Selective modulation of the glucocorticoid receptor can distinguish between transrepression of NF-κB and AP-1.

Glucocorticoids (GCs) block inflammation via interference of the liganded glucocorticoid receptor (GR) with the activity of pro-inflammatory transcription factors NF-κB and AP-1, a mechanism known as transrepression. This mechanism is believed to involve the activity of GR monomers. Here, we explored how the GR monomer-favoring Compound A (CpdA) affects AP-1 activation and activity. Our results demonstrate that non-steroidal CpdA, unlike classic steroidal GCs, blocks NF-κB- but not AP-1-driven gene expression. CpdA rather sustains AP-1-driven gene expression, a result which could mechanistically be explained by the failure of CpdA to block upstream JNK kinase activation and concomitantly also phosphorylation of c-Jun. In concordance and in contrast to DEX, CpdA maintained the expression of the activated AP-1 target gene c-jun, as well as the production of the c-Jun protein. As for the underlying mechanism, GR is a necessary intermediate in the CpdA-mediated gene expression of AP-1-regulated genes, but seems to be superfluous to CpdA-mediated JNK phosphorylation prolongation. The latter phenomenon concurs with the inability of CpdA to stimulate DUSP1 gene expression. ChIP analysis demonstrates that DEX-activated GR, but not CpdA-activated GR, is recruited to AP-1-driven promoters. Furthermore, in mice we observed that CpdA instigates a strong enhancement of TNF-induced AP-1-driven gene expression. Finally, we demonstrate that this phenomenon coincides with an increased sensitivity towards TNF lethality, and implicate again a role for JNK2. In conclusion, our data support the hypothesis that a ligand-induced differential conformation of GR yields a different transcription factor cross-talk profile.

Protein kinase and HDAC inhibitors from the endophytic fungus Epicoccum nigrum.

A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 µM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 µM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.

Identification of an H-Ras nanocluster disrupting peptide.

Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors.

An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth.

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

Long and short non-coding RNAs as regulators of hematopoietic differentiation.

Genomic analyses estimated that the proportion of the genome encoding proteins corresponds to approximately 1.5%, while at least 66% are transcribed, suggesting that many non-coding DNA-regions generate non-coding RNAs (ncRNAs). The relevance of these ncRNAs in biological, physiological as well as in pathological processes increased over the last two decades with the understanding of their implication in complex regulatory networks. This review particularly focuses on the involvement of two large families of ncRNAs, namely microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the regulation of hematopoiesis. To date, miRNAs have been widely studied, leading to a wealth of data about processing, regulation and mechanisms of action and more specifically, their involvement in hematopoietic differentiation. Notably, the interaction of miRNAs with the regulatory network of transcription factors is well documented whereas roles, regulation and mechanisms of lncRNAs remain largely unexplored in hematopoiesis; this review gathers current data about lncRNAs as well as both potential and confirmed roles in normal and pathological hematopoiesis.

Altered profiles of circulating cytokines in chronic liver diseases (NAFLD/HCC): Impact of the PNPLA3I148M risk allele.

BACKGROUND: Individuals carrying the risk variant p.I148M of patatin-like phospholipase domain-containing protein 3 (PNPLA3) have a higher susceptibility to fatty liver diseases and associated complications, including HCC, a cancer closely linked to chronic inflammation. Here, we assessed circulating cytokine profiles for patients with chronic liver diseases genotyped for PNPLA3. METHODS: Serum concentrations of 22 cytokines were measured by multiplex sandwich-ELISA. The cohort comprised 123 individuals: 67 patients with NAFLD without cirrhosis (57 steatosis, 10 NASH), 24 patients with NAFLD with cirrhosis, 21 patients with HCC (15 cirrhosis), and 11 healthy controls. Receiver operator characteristic analyses were performed to assess the suitability of the cytokine profiles for the prediction of steatosis, cirrhosis, and HCC. RESULTS: HGF, IL-6, and IL-8 levels were increased in patients, with ∼2-fold higher levels in patients with cirrhosis versus healthy, while platelet derived growth factor-BB (PDGF-BB) and regulated on activation, normal T cell expressed and secreted (RANTES) showed lower concentrations compared to controls. Migration inhibitory factor and monocyte chemoattractant protein-1 (MCP-1) were found at higher levels in NAFLD samples (maximum: NAFLD-cirrhosis) versus healthy controls and HCC samples. In receiver operator characteristic analyses, migration inhibitory factor, IL-8, IL-6, and monocyte chemoattractant protein-1 yielded high sensitivity scores for predicting noncirrhotic NAFLD (vs. healthy). The top combination to predict cirrhosis was HGF plus PDGF-BB. Migration inhibitory factor performed best to discriminate HCC from NAFLD; the addition of monokine induced gamma (MIG), RANTES, IL-4, macrophage colony-stimulating factor (M-CSF), or IL-17A as second parameters further increased the AUC values (> 0.9). No significant impact of the PNPLA3I148M allele on cytokine levels was observed in this cohort. CONCLUSIONS: Cytokines have biomarker potential in patients with fatty liver, possibly suited for early HCC detection in patients with fatty liver. Patients carrying the PNPLA3 risk allele did not present significantly different levels of circulating cytokines.

