RESUMEN

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a discouraging prognosis. Its aggressive and highly infiltrative nature renders the current standard treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the signaling pathways that regulate GBM migration/invasion and resistance is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNF receptor superfamily, increases with glial tumor grade, inversely correlates with patient overall survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Here, we have identified for the first time that TROY interacts with JAK1. Increased TROY expression increases JAK1 phosphorylation. In addition, increased TROY expression promotes STAT3 phosphorylation and STAT3 transcriptional activity that is dependent upon JAK1. TROY-mediated activation of STAT3 is independent of its ability to stimulate activity of NF-κB. Inhibition of JAK1 activity by ruxolitinib or knockdown of JAK1 expression by siRNA significantly inhibits TROY-induced STAT3 activation, GBM cell migration, and decreases resistance to temozolomide. Taken together, our data indicate that the TROY signaling complex may represent a potential therapeutic target with the distinctive capacity to exert effects on multiple pathways mediating GBM cell invasion and resistance.

Asunto(s)

Neoplasias Encefálicas/patología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Janus Quinasa 1/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Transcripción STAT3/metabolismo , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferación Celular , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Janus Quinasa 1/genética , Receptores del Factor de Necrosis Tumoral/genética , Factor de Transcripción STAT3/genética , Células Tumorales Cultivadas

RESUMEN

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults. A hallmark of GBM is aggressive invasion of tumor cells into the surrounding normal brain. Both the current standard of care and targeted therapies have largely failed to specifically address this issue. Therefore, identifying key regulators of GBM cell migration and invasion is important. The leukemia-associated Rho guanine nucleotide exchange factor (LARG) has previously been implicated in cell invasion in other tumor types; however, its role in GBM pathobiology remains undefined. Herein, we report that the expression levels of LARG and ras homolog family members C (RhoC), and A (RhoA) increase with glial tumor grade and are highest in GBM. LARG and RhoC protein expression is more prominent in invading cells, whereas RhoA expression is largely restricted to cells in the tumor core. Knockdown of LARG by siRNA inhibits GBM cell migration in vitro and invasion ex vivo in organotypic brain slices. Moreover, siRNA-mediated silencing of RhoC suppresses GBM cell migration in vitro and invasion ex vivo, whereas depletion of RhoA enhances GBM cell migration and invasion, supporting a role for LARG and RhoC in GBM cell migration and invasion. Depletion of LARG increases the sensitivity of GBM cells to temozolomide treatment. Collectively, these results suggest that LARG and RhoC may represent unappreciated targets to inhibit glioma invasion.

Asunto(s)

Movimiento Celular/fisiología , Glioblastoma/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína rhoC de Unión a GTP/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Humanos , Transducción de Señal/fisiología

RESUMEN

Glioblastoma multiforme (GBM) is the most common type of malignant brain tumors in adults and has a dismal prognosis. The highly aggressive invasion of malignant cells into the normal brain parenchyma renders complete surgical resection of GBM tumors impossible, increases resistance to therapeutic treatment, and leads to near-universal tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) plays an important role in glioblastoma cell invasion and therapeutic resistance. However, the potential downstream effectors of TROY signaling have not been fully characterized. Here, we identified PDZ-RhoGEF as a binding partner for TROY that potentiated TROY-induced nuclear factor kappa B activation which is necessary for both cell invasion and survival. In addition, PDZ-RhoGEF also interacts with Pyk2, indicating that PDZ-RhoGEF is a component of a signalsome that includes TROY and Pyk2. PDZ-RhoGEF is overexpressed in glioblastoma tumors and stimulates glioma cell invasion via Rho activation. Increased PDZ-RhoGEF expression enhanced TROY-induced glioma cell migration. Conversely, silencing PDZ-RhoGEF expression inhibited TROY-induced glioma cell migration, increased sensitivity to temozolomide treatment, and extended survival of orthotopic xenograft mice. Furthermore, depletion of RhoC or RhoA inhibited TROY- and PDZ-RhoGEF-induced cell migration. Mechanistically, increased TROY expression stimulated Rho activation, and depletion of PDZ-RhoGEF expression reduced this activation. Taken together, these data suggest that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance.

