Global Proteome and Phospho-proteome Analysis of Merlin-deficient Meningioma and Schwannoma Identifies PDLIM2 as a Novel Therapeutic Target

Abstract

Loss or mutation of the tumour suppressor Merlin predisposes individuals to develop multiple nervous system tumours, including schwannomas and meningiomas, sporadically or as part of the autosomal dominant inherited condition Neurofibromatosis 2 (NF2). These tumours display largely low grade features but their presence can lead to significant morbidity. Surgery and radiotherapy remain the only treatment options despite years of research, therefore an effective therapeutic is required.Unbiased omics studies have become pivotal in the identification of differentially expressed genes and proteins that may act as drug targets or biomarkers. Here we analysed the proteome and phospho-proteome of these genetically defined tumours using primary human tumour cells to identify upregulated/activated proteins and/or pathways. We identified over 2000 proteins in comparative experiments between Merlin-deficient schwannoma and meningioma compared to human Schwann and meningeal cells respectively. Using functional enrichment analysis we highlighted several dysregulated pathways and Gene Ontology terms. We identified several proteins and phospho-proteins that are more highly expressed in tumours compared to controls. Among proteins jointly dysregulated in both tumours we focused in particular on PDZ and LIM domain protein 2 (PDLIM2) and validated its overexpression in several tumour samples, while not detecting it in normal cells. We showed that shRNA mediated knockdown of PDLIM2 in both primary meningioma and schwannoma leads to significant reductions in cellular proliferation.To our knowledge, this is the first comprehensive assessment of the NF2-related meningioma and schwannoma proteome and phospho-proteome. Taken together, our data highlight several commonly deregulated factors, and indicate that PDLIM2 may represent a novel, common target for meningioma and schwannoma.

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.…”

“…Domingues et al [26] clearly demonstrated that it is possible to detect tumour-associated macrophages in single-cell suspensions using mechanical disaggregation by multicolour flow cytometric immunophenotyping, and we therefore decided to use this technique to quantify the immune infiltrates in our well established primary cell culture model [37] and to correlate these results to mutational hotspots.…”

miR-9-1 Is a New Circulating Biomarker for Higher-grade Meningioma Regulated via the EGFR-FOS Network

“…Proteins were separated on a Laemmli SDS-PAGE, and transferred to a polyvinylidene di uoride membrane (Immun-Blot® PVDF Membrane, Bio-Rad). Membrane blocking, antibody incubation, and washes were performed as previously described (21). (sc-59286, RRID:AB_784278) and anti-Calnexin (sc-46669, RRID:AB_626784) were purchased from Santa Cruz Biotechnology (USA), while anti-GM130 (clone 35, RRID:AB_398142) and GAPDH (#MAB374, RRID:AB_2107445) were from BD Biosciences (UK) and Millipore (USA), respectively.…”

“…The malignant meningioma cell line KT21-MG1 (RRID:CVCL_M429) was cultured as previously reported (20). The benign meningioma cell line Ben Men-1 (RRID:CVCL_1959), and the malignant meningioma cell line IOMM-Lee (RRID:CVCL_5779) were both maintained as in (21). WHO I primary meningioma cells were obtained as reported in (22) Gene expression levels were computed using the quantitative 2 -(ΔΔCt) method, employing the HMC as calibrator, in triplicate (24).…”

Proteomics in Drug Development: The Dawn of a New Era?

“…Bassiri et al . aimed at identification of novel drug targets for Merlin‐deficient meningioma and schwannoma through parallel analysis of proteome and phosphoproteome in schwannoma/schwann cells and meningioma/meningeal cells.…”