14-3-3 proteins

Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GPR30, and possible crosstalk with the aryl hydrocarbon receptor. Glioblastomas are almost invariably fatal brain tumors. Glioblastoma incidence in men is 1.5 times that of pre-menopausal women and this difference decreases postmenopause. Therefore estrogen is thought to play a protective role, although the mechanism is not well understood. Glioblastomas produce and respond to a tryptophan derivative, kynurenine, that binds and activates the aryl hydrocarbon receptor (AHR). Promiscuity of ligand binding can result in cross talk between AHR and nuclear estrogen receptors and both receptor types are known to use the p300 acetyltransferase as a cofactor. We demonstrated that inhibition of p300 resulted in a reduction of proliferation of CH157-MN meningioma cells. Treatment with βestradiol or kynurenine partially abrogated this effect, suggesting both ligands may act through another receptor that we hypothesize to be the G-protein linked estrogen receptor, GPER1/GPR30. We verified the expression of GPER1, estrogen receptor β; some estrogen receptor β isoforms and AHR by CH157-MN cells and U-87 and A-172 glioblastoma cells. Inhibition of p300 with C646 reduced proliferation of both U87 and A172 cells. In U87 cells, this effect was abrogated by co-treatment with βestradiol, the xenobiotic AHR agonist 2',3',7,8'-tetrachlorodibenzo-p-dioxin, and the GPER1 agonist G-1. Exogenous kynurenine did not abrogate the effect of p300 inhibition. However, in A172 cells, none of the agonists abrogated the effect of p300 inhibition, possibly due to the higher kynurenine production in A172 cells. Treatment of A172 and U87 cells with G-1 resulted in decreased cell proliferation which was abrogated by co-treatment with the GPER1 antagonist G-36.

Glioblastoma (GBM) is highly heterogeneous and micro-environmental diversity may necessitate therapeutically targeting specific tumor regions rather than treating the tumor as a homogenous entity. The diffusely infiltrative properties of GBM result in residual tumor at resection margins in nearly all cases, suggesting that targeting efforts should focus on this region. To identify potential druggable targets at the tumor margin, we utilized fluorescence-guided surgical resection based on 5-ALA, followed by proteomic analysis of individual tumor regions that were precisely defined during surgery. Reverse phase protein array (RPPA) was performed on 206 proteins isolated from the tumor margin, bulk, and perinecrotic regions of 13 genetically defined patient samples (10 GBM, 3AA) in order to identify differentially expressed perturbable molecular pathways or signaling networks. Using ANOVA and post-hoc Tukey-HSD, we identified 37 proteins that were differentially expressed among the three regions. We performed unsupervised hierarchical clustering on these 37 proteins and found that in general, samples from the perinecrotic and bulk regions clustered together and those from the margin clustered together. 25 of these 37 proteins were overexpressed and 12 underexpressed at the tumor margin. Among differentially expressed proteins, we focused further efforts on Akt, PAI-1, and Tyro3. In comparison to the perinecrotic region, Akt and Tyro3 were found to be overexpressed at the tumor margin while PAI-1 was underexpressed at the margin. A common signaling abnormality of GBM is upregulated Akt activity, typically downstream of EGFR, PDGFR or c-Met amplification, PTEN loss or activating mutations in PI3K subunits. Tyro3 is a promising target because of its activation of the PI3K/Akt/mTOR pathway and its role in coagulation. PAI-1 (plasminogen activator inhibitor 1) is a protein that promotes coagulation and could induce intratumoral thrombosis, a defining feature of GBMs. We present differential protein expression patterns among specific tumor microenvironments as potential therapeutic targets.