Asunto(s)

Neoplasias Encefálicas/patología , Glioblastoma/patología , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Femenino , Quinasa 2 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Ratones Desnudos , Receptores del Factor de Necrosis Tumoral/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína rhoC de Unión a GTP/genética , Proteína rhoC de Unión a GTP/metabolismo

RESUMEN

Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and assures tumor recurrence. Thus, there is an urgent need to develop innovative therapeutics to target the invasive tumor cells for improved treatment outcomes of this disease. Expression of TROY (TNFRSF19), a member of the tumor necrosis factor (TNF) receptor family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion in vitro and in vivo and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits glioblastoma cell invasion, increases temozolomide sensitivity, and prolongs survival in an intracranial xenograft model. Here, a novel complex is identified between TROY and EGFR, which is mediated predominantly by the cysteine-rich CRD3 domain of TROY. Glioblastoma tumors with elevated TROY expression have a statistically positive correlation with increased EGFR expression. TROY expression significantly increases the capacity of EGF to stimulate glioblastoma cell invasion, whereas depletion of TROY expression blocks EGF stimulation of glioblastoma cell invasion. Mechanistically, TROY expression modulates EGFR signaling by facilitating EGFR activation and delaying EGFR receptor internalization. Moreover, the association of EGFR with TROY increases TROY-induced NF-κB activation. These findings substantiate a critical role for the TROY-EGFR complex in regulation of glioblastoma cell invasion.Implications: The TROY-EGFR signaling complex emerges as a potential therapeutic target to inhibit glioblastoma cell invasion. Mol Cancer Res; 16(2); 322-32. ©2017 AACR.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Sitios de Unión , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal , Regulación hacia Arriba

RESUMEN

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

Asunto(s)

Ácido Aurintricarboxílico/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Necrosis Tumoral/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Ácido Aurintricarboxílico/química , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Citocina TWEAK , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Sinergismo Farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Ratones Desnudos , Estructura Molecular , Interferencia de ARN , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Receptor de TWEAK , Temozolomida , Factores de Necrosis Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos

RESUMEN

Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion.

RESUMEN

UNLABELLED: Of the features that characterize glioblastoma, arguably none is more clinically relevant than the propensity of malignant glioma cells to aggressively invade into the surrounding normal brain tissue. These invasive cells render complete resection impossible, confer significant resistance to chemo- and radiation-therapy, and virtually assure tumor recurrence. Expression of TROY (TNFRSF19), a member of the TNF receptor superfamily, inversely correlates with patient survival and stimulates glioblastoma cell migration and invasion in vitro. In this study, we report that TROY is overexpressed in glioblastoma tumor specimens and TROY mRNA expression is increased in the invasive cell population in vivo. In addition, inappropriate expression of TROY in mouse astrocytes in vivo using glial-specific gene transfer in transgenic mice induces astrocyte migration within the brain, validating the importance of the TROY signaling cascade in glioblastoma cell migration and invasion. Knockdown of TROY expression in primary glioblastoma xenografts significantly prolonged survival in vivo. Moreover, TROY expression significantly increased resistance of glioblastoma cells to both IR- and TMZ-induced apoptosis via activation of Akt and NF-κB. Inhibition of either Akt or NF-κB activity suppressed the survival benefits of TROY signaling in response to TMZ treatment. These findings position aberrant expression and/or signaling by TROY as a contributor to the dispersion of glioblastoma cells and therapeutic resistance. IMPLICATIONS: Targeting of TROY may increase tumor vulnerability and improve therapeutic response in glioblastoma. Mol Cancer Res; 11(8); 865-74. ©2013 AACR.

Asunto(s)

Glioblastoma/genética , Glioblastoma/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Astrocitos/fisiología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Pollos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos/genética , Epilepsia , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Ratones Transgénicos , FN-kappa B/antagonistas & inhibidores , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Temozolomida , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: Glioblastoma (GB) is the most common and lethal type of primary brain tumor. Clinical outcome remains poor and is essentially palliative due to the highly invasive nature of the disease. A more thorough understanding of the molecular mechanisms that drive glioma invasion is required to limit dispersion of malignant glioma cells. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential role of differential expression of microRNAs (miRNA) in glioma invasion by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Migratory and migration-restricted cell populations from seven glioma cell lines were isolated and profiled for miRNA expression. Statistical analyses revealed a set of miRNAs common to all seven glioma cell lines that were significantly down regulated in the migrating cell population relative to cells in the migration-restricted population. Among the down-regulated miRNAs, miR-23b has been reported to target potential drivers of cell migration and invasion in other cell types. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3' untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the protein expression level of Pyk2 in glioma cells but did not significantly alter the protein expression level of the related focal adhesion kinase FAK. Expression of Pyk2 via a transcript variant missing the 3'UTR in miR-23b-expressing cells partially rescued cell migration, whereas expression of Pyk2 via a transcript containing an intact 3'UTR failed to rescue cell migration. CONCLUSIONS/SIGNIFICANCE: Reduced expression of miR-23b enhances glioma cell migration in vitro and invasion ex vivo via modulation of Pyk2 protein expression. The data suggest that specific miRNAs may regulate glioma migration and invasion to influence the progression of this disease.

