MNK2 governs the macrophage antiinflammatory phenotype.

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.

Translational profiling of macrophages infected with Leishmania donovani identifies mTOR- and eIF4A-sensitive immune-related transcripts.

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-ß). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis.

Distinct Cancer-Promoting Stromal Gene Expression Depending on Lung Function.

Rationale: Chronic obstructive pulmonary disease is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that lung stromal cells activate pathological gene expression programs that support oncogenesis.Objectives: To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer.Methods: The study included subjects with and without lung cancer across a spectrum of lung-function values. We conducted a multiomics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome.Measurements and Main Results: Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation modulating protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms that drove these cancer-associated translation programs differed based on lung function. In subjects with normal to mildly impaired lung function, the mammalian target of rapamycin (mTOR) pathway served as an upstream driver, whereas in subjects with severe airflow obstruction, pathways downstream of pathological extracellular matrix emerged. Consistent with a role during cancer initiation, both the mTOR and extracellular matrix gene expression programs paralleled the activation of previously identified procancer secretomes. Furthermore, an in situ examination of lung tissue showed that stromal fibroblasts expressed cancer-associated proteins from two procancer secretomes: one that included IL-6 (in cases of mild or no airflow obstruction), and one that included BMP1 (in cases of severe airflow obstruction).Conclusions: Two distinct stromal gene expression programs that promote cancer initiation are activated in patients with lung cancer depending on lung function. Our work has implications both for screening strategies and for personalized approaches to cancer treatment.

Polysome-profiling in small tissue samples.

Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5-10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.

nanoCAGE reveals 5' UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs.

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.

The Protozoan Parasite Toxoplasma gondii Selectively Reprograms the Host Cell Translatome.

The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5'-terminal oligopyrimidine (5' TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection.

IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.

Genetic variants in the promoter region of the calcium-sensing receptor gene are associated with its down-regulation in neuroblastic tumors.

We have previously reported that the calcium-sensing receptor (CaSR) is expressed in benign, differentiated neuroblastic tumors, and epigenetically silenced in undifferentiated, malignant cases. Furthermore, cinacalcet, an allosteric activator of the CaSR, reduces neuroblastoma tumor growth in preclinical models. However, to identify patients that might benefit from this treatment, a complete understanding of mechanisms governing CaSR expression in these tumors would be required. We have now analyzed two polymorphisms in the promoter region of the CASR gene (rs7652589 and rs1501899) by allelic discrimination in neuroblastoma patients and cell lines. Association of genotypes and haplotypes with CaSR mRNA levels and CASR promoter P2 methylation status was determined. Data presented show that minor alleles rs7652589 and rs1501899, present either in homo- or heterozygosis, were correlated with reduced CaSR mRNA levels in matching primary tumors and this association was independent of CASR promoter P2 hypermethylation. Haplotype AA was independently associated with reduced CaSR expression after adjusting by promoter P2 methylation status. These polymorphisms were identified in some ganglioneuromas in which CaSR expression is low despite exhibiting a high degree of differentiation. Furthermore, homozygous variants rs7652589 and rs1501899 were detected in SH-SY5Y cells, which are devoid of CaSR expression in the absence of hypermethylation of CASR promoter P2. In summary, minor alleles rs7652589 and rs1501899 are associated with reduced CaSR expression in neuroblastic tumors and neuroblastoma cell lines in which the CASR gene promoter P2 is not hypermethylated. Therefore, they potentially represent an additional mechanism of CASR transcriptional regulation in this group of developmental malignancies. © 2016 Wiley Periodicals, Inc.

mTOR-sensitive translation: Cleared fog reveals more trees.

Translation is fundamental for many biologic processes as it enables cells to rapidly respond to stimuli without requiring de novo mRNA synthesis. The mammalian/mechanistic target of rapamycin (mTOR) is a key regulator of translation. Although mTOR affects global protein synthesis, translation of a subset of mRNAs appears to be exceptionally sensitive to changes in mTOR activity. Recent efforts to catalog these mTOR-sensitive mRNAs resulted in conflicting results. Whereas ribosome-profiling almost exclusively identified 5'-terminal oligopyrimidine (TOP) mRNAs as mTOR-sensitive, polysome-profiling suggested that mTOR also regulates translation of non-TOP mRNAs. This inconsistency was explained by analytical and technical biases limiting the efficiency of ribosome-profiling in detecting mRNAs showing differential translation. Moreover, genome-wide characterization of 5'UTRs of non-TOP mTOR-sensitive mRNAs revealed 2 subsets of transcripts which differ in their requirement for translation initiation factors and biologic functions. We summarize these recent advances and their impact on the understanding of mTOR-sensitive translation.

