Molecular Classification of Colorectal Cancer by microRNA Profiling: Correlation with the Consensus Molecular Subtypes (CMS) and Validation of miR-30b Targets.

Colorectal cancer consensus molecular subtypes (CMSs) are widely accepted and constitutes the basis for patient stratification to improve clinical practice. We aimed to find whether miRNAs could reproduce molecular subtypes, and to identify miRNA targets associated to the High-stroma/CMS4 subtype. The expression of 939 miRNAs was analyzed in tumors classified in CMS. TALASSO was used to find gene-miRNA interactions. A miR-mRNA regulatory network was constructed using Cytoscape. Candidate gene-miR interactions were validated in 293T cells. Hierarchical-Clustering identified three miRNA tumor subtypes (miR-LS; miR-MI; and miR-HS) which were significantly associated (p < 0.001) to the reported mRNA subtypes. miR-LS correlated with the low-stroma/CMS2; miR-MI with the mucinous-MSI/CMS1 and miR-HS with high-stroma/CMS4. MicroRNA tumor subtypes and association to CMSs were validated with TCGA datasets. TALASSO identified 1462 interactions (p < 0.05) out of 21,615 found between 176 miRs and 788 genes. Based on the regulatory network, 88 miR-mRNA interactions were selected as candidates. This network was functionally validated for the pair miR-30b/SLC6A6. We found that miR-30b overexpression silenced 3'-UTR-SLC6A6-driven luciferase expression in 293T-cells; mutation of the target sequence in the 3'-UTR-SLC6A6 prevented the miR-30b inhibitory effect. In conclusion CRC subtype classification using a miR-signature might facilitate a real-time analysis of the disease course and treatment response.

A flow cytometry-based method to screen for modulators of tumor-specific T cell cytotoxicity.

Cancer immunotherapy relies on the ability of immune cells to kill malignant cells. The cytotoxic T lymphocyte (CTL) response is perhaps the functional measure that best reflects cell-mediated immunity against cancer. Straightforward methods that facilitate quantitative evaluation of the potency of compounds that can modulate T cell-mediated, tumor antigen-specific immune responses are central in the screening cascade when searching for new immunotherapeutic agents for cancer. Here we describe a simple, sensitive method, based on flow cytometry analyses, to quantitatively measure cytotoxicity in vitro. We provide examples that validate its feasibility and specificity using CD8+ T lymphocytes specific for a surrogate tumor antigen, and a blocking antibody for the inhibitory PD-1/PD-L1 axis. This method can nonetheless be applied to the screening of virtually any cytotoxicity modulatory compound, including antibodies and small molecules or T cell-based therapies, and can be scaled up for high-throughput workflow and automation.

PD-1 signaling affects cristae morphology and leads to mitochondrial dysfunction in human CD8+ T lymphocytes.

BACKGROUND: Binding of the programmed death-1 (PD-1) receptor to its ligands (PD-L1/2) transduces inhibitory signals that promote exhaustion of activated T cells. Blockade of the PD-1 pathway is widely used for cancer treatment, yet the inhibitory signals transduced by PD-1 in T cells remain elusive. METHODS: Expression profiles of human CD8+ T cells in resting, activated (CD3 + CD28) and PD-1-stimulated cells (CD3 + CD28 + PD-L1-Fc) conditions were evaluated by RNA-seq. Bioinformatic analyses were used to identify signaling pathways differentially regulated in PD-1-stimulated cells. Metabolic analyses were performed with SeaHorse technology, and mitochondrial ultrastructure was determined by transmission electron microscopy. PD-1-regulated mitochondrial genes were silenced using short-hairpin RNA in primary cells. Blue native gel electrophoresis was used to determine respiratory supercomplex assembly. RESULTS: PD-1 engagement in human CD8+ T cells triggers a specific, progressive genetic program different from that found in resting cells. Gene ontology identified metabolic processes, including glycolysis and oxidative phosphorylation (OXPHOS), as the main pathways targeted by PD-1. We observed severe functional and structural alterations in the mitochondria of PD-1-stimulated cells, including a reduction in the number and length of mitochondrial cristae. These cristae alterations were associated with reduced expression of CHCHD3 and CHCHD10, two proteins that form part of the mitochondrial contact site and cristae organizing system (MICOS). Although PD-1-stimulated cells showed severe cristae alterations, assembly of respiratory supercomplexes was unexpectedly greater in these cells than in activated T cells. CHCHD3 silencing in primary CD8+ T cells recapitulated some effects induced by PD-1 stimulation, including reduced mitochondrial polarization and interferon-γ production following T cell activation with anti-CD3 and -CD28 activating antibodies. CONCLUSIONS: Our results suggest that mitochondria are the main targets of PD-1 inhibitory activity. PD-1 reprograms CD8+ T cell metabolism for efficient use of fatty acid oxidation; this mitochondrial phenotype might explain the long-lived phenotype of PD-1-engaged T cells.

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα-/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

Notch-regulated miR-223 targets the aryl hydrocarbon receptor pathway and increases cytokine production in macrophages from rheumatoid arthritis patients.

Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14(+) cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3'UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.

Involvement of lipid droplets in hepatic responses to lipopolysaccharide treatment in mice.

Infection and inflammation induce important changes in lipid metabolism, which result in increased free fatty acids and triacylglycerol in plasma and altered high density lipoprotein (HDL) metabolism. Our aim was to elucidate whether hepatic lipid droplets (LDs) are involved in the adaptations of lipid metabolism to endotoxemia. We characterized the lipid content and several enzymatic activities in subcellular fractions and subpopulations of LDs from livers of mice 24h after lipopolysaccharide (LPS) treatment and analyzed the expression of key genes involved in lipid management. Endotoxemic mice showed lower lipid content in LDs with decreased molar fraction of cholesteryl ester and higher diacylglycerol/triacylglycerol ratio as compared to their controls. They also showed a decrease in cytosolic triacylglycerol hydrolase activity, specifically in dense LDs, and in microsomal and cytosolic diacylglycerol hydrolase activity; concomitantly neutral lipid biosynthetic capacity and triacylglycerol levels in plasma lipoproteins increased. Together with the overexpression of genes involved in lipogenesis and HDL formation our results suggest that altered hepatic management of LD lipids in LPS-treated mice might be related to the channeled mobilization of triacylglycerol for very low density lipoprotein assembly and to the induction of cholesterol export.