Asunto(s)

Movimiento Celular/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Glioblastoma/metabolismo , MicroARNs/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Quinasa 2 de Adhesión Focal/genética , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/genética , Humanos , MicroARNs/genética

RESUMEN

The focal adhesion kinase Pyk2 integrates signals from cell adhesion receptors, growth factor receptors, and G-protein-coupled receptors leading to the activation of intracellular signaling pathways that regulate cellular phenotypes. The intrinsic mechanism for the activation of Pyk2 activity remains to be fully defined. Previously, we reported that mutations in the N-terminal FERM domain result in loss of Pyk2 activity and expression of the FERM domain as an autonomous fragment inhibits Pyk2 activity. In the present study, we sought to determine the mechanism that underlies these effects. Utilizing differentially epitope-tagged Pyk2 constructs, we observed that Pyk2 forms oligomeric complexes in cells and that complex formation correlates positively with tyrosine phosphorylation. Similarly, when expressed as an autonomous fragment, the Pyk2 FERM domain formed a complex with other Pyk2 FERM domains but not the FAK FERM domain. When co-expressed with full-length Pyk2, the autonomously expressed Pyk2 FERM domain formed a complex with full-length Pyk2 preventing the formation of Pyk2 oligomers and resulting in reduced Pyk2 phosphorylation. Deletion of the FERM domain from Pyk2 enhanced Pyk2 complex formation and phosphorylation. Together, these data indicate that the Pyk2 FERM domain is involved in the regulation of Pyk2 activity by acting to regulate the formation of Pyk2 oligomers that are critical for Pyk2 activity.

Asunto(s)

Quinasa 2 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/química , Quinasa 2 de Adhesión Focal/genética , Células HEK293 , Humanos , Mutación , Fosforilación , Multimerización de Proteína , Estructura Terciaria de Proteína

RESUMEN

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Glioblastoma/metabolismo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Proteína de Unión al GTP rac1/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Quinasa 2 de Adhesión Focal/deficiencia , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Inmunoprecipitación , Invasividad Neoplásica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transfección , Proteína de Unión al GTP rac1/deficiencia

RESUMEN

The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH(2)-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the beta5C-alpha1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Movimiento Celular/efectos de los fármacos , Quinasa 2 de Adhesión Focal/metabolismo , Glioma/tratamiento farmacológico , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular , Línea Celular Tumoral , Epítopos/química , Epítopos/metabolismo , Femenino , Quinasa 2 de Adhesión Focal/química , Quinasa 2 de Adhesión Focal/genética , Glioma/metabolismo , Glioma/patología , Humanos , Immunoblotting , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Disease progression of glioblastoma involves a complex interplay between tumor cells and the peri-tumor microenvironment. The propensity of malignant glioma cells to disperse throughout the brain typifies the disease and portends a poor response to surgical resection, radiotherapy, and current chemotherapeutics. The focal adhesion kinases FAK and Pyk2 function as important signaling effectors in glioma through stimulation of pro-migratory and proliferative signaling pathways. In the current study, we examined the importance of Pyk2 and FAK in the pathobiology of malignant glioma in an intracranial xenograft model. We show that mice with xenografts established with glioma cells with specific knockdown of Pyk2 or FAK expression by RNA interference had significantly increased survival compared to control mice. Furthermore, the effect of inhibition of Pyk2 activity in xenografts was compared to the effect of knockdown of Pyk2 expression. Inhibition of Pyk2 activity by stable expression an autonomous FERM domain in glioma cells slowed disease progression in the intracranial xenograft model. In contrast, expression of a variant FERM domain that does not inhibit Pyk2 activity did not alter survival. These results substantiate the disease relevance of both Pyk2 and FAK in glioma and suggest a novel approach to target Pyk2 for therapeutic benefit.

