TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers

Abstract

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

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

“…Following OncoFinder analysis, we conducted a small scale confirmatory study on 13 GBM and 10 LGG tissue samples from Caucasian patients (Table 2 ) and compared them to 22 reference non-tumor brain samples. Results from our previous study, TRIM28 and ACTB , were also included in the analysis [ 35 ].…”

“…An existing nanobody library was used in the present study. An adult alpaca was immunized with whole GBM cells, as indicated above and described previously [ 35 ]. The nanobody library was constructed by the Nanobody Service Facility (Vrije Universiteit Brussel, Brussels, Belgium), according to their established protocols [ 52 – 54 ].…”

“…Possible interactions between the identified proteins were analyzed using the OncoFinder software [ 59 ] and the Reactome [ 60 ], Kyoto Encyclopedia of Genes and Genomes [ 61 ], and HumanCyc [( www.humancyc.org )] molecular interactions databases. The results from our previous study for TRIM28 and ACTB [ 35 ] were also included in the present analysis. Furthermore, the genes of interest were analyzed against TCGA RNA sequencing dataset which consists of 171 GBM, 530 LGG and five reference samples.…”

Beta Actin Expression Profile in Malignant Human Glioma Tumors

“…Increase in beta-actin levels is related to invasiveness, including its effects on the carcinogen, primary and secondary GBM. 31,32 In this situation, beta-actin polymerization results in an invasive structure which affects cellular movements and can lead to invasiveness and malignancy. 33,34 There are a large number of studies in the literature on beta-actin's role in different types of cancer and gliomas, including the studies of Fages et al, Ziv-Av et al and Panopoulos and colleagues which are all confirm our results on the effect of increased beta-actin expression in different types of cancer including glioma.…”

The application of pigment-protein fraction from Nannochloropsis oculata on β-actin response of Cromileptes altivelis infected with viral nervous necrosis

“…The focus in this research was β-actin protein. Jovčevska et al (2014) stated that β-actin molecular weight was 42 kDa. According to Figure 2, there was a protein band with molecular weight of 42.4 kDa.…”