into nucleus. Hence, we conclude that glioma cells utilize the availability of cytosolic p-JNK in driving the cells towards migration. Finally, treating the cells with pUC alone or in combination with radiation induced the translocation of the MEKK-1-p-JNK complex from cytosol to nucleus, thereby inhibiting the migration of glioma cells.
Asunto(s)
Catepsina B/metabolismo , Movimiento Celular , Glioma/enzimología , MAP Quinasa Quinasa 4/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Animales , Catepsina B/genética , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/enzimología , Citoplasma/genética , Citoplasma/metabolismo , Citosol/enzimología , Citosol/metabolismo , Femenino , Glioma/genética , Glioma/metabolismo , Glioma/fisiopatología , Humanos , MAP Quinasa Quinasa 4/genética , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Transporte de Proteínas , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genéticaRESUMEN
Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2.
Asunto(s)
Catepsina B/fisiología , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/fisiología , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Femenino , Citometría de Flujo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioma/patología , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de la radiación , Péptidos/metabolismo , Complejo Represivo Polycomb 1/genética , ARN Interferente Pequeño/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Factores de Transcripción SOXB1/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína con Dedos de Zinc GLI1RESUMEN
Glioma is a highly complex brain tumor characterized by the dysregulation of proteins and genes that leads to tumor metastasis. Cathepsin B and uPAR are overexpressed in gliomas and they are postulated to play central roles in glioma metastasis. In this study, efficient downregulation of cathepsin B and uPAR by siRNA treatments significantly reduced glioma cell adhesion to laminin as compared to vitronectin, fibronectin, or collagen I in U251 and 4910 glioma cell lines. Brain glioma tissue array analysis showed high expression of CD151 in clinical samples when compared with normal brain tissue. Cathepsin B and uPAR siRNA treatment led to the downregulation of CD151 and laminin-binding integrins α3 and ß1. Co-immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR cathepsin B, and α3ß1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3ß1 integrin have shown that phosphorylation of FAK, SRC, paxillin, and expression of adaptor cytoskeletal proteins talin and vinculin were reduced with knockdown of cathepsin B, uPAR, and CD151. Treatment with the bicistronic construct reduced interactions between uPAR and CD151 as well as lowering α3ß1 integrin, talin, and vinculin expression levels in pre-established glioma tumors of nude mice. In conclusion, our results show that downregulation of cathepsin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. Taken together, the results of the present study show that targeting the uPAR-cathepsin B system has possible therapeutic potential.
Asunto(s)
Catepsina B/metabolismo , Adhesión Celular , Movimiento Celular , Glioma/patología , Integrina alfa3beta1/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Tetraspanina 24/metabolismo , Animales , Apoptosis , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Regulación hacia Abajo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Integrina alfa3beta1/antagonistas & inhibidores , Integrina alfa3beta1/genética , Ratones , Ratones Desnudos , Fosforilación , ARN Interferente Pequeño/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Tetraspanina 24/antagonistas & inhibidores , Tetraspanina 24/genética , Células Tumorales Cultivadas , KalininaRESUMEN
Glioblastomas present as diffuse tumors with invasion into normal brain tissue and frequently recur or progress after radiation as focal masses because of glioma-initiating cells. The role of the urokinase-type plasminogen activator receptor (uPAR) and cathepsin B in stem-like phenotype has been extensively studied in several solid tumors. In the present study, we demonstrated that selection of glioma-initiating cells using CD133 expression leads to a specific enrichment of CD133(+) cells in both U87 and 4910 cells. In addition, CD133(+) cells exhibited a considerable amount of other stem cell markers, such as Nestin and Sox-2. Radiation treatment significantly enhanced uPAR and cathepsin B levels in glioma-initiating cells. To downregulate radiation-induced uPAR and cathepsin B expression, we used a bicistronic shRNA construct that simultaneously targets both uPAR and cathepsin B (pCU). Downregulation of uPAR and cathepsin B using pCU decreased radiation-enhanced uPAR and cathepsin B levels and caused DNA damage-induced apoptosis in glioma cell lines and glioma-initiating cells. The most striking finding of this study is that knockdown of uPAR and cathepsin B inhibited ongoing transcription by suppressing BrUTP incorporation at γH2AX foci. In addition, uPAR and cathepsin B gene silencing inversely regulated survivin and H2AX expression in both glioma cells and glioma-initiating cells. Pretreatment with pCU reduced radiation-enhanced expression of uPAR, cathepsin B, and survivin and enhanced DNA damage in pre-established glioma in nude mice. Taken together, our in vitro and in vivo findings suggest that uPAR and cathepsin B inhibition might serve as an adjunct to radiation therapy to target glioma-initiating cells and, therefore, for the treatment of glioma.
