Differential proteome analysis of human gliomas stratified for loss of heterozygosity on chromosomal arms 1p and 19q

Abstract

Combined deletion of chromosomal arms 1p and 19q is an independent prognostic marker in patients with oligodendroglial brain tumors, including oligodendrogliomas and oligoastrocytomas. However, the relevant genes in these chromosome arms and the molecular mechanisms underlying the prognostic significance of 1p/19q deletion are yet unknown. We used two-dimensional difference gel electrophoresis followed by mass spectrometry to perform a proteome-wide profiling of low-grade oligoastrocytomas stratified for the presence or absence of 1p/19q deletions. Thereby, we identified 22 different proteins showing differential expression in tumors with or without combined deletions of 1p and 19q. Four of the differentially expressed proteins, which are vimentin, villin 2 (ezrin), annexin A1, and glial fibrillary acidic protein, were selected for further analysis. Lower relative expression levels of these proteins in 1p/19q-deleted gliomas were confirmed at the protein level by Western blot analysis and immunohistochemistry. Furthermore, sequencing of sodium bisulfite-treated tumor DNA revealed more frequent methylation of 5'-CpG islands associated with the VIM and VIL2 genes in 1p/19q-deleted gliomas when compared with gliomas without these deletions. In summary, we confirm proteome-wide profiling as a powerful means to identify candidate biomarkers in gliomas. In addition, our data support the hypothesis that 1p/19q-deleted gliomas frequently show epigenetic down-regulation of multiple genes due to aberrant methylation of the 5'-CpG islands.

Fig. 1.

Representative large 2D-DIGE gel (40 × 30 cm) showing approximately 3500 protein spots. The arrows indicate differentially regulated protein spots determined by image analysis and identified by MALDI-MS. The annotated spot numbers correspond to the numbers given in Supplementary Material, Table S2. Case number OA50 is shown in red and OA28 in green. The overlay is displayed.

Fig. 2.

Representative results obtained by 2D-DIGE profiling for four proteins that demonstrated differential expression in 1p/19q-deleted vs 1p/19q-retained oligoastrocytomas, which were selected for further validation by Western blotting and immunohistochemistry. The left and middle columns show 2D-DIGE images from individual tumors with (left column) or without (middle column) 1p/19q deletion. The right column displays box plot diagrams illustrating significantly different expression levels of the respective protein in oligoastrocytomas with 1p/19q deletion when compared with oligoastrocytomas with 1p/19 retention. (A) Annexin A1 (left, case OA50; middle, case OA28). (B) Glial fibrillary acidic protein (GFAP) (left, case OA40; middle, case OA51). (C) Villin 2/ezrin (left, case OA50; middle, case OA28). (D) Vimentin (left, case OA40; middle, case OA51).

Fig. 3.

Validation of selected candidate proteins using Western blot analysis. Protein abundances for annexin A1, GFAP, villin 2/ezrin, and vimentin obtained in 10 different gliomas (see Supplementary Material, Table S1) with 1p/19q deletion (lanes 1–10) and 9 different gliomas without 1p/19q deletion (lanes 11–19) are shown. β-Tubulin was used as a control for protein loading. The molecular weight of each protein is indicated on the right side of the blots.

Fig. 4.

Selected examples of the immunohistochemical validation of the differential expression of annexin A1 (left, case OA50; middle, case OA51), GFAP (left, case OA29; middle, case O64), villin 2/ezrin (left, case OA29; middle, case OA20), and vimentin (left, case OA29; middle, case OA28) in formalin-fixed and paraffin-embedded primary glioma tissue samples with and without 1p/19q deletion. All sections are counterstained with hematoxylin. The original microscopic magnification of each picture was ×400. On the right-hand side of each immunohistochemical panel, box plot diagrams are shown illustrating the results of immunohistochemical evaluation of each protein in 43 different gliomas, including 16 tumors without 1p/19q deletion and 27 tumors with 1p/19q deletion. All four proteins showed significantly lower immunoreactivity scores in the 1p/19q-deleted tumors.

Fig. 5.

Receiver operation characteristic curves indicating sensitivity and specificity of the immunohistochemical stainings for vimentin, GFAP, villin 2/ezrin, and annexin A1 with respect to the distinction of 1p/19q-deleted vs 1p/19q-retained gliomas. The results are based on the immunohistochemical evaluation of 43 gliomas. ROC analysis revealed that each analyzed protein has the potential to determine 1p/19q status in glioma with high diagnostic accuracy (AUC = 0.91 for vimentin, 0.84 for annexin A1, 0.83 for GFAP, and 0.82 for villin 2/ezrin staining) using immunohistochemistry.

Fig. 6.

Summary of the results obtained in 33 gliomas concerning DNA hypermethylation of VIM (A), VIL2 (C), and GFAP (D) in relation to the 1p/19q allelic status. The chromosomal location and genomic structure of the investigated genes, including the location of associated CpG islands is indicated on top of each scheme (derived from UCSC genome browser at http://genome.ucsc.edu/). The results of the DNA methylation analyses are represented in a 4-tiered semiquantitative grey-scale pattern: white square, not methylated; light grey square, weakly methylated; grey square, moderately methylated; black square, strongly methylated. Each number represents a single CpG dinucleotide analyzed for methylation by sodium bisulfite sequencing in the respective CpG islands. The total number of investigated CpG sites was 29 (VIM), 16 (VIL2), and 28 (GFAP), respectively. (B) Examples of sequencing of parts of the VIM 5′-CpG–rich region after sodium bisulfite modification showing methylation of CpG sites in the 1p/19q-deleted tumor AO29 (arrowheads in upper lane) but not in tumor OA50, which lacked detectable 1p/19q losses (lower lane) (shown is the reverse sequence). n.d., not determined.