Proteomic analysis discovers the differential expression of novel proteins and phosphoproteins in meningioma including NEK9, HK2 and SET and deregulation of RNA metabolism

Abstract

BackgroundMeningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is hampered by poor knowledge of their proteomic signature. There is an urgent need to identify new therapeutic targets and biomarkers of meningioma.MethodsWe performed proteomic profiling of grade I, II and III frozen meningioma specimens and three normal healthy human meninges 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. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry.FindingsWe quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades and normal meninges. Bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation of the total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins identified and validated in meningioma included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, HK2, involved in cellular metabolism.InterpretationOverall, we generated a proteomic thesaurus of meningiomas for the identification of potential biomarkers and therapeutic targets.FundThis study was supported by Brain Tumour Research.

Comprehending Meningioma Signaling Cascades Using Multipronged Proteomics Approaches & Targeted Validation of Potential Markers

“…We earlier reported an elevation in the levels of AHNAK, Gelsolin, S100 family of proteins, and several other proteins like CKAP4 that were specific to particular grades of meningiomas ( 9 ). Several of these candidates which earlier identified via our quantitative proteomics study were also found to be involved in meningioma pathobiology via the current study and independent studies on larger patient cohorts as well ( 7 , 10 ).…”

Checking NEKs: Overcoming a Bottleneck in Human Diseases

“…NEK9 was shown to be up-regulated in grade I, II and III meningioma. These data were also validated by Western blotting analyses [189].…”

A Rapid Robust Method for Subgrouping Non-NF2 Meningiomas According to Genotype and Detection of Lower Levels of M2 Macrophages in AKT1 E17K Mutated Tumours

“…Future work is required to elucidate the role of M2 macrophages in meningiomas, and to uncover the mechanisms in which tumour cells harbouring a mutation such as AKT1 E17K can directly affect the tumour microenvironment. From proteomics studies [36,37] published by our group and others, it is clear that the tumour microenvironment of meningiomas is highly heterogeneous, and that linking cell population, gene expression and/or signalling pathway activation data to genetically defined non-NF2 and NF2 groups would be very beneficial. The simple, cost-effective endpoint genotyping technique used here could aid such stratification in any research or clinical laboratory with a real-time PCR instrument.…”

“…Due to the complex nature of NF2 loss by pathogenic single mutations occurring across all exons or partial/complete deletions of the NF2 gene, KASP™ genotyping was not suitable. Next-generation sequencing and multiplex ligation-dependent probe amplification were used on a small number of tumours to validate loss of the Merlin (NF2) protein by Western blotting [36]. NF2 status was then assessed by either intact Merlin protein (non-NF2 meningiomas) or Merlin loss (NF2 meningiomas).…”