Asunto(s)
Apoptosis/efectos de la radiación , Catepsina B/antagonistas & inhibidores , Glioma/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Animales , Western Blotting , Catepsina B/genética , Catepsina B/metabolismo , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Glioma/metabolismo , Glioma/radioterapia , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Desnudos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Transcripción GenéticaRESUMEN
Despite advances in radiotherapeutic and chemotherapeutic techniques and aggressive surgical resection, the prognosis of glioblastoma patients is dismal. Accumulation of evidence indicates that some cancer cells survive even the most aggressive treatments, and these surviving cells, which are resistant to therapy and are perhaps essential for the malignancy, may be cancer stem cells. The CD133 surface marker is commonly used to isolate these extremely resistant glioma-initiating cells (GICs). In the present study, GICs which tested positive for the CD133 marker (CD133+) were isolated from both the established U251 cell line and the 5310 xenograft glioma cell line to study the events related to the molecular pathogenesis of these cells. Simultaneous down-regulation of uPAR and cathepsin B by shRNA (pUC) treatment caused the disruption of radiation-induced complex formation of pPKC θ/δ, integrin ß1 and PKC ζ, integrin ß1 in glioma cells. Further, pUC treatment inhibited PKC/integrin signaling via FAK by causing disassociation of FAK and the cytoskeletal molecules vinculin and α-actinin. Also, we observed the inhibition of ERK phosphorylation. This inhibition was mediated by pUC and directed a negative feedback mechanism over the FAK signaling molecules, which led to an extensive reduction in the signal for cytoskeletal organization generating migratory arrest. Altogether, it can be hypothesized that knockdown of uPAR and cathepsin B using shRNA is an effective strategy for controlling highly invasive glioma cells and extremely resistant glioma-initiating cells.
Asunto(s)
Catepsina B/genética , Citoesqueleto/metabolismo , Glioma/patología , Integrina beta1/metabolismo , Células Madre Neoplásicas/metabolismo , Proteína Quinasa C/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Acetofenonas/farmacología , Animales , Antígenos de Diferenciación/metabolismo , Benzopiranos/farmacología , Catepsina B/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Movimiento Celular , Transformación Celular Neoplásica , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Expresión Génica/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , Integrinas/metabolismo , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de la radiación , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal , Esferoides Celulares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date. METHODOLOGY/PRINCIPLE FINDINGS: In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling proteins in glioma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies.
Asunto(s)
Receptores ErbB/metabolismo , Sangre Fetal/citología , Glioblastoma/metabolismo , Mitocondrias/metabolismo , Células Madre/fisiología , Animales , Proteína Tirosina Quinasa CSK , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Ratones , Fosforilación , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Familia-src QuinasasRESUMEN
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ and increased the interaction of BAD with Bcl-xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD, activation of caspase 3 as well as PARP and cytochrome c and SMAC release. These effects were inhibited by FK506 (10 µM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 µg/ml). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.
Asunto(s)
Apoptosis , Calcineurina/metabolismo , Catepsina B/metabolismo , Glioma/metabolismo , Glioma/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Western Blotting , Caspasa 3/metabolismo , Catepsina B/antagonistas & inhibidores , Línea Celular Tumoral , Citocromos c/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Potencial de la Membrana Mitocondrial , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Transducción de Señal , Fracciones SubcelularesRESUMEN
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. p27(Kip1) (p27) is one of the major cyclin-CDK regulators in the G1 phase. uPAR and cathepsin B downregulation was recently shown to induce p27 expression through PI3K/Akt/FOXO3a signaling. Since uPAR and cathepsin B knockdown also decreased phosphorylation of ERK, we hypothesized that ERK also has a role to play in p27 induction. As induction of p27 is due to an increase in gene transcription, we investigated the roles of c-Myc and E2F1 transcription factors which have been shown to potently affect p27 promoter activity. In the present study, shRNA against cathepsin B and uPAR as well as specific inhibitors, Wortmannin (10 µM) and U0126 (10 µM), were used to determine the roles of AKT and ERK signaling on p27 expression. Immunoblot analysis demonstrated that downregulation of both p-ERK and p-AKT downstream of EGFR and ß1 integrin are involved in the p27 upregulation. Cathepsin B and uPAR downregulation induced E2F1 and decreased phosphorylaion of pocket proteins and c-Myc expression. CHIP analysis and luciferase expression studies confirmed the functional association of transcription factor E2F1 to the p27 promoter. Further, c-Myc-Max interaction inhibitor studies showed an inverse pattern of c-Myc and p27 expression. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, cathepsin B and uPAR downregulation reduced p-ERK levels and c-Myc expression, increased expression of E2F1 and FOXO3a, decreased phosphorylation of pocket proteins and thus upregulated p27 expression in glioma cells.
Asunto(s)
Catepsina B/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , Glioma/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Regulación hacia Arriba , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Receptores ErbB/metabolismo , Fase G1 , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioma/genética , Glioma/patología , Humanos , Integrina alfa5beta1/metabolismo , Ratones , Proteínas de Neoplasias/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Fase de Descanso del Ciclo Celular , Transducción de SeñalRESUMEN
BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-ß, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Catepsina B/metabolismo , Regulación hacia Abajo , Glioblastoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Proteína X Asociada a bcl-2/fisiología , Western Blotting , Neoplasias Encefálicas/patología , Catepsina B/genética , Línea Celular Tumoral , Glioblastoma/patología , Humanos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Transducción de SeñalRESUMEN
BACKGROUND: Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined. METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly. CONCLUSION/SIGNIFICANCE: Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.