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 XenoinjertoRESUMEN
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.