Genetic variants in glutamine metabolic pathway genes predict cutaneous melanoma-specific survival.

Glutamine dependence is a unique metabolic defect seen in cutaneous melanoma (CM), directly influencing the treatment and prognosis. Here, we investigated the associations between 6025 common single-nucleotide polymorphisms (SNPs) in 77 glutamine metabolic pathway genes with CM-specific survival (CMSS) using genotyping datasets from two published genome-wide association studies (GWASs). In the single-locus analysis, 76 SNPs were found to be significantly associated with CMSS (P < .050, false-positive report probability < 0.2 and Bayesian false discovery probability < 0.8) in the discovery dataset, of which seven SNPs were replicated in the validation dataset and three SNPs (HAL rs17676826T > C, LGSN rs12663017T > A, and NOXRED1 rs8012548A > G) independently predicted CMSS, with an effect-allele attributed adjusted hazards ratio of 1.52 (95% confidence interval = 1.19-1.93) and P < .001, 0.68 (0.54-0.87) and P = .002 and 0.62 (0.46-0.83) and P = .002, respectively. The model including the number of unfavorable genotypes (NUGs) of these three SNPs and covariates improved the five-year CMSS prediction (P = .012) than the one with other covariates only. Further expression quantitative trait loci (eQTL) analysis found that the LGSN rs12663017 A allele was significantly associated with increased messenger RNA (mRNA) expression levels (P = 8.89 × 10 -11 ) in lymphoblastoid cell lines of the 1000 Genomes Project database. In the analysis of the genotype tissue expression (GTEx) project datasets, HAL rs17676826 C and NOXRED1 rs8012548 G alleles were significantly associated with their mRNA expression levels in sun-exposed skin of the lower leg (P = 6.62 × 10-6 and 1.37 × 10-7 , respectively) and in sun-not-exposed suprapubic skin (P < .001 and 1.43 × 10-8 , respectively). Taken together, these genetic variants of glutamine-metabolic pathway genes may be promising predictors of survival in patients with CM.

Genetic variants in RORA and DNMT1 associated with cutaneous melanoma survival.

Cutaneous melanoma (CM) is considered as a steroid hormone-related malignancy. However, few studies have evaluated the roles of genetic variants encoding steroid hormone receptor genes and their related regulators (SHR-related genes) in CM-specific survival (CMSS). Here, we performed a pathway-based analysis to evaluate genetic variants of 191 SHR-related genes in 858 CMSS patients using a dataset from a genome-wide association study (GWAS) from The University of Texas MD Anderson Cancer Center (MDACC), and then validated the results in an additional dataset of 409 patients from the Harvard GWAS. Using multivariate Cox proportional hazards regression analysis, we identified three-independent SNPs (RORA rs782917 G > A, RORA rs17204952 C > T and DNMT1 rs7253062 G > A) as predictors of CMSS, with a variant-allele attributed hazards ratio (HR) and 95% confidence interval of 1.62 (1.25-2.09), 1.60 (1.20-2.13) and 1.52 (1.20-1.94), respectively. Combined analysis of risk genotypes of these three SNPs revealed a decreased CMSS in a dose-response manner as the number of risk genotypes increased (ptrend < 0.001); however, no improvement in the prediction model was observed (area under the curve [AUC] = 79.6-80.8%, p = 0.656), when these risk genotypes were added to the model containing clinical variables. Our findings suggest that genetic variants of RORA and DNMT1 may be promising biomarkers for CMSS, but these results needed to be validated in future larger studies.

Arc/Arg3.1 translation is controlled by convergent N-methyl-D-aspartate and Gs-coupled receptor signaling pathways.

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arc's molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the betaIV spectrin splice variant betaSpIVSigma5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to beta-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and betaSpIVSigma5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence,(457)KIKF(460). Mutation of the PP1-binding motif or PP1 inhibition by okadaic acid and calyculin A abolished filopodia and restored stress fibers in cells expressing neurabin I. In vitro and in vivo studies suggested that the actin-binding domain attenuated protein kinase A (PKA) phosphorylation of neurabin I. Modification of a major PKA site, serine-461, impaired PP1 binding. Finally, p70S6K was excluded from neurabin I/PP1 complexes and required the displacement of PP1 for recruitment to neurabin I. These studies provided new insights into the assembly and regulation of a neurabin I/PP1 complex that controls actin rearrangement to promote spine development in mammalian neurons.