miRNA-132-5p mediates a negative feedback regulation of IL-8 secretion through S100A8/A9 downregulation in neutrophil-like HL-60 cells.

Background: Neutrophils are an important source of pro-inflammatory and immunomodulatory cytokines. This makes neutrophils efficient drivers of interactions with immune and non-immune cells to maintain homeostasis and modulate the inflammatory process by notably regulating the release of cytokines. Ca2+-dependent regulatory mechanism encompassing cytokine secretion by neutrophils are not still identified. In this context, we propose to define new insights on the role of Ca2+-binding proteins S100A8/A9 and on the regulatory role of miRNA-132-5p, which was identified as a regulator of S100A8/A9 expression, on IL-8 secretion. Methods: Differentiated HL-60 cells, a human promyelocytic leukemia cell line that can be induced to differentiate into neutrophil-like cells, were used as a model of human neutrophils and treated with N- formyl-methionyl-leucyl-phenylalanine (fMLF), a bacterial peptide that activates neutrophils. shRNA knockdown was used to define the role of selected targets (S100A8/A9 and miRNA-132-5p) on IL-8 secretion. Results and discussion: Different types of cytokines engage different signaling pathways in the secretion process. IL-8 release is tightly regulated by Ca2+ binding proteins S100A8/A9. miRNA-132-5p is up-regulated over time upon fMLF stimulation and decreases S100A8/A9 expression and IL-8 secretion. Conclusion: These findings reveal a novel regulatory loop involving S100A8/A9 and miRNA-132-5p that modulates IL-8 secretion by neutrophils in inflammatory conditions. This loop could be a potential target for therapeutic intervention in inflammatory diseases.

A multi-omics integrative approach unravels novel genes and pathways associated with senescence escape after targeted therapy in NRAS mutant melanoma.

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.

The gut microbial metabolite formate exacerbates colorectal cancer progression.

The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.

Stromal fibroblasts shape the myeloid phenotype in normal colon and colorectal cancer and induce CD163 and CCL2 expression in macrophages.

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.

Quantitative trait locus mapping identifies a locus linked to striatal dopamine and points to collagen IV alpha-6 chain as a novel regulator of striatal axonal branching in mice.

Dopaminergic neurons (DA neurons) are controlled by multiple factors, many involved in neurological disease. Parkinson's disease motor symptoms are caused by the demise of nigral DA neurons, leading to loss of striatal dopamine (DA). Here, we measured DA concentration in the dorsal striatum of 32 members of Collaborative Cross (CC) family and their eight founder strains. Striatal DA varied greatly in founders, and differences were highly heritable in the inbred CC progeny. We identified a locus, containing 164 genes, linked to DA concentration in the dorsal striatum on chromosome X. We used RNAseq profiling of the ventral midbrain of two founders with substantial difference in striatal DA-C56BL/6 J and A/J-to highlight potential protein-coding candidates modulating this trait. Among the five differentially expressed genes within the locus, we found that the gene coding for the collagen IV alpha 6 chain (Col4a6) was expressed nine times less in A/J than in C57BL/6J. Using single cell RNA-seq data from developing human midbrain, we found that COL4A6 is highly expressed in radial glia-like cells and neuronal progenitors, indicating a role in neuronal development. Collagen IV alpha-6 chain (COL4A6) controls axogenesis in simple model organisms. Consistent with these findings, A/J mice had less striatal axonal branching than C57BL/6J mice. We tentatively conclude that DA concentration and axonal branching in dorsal striatum are modulated by COL4A6, possibly during development. Our study shows that genetic mapping based on an easily measured Central Nervous System (CNS) trait, using the CC population, combined with follow-up observations, can parse heritability of such a trait, and nominate novel functions for commonly expressed proteins.

PathEx: a novel multi factors based datasets selector web tool.