Asunto(s)

Neoplasias Encefálicas/mortalidad , Quinasa 1 de Adhesión Focal/deficiencia , Quinasa 2 de Adhesión Focal/deficiencia , Glioma/mortalidad , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , Modelos Animales de Enfermedad , Femenino , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias/métodos , ARN Interferente Pequeño/uso terapéutico , Análisis de Supervivencia , Transducción Genética

RESUMEN

The strong tendency of malignant glioma cells to invade locally into surrounding normal brain precludes effective surgical resection, reduces the efficacy of radiotherapy, and is associated with increased resistance to chemotherapy regimens. We report that the N-terminal FERM domain of Pyk2 regulates its promigratory function. A 3-dimensional model of the Pyk2 FERM domain was generated and mutagenesis studies identified residues essential for Pyk2 promigratory function. Model-based targeted mutations within the FERM domain decreased Pyk2 phosphorylation and reduced the capacity of Pyk2 to stimulate glioma cell migration but did not significantly alter the intracellular distribution of Pyk2. Expression of autonomous Pyk2 FERM domain fragments containing analogous mutations exhibited reduced capacity to inhibit glioma cell migration and Pyk2 phosphorylation relative to expression of an autonomous wild type FERM domain fragment. These results indicate that the FERM domain plays an important role in regulating the functional competency of Pyk2 as a promigratory factor in glioma.

Asunto(s)

Movimiento Celular , Quinasa 2 de Adhesión Focal/química , Quinasa 2 de Adhesión Focal/metabolismo , Glioma/enzimología , Glioma/patología , Animales , Línea Celular , Chlorocebus aethiops , Quinasa 2 de Adhesión Focal/genética , Regulación Enzimológica de la Expresión Génica , Glioma/genética , Modelos Moleculares , Mutación/genética , Fosforilación , Estructura Terciaria de Proteína

RESUMEN

Glioblastoma multiforme is extraordinarily aggressive due to the propensity of cells to migrate away from the tumor core into the surrounding normal brain. In this report, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2) and FAK with regard to influencing glioma cell phenotypes. Expression of Pyk2 stimulated glioma cell migration, whereas expression of FAK inhibited glioma cell migration and stimulated cell cycle progression. Pyk2 autophosphorylation was necessary, but not sufficient, to stimulate cellular migration. The N-terminal domain of Pyk2 is required for stimulation of migration as an N-terminally deleted variant of Pyk2 failed to stimulate migration, whereas expression of an autonomous Pyk2 N-terminal domain inhibited cell migration. Substitution of the C-terminal domain of Pyk2 with the corresponding domain of FAK stimulated cell migration as effectively as wild-type Pyk2; however, substitution of the N-terminal domain of Pyk2 with that of FAK inhibited cell migration, substantiating that the N-terminal domain of Pyk2 was required to stimulate migration. Silencing of Pyk2 expression by RNA interference significantly inhibited glioma migration. Cell migration was restored on re-expression of Pyk2, but expression of FAK in Pyk2 knockdown cells failed to restore migration. We conclude that Pyk2 plays a central role in the migratory behavior of glioblastomas.

Asunto(s)

Glioma/patología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Relación Dosis-Respuesta a Droga , Epítopos/química , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Eliminación de Gen , Silenciador del Gen , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Invasividad Neoplásica , Fenotipo , Fosforilación , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Retroviridae/genética , Transducción de Señal , Factores de Tiempo

RESUMEN

The propensity of malignant gliomas to invade surrounding brain tissue contributes to poor clinical outcome. Integrin-mediated adhesion to extracellular matrix regulates the migration and proliferation of many cell types, but its role in glioma progression is undefined. We investigated the role of the cytoplasmic tyrosine kinases FAK and Pyk2, potential integrin effectors, in the phenotypic determination of four different human glioblastoma cell lines. While FAK expression was similar between the four cell lines, increased FAK activity correlated with high proliferation and low migratory rates. In contrast, Pyk2 activity was significantly increased in migratory cell lines and depressed in proliferative cell lines. Overexpression of Pyk2 stimulated migration, whereas FAK overexpression inhibited cell migration and stimulated cellular proliferation. These data suggest that FAK and Pyk2 function as important signaling effectors in gliomas and indicate that their differential regulation may be determining factors in the temporal development of proliferative or migrational phenotypes.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Proteínas Tirosina Quinasas/genética , División Celular/fisiología , Movimiento Celular/fisiología , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Fenotipo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales Cultivadas