CFTR rearrangements in Spanish cystic fibrosis patients: first new duplication (35kb) characterised in the Mediterranean countries.

Developments in quantitative PCR technologies have greatly improved our ability to detect large genome rearrangements. In particular oligonucleotide-based array comparative genomic hybridisation has become a useful tool for appropriate and rapid detection of breakpoints. In this work, we have analysed 80 samples (42 unknown CF alleles) applying three quantitative technologies (MLPA, qPCR and array-CGH) to detect recurrent as well as novel large rearrangements in the Spanish CF population. Three deletions and one duplication have been identified in five alleles (12%). Interestingly, we provide the comprehensive characterisation of the first duplication in our CF cohort. The new CFTRdupProm-3 mutation spans 35.7 kb involving the 5'-end of the CFTR gene. Additionally, the RNA analysis has revealed a cryptic sequence with a premature termination codon leading to a disrupted protein. This duplication has been identified in five unrelated families from Spain, France and Italy with all patients showing the same associated haplotype, which is further evidence for its likely common Mediterranean origin. Overall, considering this and other previous studies, CFTR rearrangements account for 1.3% of the Spanish CF alleles.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2ß phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2ß mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2ß and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis.

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.

Polymorphisms in the calcium-sensing receptor gene are associated with clinical outcome of neuroblastoma.

BACKGROUND: Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) to assess if genetic variants producing a less active receptor are associated with more aggressive disease course. METHODS: Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays. RESULTS: Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002) and the histological subgroup of undifferentiated neuroblastomas (P = 0.046). Patients harboring this polymorphism had significantly lower overall (P = 0.022) and event-free survival (P = 0.01) rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14-5.29]; P = 0.022) and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20-6.25]; P = 0.016). CONCLUSIONS: The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636 polymorphism. Thus, its association with metastatic disease and poor outcome would add to our previous data and further support that inactivation of the CaSR gene is a mechanism associated with neuroblastoma malignant behavior.

GUSB and ATP2B4 are suitable reference genes for CFTR gene expression data normalization in nasal epithelium cells.

BACKGROUND: CFTR expression studies contribute in understanding the relationship between CFTR transcripts and clinical outcomes. Normalization of qPCR data is an essential step to determine target gene expression. Consequently, appropriate reference genes must be selected for each gene/tissue. In this work, we have assessed the suitability of four potential reference genes for CFTR expression analysis in nasal epithelium. METHODS: B2M, GUSB, HPRT1 and ATP2B4 expression was evaluated in nasal epithelium samples (CFTR-wt controls, n=21; CFTR-splicing group, n=18) by RT-qPCR. Calibration curves were built and different analyses (geNorm, NormFinder, Mann-Whitney) were performed to evaluate gene expression stability between samples as well as between groups. RESULTS AND CONCLUSIONS: We have applied an accurate approach to select reference genes for CFTR expression analysis in nasal epithelium. From the four genes assessed, GUSB and ATP2B4 have been validated as a reliable gene combination for CFTR gene qPCR data normalization.

The p.Arg258Gly mutation in intracellular loop 2 of CFTR is associated with CFTR-related disorders.

Missense mutations account for approximately 50% of the mutations described in the CFTR gene. However, their proportion is higher in CFTR-related disorders (CFTR-RD) than in cystic fibrosis (CF), suggesting a different mutational spectrum. The uncertainty surrounding many of these mutations prevents suitable genetic counseling. Thus, it is crucial to determine whether a missense mutation has clinical expression, and if it does, to then define the associated phenotype. Herein we have assessed the phenotype associated with the p.Arg258Gly (R258G) mutation, checking our cohorts of patients (CF and CFTR-RD) and control subjects (CF carriers, fertile males, and general population). We also performed in silico predictive studies on the possible consequences of this mutation at the protein level. Lastly, we exhaustively reviewed the literature on this mutation. To date, R258G has only been found in six patients: a French congenital bilateral absence of vas deferens patient, reported in 1995 and five unrelated subjects from our cohort of non-CF patients, described here. Based on these findings, we postulate that R258G is primarily a CFTR-RD-associated mutation.