OS08.5.A Proteomic analysis of meningioma

Abstract

Abstract BACKGROUND Meningioma is the most common primary intracranial tumor. Although ~80% are benign some WHO grade I are clinically aggressive. Chemotherapies are ineffective and biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbor mutations in the NF2 gene andutations in TRAF7, KLF4, AKT1, SMO and PIK3CA have been identified in the majority NF2-positive tumors esp lower grade. However, the molecular mechanisms behind meningioma tumourigenesis is still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterizing the proteomic landscape. MATERIAL AND METHODS We analysed grade I, II and III frozen meningioma specimens and three different mutational groups: AKT1/TRAF7, KLF4/TRAF7 and NF2 -/- using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. For mutational subtypes quantitative phosphoproteomics was performed using TMT 10plex labeling approach followed by motif analysis using motif-X algorithm. We validated differential expression of proteins and phosphoproteins by Western blot and immunohistochemistry. RESULTS We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades. Bioinformatics analysis revealed commonly upregulated (phospho)proteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation confirmed significant overexpression of proteins such as EGFR, CKAP4, the nuclear proto-oncogene SET, the splicing factor SF2/ASF as well as total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Hexokinase 2 was overexpressed in higher grades. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups. Analysis showed distinct proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. 14 and 7 proteins were commonly significantly upregulated in KLF4/TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were verified via WB. Lastly, analyses of 6600 phosphosites predicted regulatory kinases CONCLUSION We show extensive proteomic and phospophoproteomics analysis of meningioma and suggest new therapeutic and biomarker candidates.