BACKGROUND Hypoxia is a major driver of invasiveness and resistance in glioblastoma (GBM). We hypothesize that GBM adapts to chronic hypoxic conditions through utilization of peroxisomal fatty acid oxidation (FAO). We explored the contribution of acyl coenzyme A oxidase 1 (ACOX-1), a rate-limiting enzyme for peroxisomal FAO, in metabolic adaptation to hypoxia and as a potential therapeutic target. METHODS A primary cell line from a bevacizumab-resistant tumor (012015) was cultured under chronic hypoxia (2%O2) or normoxia (21% O2). We used UPLC-MS/MS and GC-MS to determine lipid and polar metabolites. RNA seq was performed on U251 ACOX-1 (KO) cells under normoxic and hypoxic conditions. Differential gene expression was obtained using TopHat2 (genome alignment), HTSeq, and DEseq. Gene set enrichment analysis was used to compare biological pathways between conditions. ACOX-1 was deleted using CRISPR-Cas9 and orthotopically implanted in NCr/SCID nude mice. Animals were treated with 30 m...

h i g h l i g h t s AHR is over-expressed and hyperactivated in carcinoma tissues of IBC patients. AHR knockdown inhibits expression of CYP1B1 and Wnt5a in IBC cells. AHR and CYP1B1 expression correlates with Wnt5 a/b and b-catenin expression levels. AHR and CYP1B1 expression correlates with percentage of CD44 (+) / CD24 (À/low) subset in IBC. AHR and its surrogate molecules correlate with IBC poor prognosis.

Glioblastoma (GBM) is the most aggressive form of glioma tumor in adult brain. Among the numerous factors responsible for GBM cell proliferation and invasion, neurotransmitters such as dopamine, serotonin and glutamate can play key roles. Studies performed in mice housed in germ-free (GF) conditions demonstrated the relevance of the gut-brain axis in a number of physiological and pathological conditions. The gut–brain communication is made possible by vagal/nervous and blood/lymphatic routes and pave the way for reciprocal modulation of functions. The gut microbiota produces and consumes a wide range of molecules, including neurotransmitters (dopamine, norepinephrine, serotonin, gamma-aminobutyric acid [GABA], and glutamate) that reach their cellular targets through the bloodstream. Growing evidence in animals suggests that modulation of these neurotransmitters by the microbiota impacts host neurophysiology and behavior, and affects neural cell progenitors and glial cells, along wit...

Glioblastoma (GBM) is the most aggressive form of glioma tumor in adult brain. Among the numerous factors responsible for GBM cell proliferation and invasion, neurotransmitters such as dopamine, serotonin and glutamate can play key roles. Studies performed in mice housed in germ-free (GF) conditions demonstrated the relevance of the gut-brain axis in a number of physiological and pathological conditions. The gut–brain communication is made possible by vagal/nervous and blood/lymphatic routes and pave the way for reciprocal modulation of functions. The gut microbiota produces and consumes a wide range of molecules, including neurotransmitters (dopamine, norepinephrine, serotonin, gamma-aminobutyric acid [GABA], and glutamate) that reach their cellular targets through the bloodstream. Growing evidence in animals suggests that modulation of these neurotransmitters by the microbiota impacts host neurophysiology and behavior, and affects neural cell progenitors and glial cells, along wit...

New avenues for glioblastoma therapy are required due to the limited mortality benefit of the current treatments. The renin-angiotensin system (RAS) exhibits local actions and works as a paracrine system in different tissues and tumors, including glioma. The glioblastoma cell lines U-87 MG and T98G overexpresses Angiotensin II (Ang II)/Angiotensin II type I receptor (AGTR1) signaling, which enhances in vitro and in vivo local estrogen production through a direct up-regulation of the aromatase gene promoters p I.f and p I.4. In addition, Ang II/AGTR1 signaling transactivates estrogen receptor-α in a ligand-independent manner through mitogen-activated protein kinase (MAPK) activation. The higher aromatase mRNA expression in patients with glioblastoma was associated with the worst survival prognostic, according to The Cancer Genome Atlas (TCGA). An intrinsic immunosuppressive glioblastoma tumor milieu has been previously documented. We demonstrate how Ang II treatment in glioblastoma c...

