Translational Control of Metabolism and Cell Cycle Progression in Hepatocellular Carcinoma.

The liver is a metabolic hub characterized by high levels of protein synthesis. Eukaryotic initiation factors, eIFs, control the first phase of translation, initiation. Initiation factors are essential for tumor progression and, since they regulate the translation of specific mRNAs downstream of oncogenic signaling cascades, may be druggable. In this review, we address the issue of whether the massive translational machinery of liver cells contributes to liver pathology and to the progression of hepatocellular carcinoma (HCC); it represents a valuable biomarker and druggable target. First, we observe that the common markers of HCC cells, such as phosphorylated ribosomal protein S6, belong to the ribosomal and translational apparatus. This fact is in agreement with observations that demonstrate a huge amplification of the ribosomal machinery during the progression to HCC. Some translation factors, such as eIF4E and eIF6, are then harnessed by oncogenic signaling. In particular, the action of eIF4E and eIF6 is particularly important in HCC when driven by fatty liver pathologies. Indeed, both eIF4E and eIF6 amplify at the translational level the production and accumulation of fatty acids. As it is evident that abnormal levels of these factors drive cancer, we discuss their therapeutic value.

Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle.

Macrophages are characterized by a high plasticity in response to changes in tissue microenvironment, which allows them to acquire different phenotypes and to exert essential functions in complex processes, such as tissue regeneration. Here, we report that the membrane protein Cripto plays a key role in shaping macrophage plasticity in skeletal muscle during regeneration and disease. Conditional deletion of Cripto in the myeloid lineage (CriptoMy-LOF ) perturbs MP plasticity in acutely injured muscle and in mouse models of Duchenne muscular dystrophy (mdx). Specifically, CriptoMy-LOF macrophages infiltrate the muscle, but fail to properly expand as anti-inflammatory CD206+ macrophages, which is due, at least in part, to aberrant activation of TGFß/Smad signaling. This reduction in macrophage plasticity disturbs vascular remodeling by increasing Endothelial-to-Mesenchymal Transition (EndMT), reduces muscle regenerative potential, and leads to an exacerbation of the dystrophic phenotype. Thus, in muscle-infiltrating macrophages, Cripto is required to promote the expansion of the CD206+ anti-inflammatory macrophage type and to restrict the EndMT process, providing a direct functional link between this macrophage population and endothelial cells.

Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

ACOD1 deficiency offers protection in a mouse model of diet-induced obesity by maintaining a healthy gut microbiota.

Aconitate decarboxylase 1 (ACOD1) is the enzyme synthesizing itaconate, an immuno-regulatory metabolite tuning host-pathogen interactions. Such functions are achieved by affecting metabolic pathways regulating inflammation and microbe survival. However, at the whole-body level, metabolic roles of itaconate remain largely unresolved. By using multiomics-integrated approaches, here we show that ACOD1 responds to high-fat diet consumption in mice by promoting gut microbiota alterations supporting metabolic disease. Genetic disruption of itaconate biosynthesis protects mice against obesity, alterations in glucose homeostasis and liver metabolic dysfunctions by decreasing meta-inflammatory responses to dietary lipid overload. Mechanistically, fecal metagenomics and microbiota transplantation experiments demonstrate such effects are dependent on an amelioration of the intestinal ecosystem composition, skewed by high-fat diet feeding towards obesogenic phenotype. In particular, unbiased fecal microbiota profiling and axenic culture experiments point towards a primary role for itaconate in inhibiting growth of Bacteroidaceae and Bacteroides, family and genus of Bacteroidetes phylum, the major gut microbial taxon associated with metabolic health. Specularly to the effects imposed by Acod1 deficiency on fecal microbiota, oral itaconate consumption enhances diet-induced gut dysbiosis and associated obesogenic responses in mice. Unveiling an unrecognized role of itaconate, either endogenously produced or exogenously administered, in supporting microbiota alterations underlying diet-induced obesity in mice, our study points ACOD1 as a target against inflammatory consequences of overnutrition.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.

Targeting of eIF6-driven translation induces a metabolic rewiring that reduces NAFLD and the consequent evolution to hepatocellular carcinoma.

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.

The Tricarboxylic Acid Cycle at the Crossroad Between Cancer and Immunity.

