Constitutive activation of the EGFR-STAT1 axis increases proliferation of meningioma tumor cells.

BACKGROUND: Meningiomas are the most frequent primary brain tumors of the central nervous system. The standard of treatment is surgery and radiotherapy, but effective pharmacological options are not available yet. The well-characterized genetic background stratifies these tumors in several subgroups, thus increasing diversification. We identified epidermal growth factor receptor-signal transducer and activator of transcription 1 (EGFR-STAT1) overexpression and activation as a common identifier of these tumors. METHODS: We analyzed STAT1 overexpression and phosphorylation in 131 meningiomas of different grades and locations by utilizing several techniques, including Western blots, qPCR, and immunocytochemistry. We also silenced and overexpressed wild-type and mutant forms of the gene to assess its biological function and its network. Results were further validated by drug testing. RESULTS: STAT1 was found widely overexpressed in meningioma but not in the corresponding healthy controls. The protein showed constitutive phosphorylation not dependent on the JAK-STAT pathway. STAT1 knockdown resulted in a significant reduction of cellular proliferation and deactivation of AKT and ERK1/2. STAT1 is known to be activated by EGFR, so we investigated the tyrosine kinase and found that EGFR was also constitutively phosphorylated in meningioma and was responsible for the aberrant phosphorylation of STAT1. The pharmaceutical inhibition of EGFR caused a significant reduction in cellular proliferation and of overall levels of cyclin D1, pAKT, and pERK1/2. CONCLUSIONS: STAT1-EGFR-dependent constitutive phosphorylation is responsible for a positive feedback loop that causes its own overexpression and consequently an increased proliferation of the tumor cells. These findings provide the rationale for further studies aiming to identify effective therapeutic options in meningioma.

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.

The majority of meningiomas are grade I, but some grade I tumours are clinically more aggressive. Recent advances in the genetic study of meningiomas has allowed investigation into the influence of genetics on the tumour microenvironment, which is important for tumorigenesis. We have established that the endpoint genotyping method Kompetitive Allele Specific PCR (KASP™) is a fast, reliable method for the screening of meningioma samples into different non-NF2 mutational groups using a standard real-time PCR instrument. This genotyping method and four-colour flow cytometry has enabled us to assess the variability in the largest immune cell infiltrate population, M2 macrophages (CD45+HLA-DR+CD14+CD163+) in 42 meningioma samples, and to suggest that underlying genetics is relevant. Further immunohistochemistry analysis comparing AKT1 E17K mutants to WHO grade I NF2-negative samples showed significantly lower levels of CD163-positive activated M2 macrophages in meningiomas with mutated AKT1 E17K, signifying a more immunosuppressive tumour microenvironment in NF2 meningiomas. Our data suggested that underlying tumour genetics play a part in the development of the immune composition of the tumour microenvironment. Stratifying meningiomas by mutational status and correlating this with their cellular composition will aid in the development of new immunotherapies for patients.

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

BACKGROUND: Meningioma 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. METHODS: We 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. FINDINGS: We 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. INTERPRETATION: Overall, we generated a proteomic thesaurus of meningiomas for the identification of potential biomarkers and therapeutic targets. FUND: This study was supported by Brain Tumour Research.

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

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.

Simultaneous targeting of Eph receptors in glioblastoma.

Eph tyrosine kinase receptors are frequently overexpressed and functional in many cancers, and they are attractive candidates for targeted therapy. Here, we analyzed the expression of Eph receptor A3, one of the most up-regulated factors in glioblastoma cells cultured under tumorsphere-forming conditions, together with EphA2 and EphB2 receptors. EphA3 was overexpressed in up to 60% of glioblastoma tumors tested, but not in normal brain. EphA3 was localized in scattered areas of the tumor, the invasive ring, and niches near tumor vessels. EphA3 co-localized with macrophage/leukocyte markers, suggesting EphA3 expression on tumor-infiltrating cells of bone marrow origin. We took advantage of the fact that ephrinA5 (eA5) is a ligand that binds EphA3, EphA2 and EphB2 receptors, and used it to construct a novel targeted anti-glioblastoma cytotoxin. The eA5-based cytotoxin potently and specifically killed glioblastoma cells with an IC50 of at least 10-11 M. This and similar cytotoxins will simultaneously target different compartments of glioblastoma tumors while mitigating tumor heterogeneity.

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37.

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.

Ephs and Ephrins in malignant gliomas.

Eph receptor tyrosine kinases and the corresponding ephrin ligands play a pivotal role in the glioma development and progression. Aberrant protein expression levels of the Eph receptors and ephrins are often associated with higher tumor grade and poor prognosis. Their function in tumorigenesis is complex due to the intricate network of possible co-occurring interactions between neighboring tumor cells and tumor microenvironment. Both Ephs and ephrins localize on the surface of tumor cells, tumor vasculature, glioma stem cells, tumor cells infiltrating brain, and immune cells infiltrating tumors. They can both promote and inhibit tumorigenicity depending on the downstream forward and reverse signalling generated. All the above-mentioned features make the Ephs/ephrins system an intriguing candidate for the development of new therapeutic strategies in glioma treatment. This review will give a general overview on the structure and the function of Ephs and ephrins, with a particular emphasis on the state of the knowledge of their role in malignant gliomas.

Biological and structural characterization of glycosylation on ephrin-A1, a preferred ligand for EphA2 receptor tyrosine kinase.

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand's carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.

Structural and functional characterization of monomeric EphrinA1 binding site to EphA2 receptor.

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.

The plant pathogen Pseudomonas fuscovaginae contains two conserved quorum sensing systems involved in virulence and negatively regulated by RsaL and the novel regulator RsaM.

Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.

OryR is a LuxR-family protein involved in interkingdom signaling between pathogenic Xanthomonas oryzae pv. oryzae and rice.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight in rice, contains a regulator that is encoded in the genome, designated OryR, which belongs to the N-acyl homoserine lactone (AHL)-dependent quorum-sensing LuxR subfamily of proteins. However, we previously reported that X. oryzae pv. oryzae does not make AHLs and does not possess a LuxI-family AHL synthase and that the OryR protein is solubilized by a compound present in rice. In this study we obtained further evidence that OryR interacts with a rice signal molecule (RSM) and that the OryR concentration increases when rice is infected with X. oryzae pv. oryzae. We also describe three OryR target promoters which are regulated differently: (i) the neighboring proline iminopeptidase (pip) virulence gene, which is positively regulated by OryR in the presence of the RSM; (ii) the oryR promoter, which is negatively autoregulated independent of the RSM; and (iii) the 1,4-beta-cellobiosidase cbsA gene, which is positively regulated by OryR independent of the RSM. We also found that the RSM for OryR is small, is not related to AHLs, and is not able to activate the broad-range AHL biosensor Agrobacterium tumefaciens NT1(pZLQR). Furthermore, OryR does not regulate production of the quorum-sensing diffusible signal factor present in the genus Xanthomonas. Therefore, OryR has unique features and is an important regulator involved in interkingdom communication between the host and the pathogen.

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence.

SUMMARY: In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae (Xoo), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri. Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.