BACKGROUND: Microarray experiments have become very popular in life science research. However, if such experiments are only considered independently, the possibilities for analysis and interpretation of many life science phenomena are reduced. The accumulation of publicly available data provides biomedical researchers with a valuable opportunity to either discover new phenomena or improve the interpretation and validation of other phenomena that partially understood or well known. This can only be achieved by intelligently exploiting this rich mine of information. DESCRIPTION: Considering that technologies like microarrays remain prohibitively expensive for researchers with limited means to order their own experimental chips, it would be beneficial to re-use previously published microarray data. For certain researchers interested in finding gene groups (requiring many replicates), there is a great need for tools to help them to select appropriate datasets for analysis. These tools may be effective, if and only if, they are able to re-use previously deposited experiments or to create new experiments not initially envisioned by the depositors. However, the generation of new experiments requires that all published microarray data be completely annotated, which is not currently the case. Thus, we propose the PathEx approach. CONCLUSION: This paper presents PathEx, a human-focused web solution built around a two-component system: one database component, enriched with relevant biological information (expression array, omics data, literature) from different sources, and another component comprising sophisticated web interfaces that allow users to perform complex dataset building queries on the contents integrated into the PathEx database.

Functional analysis: evaluation of response intensities--tailoring ANOVA for lists of expression subsets.

BACKGROUND: Microarray data is frequently used to characterize the expression profile of a whole genome and to compare the characteristics of that genome under several conditions. Geneset analysis methods have been described previously to analyze the expression values of several genes related by known biological criteria (metabolic pathway, pathology signature, co-regulation by a common factor, etc.) at the same time and the cost of these methods allows for the use of more values to help discover the underlying biological mechanisms. RESULTS: As several methods assume different null hypotheses, we propose to reformulate the main question that biologists seek to answer. To determine which genesets are associated with expression values that differ between two experiments, we focused on three ad hoc criteria: expression levels, the direction of individual gene expression changes (up or down regulation), and correlations between genes. We introduce the FAERI methodology, tailored from a two-way ANOVA to examine these criteria. The significance of the results was evaluated according to the self-contained null hypothesis, using label sampling or by inferring the null distribution from normally distributed random data. Evaluations performed on simulated data revealed that FAERI outperforms currently available methods for each type of set tested. We then applied the FAERI method to analyze three real-world datasets on hypoxia response. FAERI was able to detect more genesets than other methodologies, and the genesets selected were coherent with current knowledge of cellular response to hypoxia. Moreover, the genesets selected by FAERI were confirmed when the analysis was repeated on two additional related datasets. CONCLUSIONS: The expression values of genesets are associated with several biological effects. The underlying mathematical structure of the genesets allows for analysis of data from several genes at the same time. Focusing on expression levels, the direction of the expression changes, and correlations, we showed that two-step data reduction allowed us to significantly improve the performance of geneset analysis using a modified two-way ANOVA procedure, and to detect genesets that current methods fail to detect.

A benchmark for statistical microarray data analysis that preserves actual biological and technical variance.

BACKGROUND: Recent reanalysis of spike-in datasets underscored the need for new and more accurate benchmark datasets for statistical microarray analysis. We present here a fresh method using biologically-relevant data to evaluate the performance of statistical methods. RESULTS: Our novel method ranks the probesets from a dataset composed of publicly-available biological microarray data and extracts subset matrices with precise information/noise ratios. Our method can be used to determine the capability of different methods to better estimate variance for a given number of replicates. The mean-variance and mean-fold change relationships of the matrices revealed a closer approximation of biological reality. CONCLUSIONS: Performance analysis refined the results from benchmarks published previously.We show that the Shrinkage t test (close to Limma) was the best of the methods tested, except when two replicates were examined, where the Regularized t test and the Window t test performed slightly better. AVAILABILITY: The R scripts used for the analysis are available at http://urbm-cluster.urbm.fundp.ac.be/~bdemeulder/.

Global analysis of quorum sensing targets in the intracellular pathogen Brucella melitensis 16 M.

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A DeltavjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.

Meta-analysis of archived DNA microarrays identifies genes regulated by hypoxia and involved in a metastatic phenotype in cancer cells.