Schizophrenia is a complex mental disorder with a fairly high degree of heritability. Although the causes of schizophrenia remain unclear, it is now widely accepted that it is a neurodevelopmental and neurodegenerative disorder involving disconnectivity and disorder of the synapses. Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene involved in neurodevelopment, including maturation of the cerebral cortex. To identify other susceptibility genes for schizophrenia, we screened for DISC1-interacting molecules [NudE-like (NUDEL), Lissencephaly-1 (LIS1), 14-3-3epsilon (YWHAE), growth factor receptor bound protein 2 (GRB2) and Kinesin family 5A of Kinesen1 (KIF5A)], assessing a total of 25 tagging single-nucleotide polymorphisms (SNPs) in a Japanese population. We identified a YWHAE SNP (rs28365859) that showed a highly significant difference between case and control samples, with higher minor allele frequencies in controls (Pallele 5 1.01 3 1025 and Pgenotype 5 4.08 3 1025 in 1429 cases and 1728 controls). Both messenger RNA transcription and protein expression of 14-3-3epsilon were also increased in the lymphocytes of healthy control subjects harboring heterozygous and homozygous minor alleles compared with homozygous major allele subjects. To further investigate a potential role for YWHAE in schizophrenia, we studied Ywhae1/2 mice in which the level of 14-3-3epsilon protein is reduced to 50% of that in wild-type littermates. These mice displayed weak defects in working memory in the eight-arm radial maze and moderately enhanced anxiety-like behavior in the elevated plus-maze. Our results suggest that YWHAE is a possible susceptibility gene that functions protectively in schizophrenia.

Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller–Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3ε (YWHAE), one of a family of ubiquitous phosphoserine/threonine–binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3ε binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3ε results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3ε in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.

Previous studies show that mice with Ywhae deficiency show abnormalities in brain development including defects in neuronal migration of post-mitotic pyramidal neurons as well as neuronal differentiation and proliferation in neuronal progenitor cells. Also, our previous research indicated that the Ywhae knockout mice show moderate defects in working memory and anxiety-like behavior. This previous work was performed using heterozygous mutant mice. Here we performed behavioral analyses using homozygous Ywhae knockout mice and found that the homozygous Ywhae knockout mice have increased locomotor activity, decreased working memory, and increased sociability. Taken together with the results obtained from the previous pathophysiological analyses in the Ywhae knockout mice, the Ywhae knockout mouse is useful for pathophysiological analyses of neuropsychiatric disorders caused by defects during neurodevelopment.

The 14-3-3 protein family is a group of multifunctional proteins that are highly expressed in the brain; however, their functions in brain development are largely unknown. Williams Syndrome is a neurodevelopmental disorder caused by a deletion in the 7q11.23 chromosome locus, including the gene encoding 14-3-3gamma, resulting in developmental delay, intellectual disabilities and epilepsy. We have previously shown that knocking down the 14-3-3gamma protein in utero in mice results in delays in neuronal migration of pyramidal neurons in the cortex. Importantly, there is a reciprocal duplication syndrome to
Williams Syndrome where the 7q11.23 locus is duplicated, resulting in epilepsy and intellectual disabilities. Thus, the deletion or the duplication of the 7q11.23 chromosome locus results in epilepsy. Taken together with the fact that defects in neuronal migration are one of main causes for epilepsy, we analyzed if the overexpression of 14-3-3gamma causes neuronal migration defects. In this work, we found that the overexpression of 14-3-3gamma in utero in the developing mouse cortex results in delays in pyramidal neuron migration, similar to what was previously observed when 14-3-3gamma was knocked down. These results, in conjunction with our previous research, indicate that a balance of 14-3-3gamma expression is required during cortical development to prevent delays in neuronal migration. This work provides clear evidence as to the involvement of 14-3-3gamma in neurodevelopmental disorders and how a disruption in 14-3-3gamma expression may contribute to the neurodevelopmental disorders that manifest when the 7q11.23 locus is altered.

14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular , radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multi-polar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects.

17p13.3 microduplication syndrome is a newly identified genetic disorder characterized by duplications in the 17p13.3 chromosome locus, resulting in a variety of disorders including autism spectrum disorder (ASD). Importantly, a minimum duplication region has been defined, and this region exclusively contains the gene encoding 14-3-3e. Furthermore, duplication of this minimum region is strongly associated with the appearance of ASD in human patients, thus implicating the overexpression of 14-3-3e in ASD. Using in vitro and in vivo techniques, we have found that 14-3-3e binds to the microtubule binding protein doublecortin preventing its degradation. We also found that 14-3-3e overexpression disrupts neurite formation by preventing the invasion of microtubules into primitive neurites, which can be rescued by the knockdown of doublecortin. To analyse the function of 14-3-3e in neurite formation, we used 14-3-3e flox mice and found that 14-3-3e deficiency results in an increase in neurite formation. Our findings provide the first evidence of cellular pathology in 17p13.3 microduplication syndrome.

