Differentially expressed proteins in glioblastoma multiforme identified with a nanobody-based anti-proteome approach and confirmed by OncoFinder as possible tumor-class predictive biomarker candidates

Abstract

Glioblastoma multiforme is the most frequent primary malignancy of the central nervous system. Despite remarkable progress towards an understanding of tumor biology, there is no efficient treatment and patient outcome remains poor. Here, we present a unique anti-proteomic approach for selection of nanobodies specific for overexpressed glioblastoma proteins. A phage-displayed nanobody library was enriched in protein extracts from NCH644 and NCH421K glioblastoma cell lines. Differential ELISA screenings revealed seven nanobodies that target the following antigens: the ACTB/NUCL complex, VIM, NAP1L1, TUFM, DPYSL2, CRMP1, and ALYREF. Western blots showed highest protein up-regulation for ALYREF, CRMP1, and VIM. Moreover, bioinformatic analysis with the OncoFinder software against the complete “Cancer Genome Atlas” brain tumor gene expression dataset suggests the involvement of different proteins in the WNT and ATM pathways, and in Aurora B, Sem3A, and E-cadherin signaling. We demonstrate the potential use of NAP1L1, NUCL, CRMP1, ACTB, and VIM for differentiation between glioblastoma and lower grade gliomas, with DPYSL2 as a promising “glioma versus reference” biomarker. A small scale validation study confirmed significant changes in mRNA expression levels of VIM, DPYSL2, ACTB and TRIM28. This work helps to fill the information gap in this field by defining novel differences in biochemical profiles between gliomas and reference samples. Thus, selected genes can be used to distinguish glioblastoma from lower grade gliomas, and from reference samples. These findings should be valuable for glioblastoma patients once they are validated on a larger sample size.

Glioblastoma-specific anti-TUFM nanobody for in-vitro immunoimaging and cancer stem cell targeting

“…With the in-silico analysis, among several targets of the nanobodies tested, Nb206 (this study) and Nb225 (our previous study) [ 30 ] were seen to interact with the human mitochondrial elongation factor EF-TU (i.e., TUFM). As the 3D structures of these nanobodies had not been solved, suitable homology building templates were searched for.…”

“…TUFM) was identified because of lysis of the glioma cells that would have occurred during the immunization and panning procedures (which was performed with whole GSCs), and also because the screening was performed on GSC protein lysates. TUFM was recognized among seven possible glioma tumor-class predictive biomarker candidates in our previous study [ 30 ].…”

PRDM8 exhibits antitumor activities toward hepatocellular carcinoma by targeting NAP1L1

“…In pancreatic neuroendocrine neoplasms, NAP1L1 promotes tumor cell proliferation and regulates cell entry into the S phase via inhibition of the mTOR pathway and epigenetic regulation of p57 promoter methylation . The potential use of NAP1L1 for differentiation between glioblastoma and lower grade gliomas has also been demonstrated . Reportedly, Nap1l1 promotes the proliferation of murine induced pluripotent stem cell attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, and the activation of AKT signaling is involved in the process .…”

The Emerging Roles of RNA Modifications in Glioblastoma

“…NSUN5, another RNA methyltransferase that is responsible for m 5 C in the C3782 position of human 28S rRNA, undergoes epigenetic loss in gliomas, which drives an overall depletion of protein synthesis, resulting in an adaptive translational program for survival under cellular stress and renders gliomas sensitive to bio-activatable substrates of the stress-related enzyme NAD(P)H quinone dehydrogenase 1 (NQO1) [81,82]. The m 5 C reader, ALYREF, may be an overexpressed antigen in GBM, according to a study with a nanobody-based anti-proteome approach [83] and is upregulated in recurrent GBM, according to a transcriptome analysis [84], implying it may be a promising drug target for GBM. These evidences show m 5 C and its RMPs may also play important roles during the tumorigenesis of GBM (Figure 2, m 5 C part), indicating they are promising pharmaceutical targets for GBM treatment.…”