BACKGROUND: Metastasis is a major cancer-related cause of death. Recent studies have described metastasis pathways. However, the exact contribution of each pathway remains unclear. Another key feature of a tumor is the presence of hypoxic areas caused by a lack of oxygen at the center of the tumor. Hypoxia leads to the expression of pro-metastatic genes as well as the repression of anti-metastatic genes. As many Affymetrix datasets about metastasis and hypoxia are publicly available and not fully exploited, this study proposes to re-analyze these datasets to extract new information about the metastatic phenotype induced by hypoxia in different cancer cell lines. METHODS: Affymetrix datasets about metastasis and/or hypoxia were downloaded from GEO and ArrayExpress. AffyProbeMiner and GCRMA packages were used for pre-processing and the Window Welch t test was used for processing. Three approaches of meta-analysis were eventually used for the selection of genes of interest. RESULTS: Three complementary approaches were used, that eventually selected 183 genes of interest. Out of these 183 genes, 99, among which the well known JUNB, FOS and TP63, have already been described in the literature to be involved in cancer. Moreover, 39 genes of those, such as SERPINE1 and MMP7, are known to regulate metastasis. Twenty-one genes including VEGFA and ID2 have also been described to be involved in the response to hypoxia. Lastly, DAVID classified those 183 genes in 24 different pathways, among which 8 are directly related to cancer while 5 others are related to proliferation and cell motility. A negative control composed of 183 random genes failed to provide such results. Interestingly, 6 pathways retrieved by DAVID with the 183 genes of interest concern pathogen recognition and phagocytosis. CONCLUSION: The proposed methodology was able to find genes actually known to be involved in cancer, metastasis and hypoxia and, thus, we propose that the other genes selected based on the same methodology are of prime interest in the metastatic phenotype induced by hypoxia.

Human telomerase reverse transcriptase depletion potentiates the growth-inhibitory activity of imatinib in chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors (TKIs) revolutionized the management of chronic myeloid leukemia (CML), resistance against TKIs and leukemia stem cell (LSC) persistence remain a clinical concern. Therefore, new therapeutic strategies combining conventional and novel therapies are urgently needed. Since telomerase is involved in oncogenesis and tumor progression but is silent in most human normal somatic cells, it may be an interesting target for CML therapy by selectively targeting cancer cells while minimizing effects on normal cells. Here, we report that hTERT expression is associated with CML disease progression. We also provide evidence that hTERT-deficient K-562 cells do not display telomere shortening and that telomere length is maintained through the ALT pathway. Furthermore, we show that hTERT depletion exerts a growth-inhibitory effect in K-562 cells and potentiates imatinib through alteration of cell cycle progression leading to a senescence-like phenotype. Finally, we demonstrate that hTERT depletion potentiates the imatinib-induced reduction of the ALDH+-LSC population. Altogether, our results suggest that the combination of telomerase and TKI should be considered as an attractive strategy to treat CML patients to eradicate cancer cells and prevent relapse by targeting LSCs.

Hypoxia-Induced Adaptations of miRNomes and Proteomes in Melanoma Cells and Their Secreted Extracellular Vesicles.

Reduced levels of intratumoural oxygen are associated with hypoxia-induced pro-oncogenic events such as invasion, metabolic reprogramming, epithelial-mesenchymal transition, metastasis and resistance to therapy, all favouring cancer progression. Small extracellular vesicles (EV) shuttle various cargos (proteins, miRNAs, DNA and others). Tumour-derived EVs can be taken up by neighbouring or distant cells in the tumour microenvironment, thus facilitating intercellular communication. The quantity of extracellular vesicle secretion and their composition can vary with changing microenvironmental conditions and disease states. Here, we investigated in melanoma cells the influence of hypoxia on the content and number of secreted EVs. Whole miRNome and proteome profiling revealed distinct expression patterns in normoxic or hypoxic growth conditions. Apart from the well-known miR-210, we identified miR-1290 as a novel hypoxia-associated microRNA, which was highly abundant in hypoxic EVs. On the other hand, miR-23a-5p and -23b-5p were consistently downregulated in hypoxic conditions, while the protein levels of the miR-23a/b-5p-predicted target IPO11 were concomitantly upregulated. Furthermore, hypoxic melanoma EVs exhibit a signature consisting of six proteins (AKR7A2, DDX39B, EIF3C, FARSA, PRMT5, VARS), which were significantly associated with a poor prognosis for melanoma patients, indicating that proteins and/or miRNAs secreted by cancer cells may be exploited as biomarkers.

Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in human hematopoietic stem/progenitor cells.

Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNFα is also a potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNFα/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34+ hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNFα, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNFα inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNFα- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (TFs) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNFα and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNFα/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNFα- and ceramide-induced phosphorylation of mTORS2448 and ULK1S758, inhibited Atg13S355 phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNFα and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNFα/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic TF/miR network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.