The 14-3-3 proteins are a family of highly conserved, multifunctional proteins that are highly expressed in the brain during development. Cumulatively, the seven 14-3-3 isoforms make up approximately 1% of total soluble brain protein. Over the last decade, evidence has accumulated implicating the importance of the 14-3-3 protein family in the development of the nervous system, in particular cortical development, and have more recently been recognized as key regulators in a number
of neurodevelopmental processes. In this review we will discuss the known roles of each 14-3-3 isoform in the development of the cortex, their relation to human neurodevelopmental disorders, as well as the challenges and questions that are left to be answered. In particular, we focus on the 14-3-3 isoforms and their involvement in
the three key stages of cortical development; neurogenesis and differentiation, neuronal migration and neuromorphogenesis and synaptogenesis.

Primary glioma, as well as secondary metastases, provide significant treatment challenges. An understanding of the biological underpinnings of glioma is likely to provide new pharmaceutical targets that will improve patient survival. Here, we look at the role that the kynurenine pathways and associated tyrptophan catabolites (TRYCATs) play in glioma, linking this to changes in oxidative and nitrosative stress (O&NS), immune-inflammatory activity, the aryl hydrocarbon receptor (AhR), and the melatoninergic pathways. It is suggested that the interactions of O&NS and the immune-inflammatory processes in glioma contribute to the induction of the TRYCATs via the kynurenine activation of the AhR, leading to increased indoleamine 2,3-dioxygenase, which deprives tryptophan for the necessary serotonin that is required as a precursor for the melatoninergic pathways. A diverse array of data pertaining to glioma can be linked to these pathways, including changes in miRNAs, epigenetic processes, estrogen receptors, 14-3-3, chromosome 4q35, neurotrophins, tristetraprolin and the N-acetylserotonin (NAS)/melatonin ratio. As many of these factors directly or indirectly act on the melatoninergic pathways, including variations in the NAS/melatonin ratio, it is suggested that the melatoninergic pathways may act as a hub that coordinate the multitude of changes occurring in glioma. Consequently , the melatoninergic pathways may be a significant pharmaceutical target for the treatment of this still very poorly managed condition .

Primary glioma, as well as secondary metastases, provide significant treatment challenges. An understanding of the biological underpinnings of glioma is likely to provide new pharmaceutical targets that will improve patient survival. Here, we look at the role that the kynurenine pathways and associated tyrptophan catabolites (TRYCATs) play in glioma, linking this to changes in oxidative and nitrosative stress (O&NS), immune-inflammatory activity, the aryl hydrocarbon receptor (AhR), and the melatoninergic pathways. It is suggested that the interactions of O&NS and the immune-inflammatory processes in glioma contribute to the induction of the TRYCATs via the kynurenine activation of the AhR, leading to increased indoleamine 2,3-dioxygenase, which deprives tryptophan for the necessary serotonin that is required as a precursor for the melatoninergic pathways. A diverse array of data pertaining to glioma can be linked to these pathways, including changes in miRNAs, epigenetic processes, estrogen receptors, 14-3-3, chromosome 4q35, neurotrophins, tristetraprolin and the N-acetylserotonin (NAS)/melatonin ratio. As many of these factors directly or indirectly act on the melatoninergic pathways, including variations in the NAS/melatonin ratio, it is suggested that the melatoninergic pathways may act as a hub that coordinate the multitude of changes occurring in glioma. Consequently , the melatoninergic pathways may be a significant pharmaceutical target for the treatment of this still very poorly managed condition. INTRODUCTION In this article, we review the role of the diverse array of factors associated with brain cancers, especially primary glioma, suggesting that there is a significant role for the tryptophan catabolite (TRYCATs) and melatoninergic pathways in coordinating the biological underpinnings of glioma. Firstly, we say some about the nature of brain cancers and their classification.