Significance: The tricarboxylic acid (TCA) cycle is a housekeeping metabolic pathway essential for generation of energy and biosynthetic intermediates. Alterations of the TCA cycle play a pivotal role in oncogenesis and inflammation. As such, some metabolic vulnerabilities, imposed by TCA cycle dysfunction in cancer, have been identified. Similarly, the TCA cycle appeared as an actionable pathway in immunopathologies. Recent Advances: Metabolic changes accompanying cell transformation have been usually considered as adaptive mechanisms to malignant transformation. The identification of oncogenic mutations in some TCA cycle enzymes changed this view, indicating altered mitochondrial metabolism as an instrumental mechanism for cancer initiation. Similarly, the observation that TCA cycle-derived metabolites have multiple signaling roles in immune cells supports the idea of this pathway as a metabolic rheostat of immune responses. Critical Issues: This review summarizes the crucial role of the TCA cycle in pathophysiology describing the post-translational and epigenetic impact of oncometabolites accumulation in cancer and immune cells. Future Directions: Additional studies will be necessary to further explore the role of oncometabolites in paracrine signaling and to identify genuine metabolic and nutritional liabilities imposed by TCA cycle dysfunction in cancer, hardly to be escaped by resistance mechanisms.

D-mannose suppresses macrophage IL-1ß production.

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1ß production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions.

Translating the Game: Ribosomes as Active Players.

Ribosomes have been long considered as executors of the translational program. The fact that ribosomes can control the translation of specific mRNAs or entire cellular programs is often neglected. Ribosomopathies, inherited diseases with mutations in ribosomal factors, show tissue specific defects and cancer predisposition. Studies of ribosomopathies have paved the way to the concept that ribosomes may control translation of specific mRNAs. Studies in Drosophila and mice support the existence of heterogeneous ribosomes that differentially translate mRNAs to coordinate cellular programs. Recent studies have now shown that ribosomal activity is not only a critical regulator of growth but also of metabolism. For instance, glycolysis and mitochondrial function have been found to be affected by ribosomal availability. Also, ATP levels drop in models of ribosomopathies. We discuss findings highlighting the relevance of ribosome heterogeneity in physiological and pathological conditions, as well as the possibility that in rate-limiting situations, ribosomes may favor some translational programs. We discuss the effects of ribosome heterogeneity on cellular metabolism, tumorigenesis and aging. We speculate a scenario in which ribosomes are not only executors of a metabolic program but act as modulators.

A Polysome-Based microRNA Screen Identifies miR-24-3p as a Novel Promigratory miRNA in Mesothelioma.

The expression of miRNAs in cancer has been widely studied and has allowed the definition of oncomirs and oncosuppressors. We note that it is often underestimated that many mRNAs are expressed, but translationally silent. In spite of this, systematic identification of miRNAs in equilibrium with their target mRNAs on polysomes has not been widely exploited. To identify biologically active oncomirs, we performed a screen for miRNAs acting on the polysomes of malignant mesothelioma (MPM) cells. Only a small percentage of expressed miRNAs physically associated with polysomes. On polysomes, we identified miRNAs already characterized in MPM, as well as novel ones like miR-24-3p, which acted as a promigratory miRNA in all cancer cells tested. miR-24-3p positively regulated Rho-GTP activity, and inhibition of miR-24-3p reduced growth in MPM cells. Analysis of miR-24-3p common targets, in two mesothelioma cell lines, identified a common subset of downregulated genes. These same genes were downregulated during the progression of multiple cancer types. Among the specific targets of miR-24-3p was cingulin, a tight junction protein that inhibits Rho-GTP activity. Overexpression of miR-24-3p only partially abrogated cingulin mRNA, but completely abrogated cingulin protein, confirming its action via translational repression. We suggest that miR-24-3p is an oncomir and speculate that identification of polysome-associated miRNAs efficiently sorts out biologically active miRNAs from inactive ones.Significance: Subcellular localization of miRNAs may predict their role in cancer and identify novel oncogenic miRNAs involved in cancer progression.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5741/F1.large.jpg Cancer Res; 78(20); 5741-53. ©2018 AACR.

Data on the effects of eIF6 downmodulation on the proportions of innate and adaptive immune system cell subpopulations and on thymocyte maturation.

The data described in this article are related to "High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans" (Manfrini et al., in press) [1]. eIF6 is a translation initiation factor required for ribosomal biogenesis (Sanvito et al., 1999) [2] and for proper translational initiation (Gallo and Manfrini, 2015; Miluzio et al., 2016) [3], [4] whose protein abundance requires tight regulation. Here we analyze by flow cytometry the effects of eIF6 depletion on proportions of specific innate and adaptive immune system subpopulations and on thymocyte maturation in mice.

High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans.

Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4+ Effector Memory T cells. In human CD4+ T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4+ T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery.

The Role of Eif6 in Skeletal Muscle Homeostasis Revealed by Endurance Training Co-expression Networks.

Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.