Heterogeneity of molecular forms of dipeptidyl peptidase-IV and fibroblast activation protein in human glioblastomas

Abstract

Background and Aims. Proteolytic enzymes contribute to the progression of various cancers. We previously reported increased expression of the proline specific peptidases dipeptidyl peptidase-IV (DPP-IV) and its closest paralogue fibroblast activation protein (FAP) in human glioblastomas. Here we analyze the molecular heterogeneity of DPP-IV and FAP in glioblastomas. Methods. ELISA, isoelectric focusing, 1D and 2D electrophoresis followed by WB or enzyme overlay assay were utilized to analyze DPP-IV and FAP isoforms. Cell fractionation using a Percoll gradient and deglycosylation with PNGase F were performed to analyze the possible basis of DPP-IV and FAP microheterogeneity. Results. Molecular forms of DPP-IV with an estimated molecular weight of 140-160 kDa and a pI predominantly 5.8 were detected in human glioblastoma; in some tumors additional isoforms with a more acidic (3.5-5.5) as well as alkaline (8.1) pI were revealed. Using 2D electrophoresis, two to three molecular forms of FAP with an alkaline (7.0-8.5) pI and an estimated MW of 120-140 kDa were identified in glioblastoma tissues. In glioma cell lines in vitro, several isoforms of both enzymes were expressed, however the alkalic forms present in glioblastoma tissues were not detected. Removal of N-linked oligosaccharides decreased the estimated molecular weight of both enzymes; the overall pattern of molecular forms nevertheless remained unchanged. Conclusion. Several isoforms of DPP-IV and FAP are present in glioblastoma tissue. The absence of alkaline isoforms of both enzymes in glioma cell lines however suggests that isoforms from other, most likely stromal, cell types contribute to the overall pattern seen in glioblastoma tissues.

Multicellular gene network analysis identifies a macrophage-related gene signature predictive of therapeutic response and prognosis of gliomas

“…ANPEP and TMEM26 were associated with drug response [49, 50]. Remarkably, the important role of DPP4 in anti-tumor immunity [51] has been revealed in many cancers including glioblastomas [52]. The inhibition of DPP4 was found to improve both naturally occurring tumor immunity and immunotherapy by enhancing lymphocyte trafficking [53].…”

FAP and FAPI-PET/CT in Malignant and Non-Malignant Diseases: A Perfect Symbiosis?

“…Non-malignant lesions and low-grade astrocytoma seem to lack FAP expression. However, in glial tumors, FAP levels correlate with the grade and thus the overexpression is associated with a poor prognosis [ 81 , 82 , 83 , 84 ]. Röhrich et al investigated FAP and FAPI-PET/CT in gliomas in vitro, in vivo and in a clinical study including 18 glioma patients (five IDH-mutant gliomas and 13 IDH-wildtype glioblastomas).…”

Regulation of Fibroblast Activation Protein by Transforming Growth Factor Beta-1 in Glioblastoma Microenvironment

“…The Western blot procedure was performed in a similar way to that described previously [65] with some modifications specified below. The lysate samples, 35 µg of total protein per one gel lane, were electrophoresed under denaturing and reducing conditions in discontinuous (5% stacking, 10% resolving) 1.5 mm polyacrylamide gels.…”