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BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA.METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters.RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence.CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features.LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.

BACKGROUND: The paternal diet affects lipid metabolism in offspring for at least two generations through nutritional programming. However, we do not know how this is propagated to the offspring. OBJECTIVES: We tested the hypothesis that the changes in lipid metabolism that are driven by paternal diet are propagated through spermatozoa and not seminal plasma. METHODS: We applied an updated, purpose-built computational network analysis tool to characterise control of lipid metabolism systemically (Lipid Traffic Analysis v2.3) on a known mouse model of paternal nutritional programming. RESULTS: The analysis showed that the two possible routes for programming effects, the sperm (genes) and seminal plasma (influence on the uterine environment), both have a distinct effect on the offspring's lipid metabolism. Further, the programming effects in offspring suggest that changes in lipid distribution are more important than alterations in lipid biosynthesis. CONCLUSIONS: These results show how the uterine environment and genes both affect lipid metabolism in offspring, enhancing our understanding of the link between parental diet and metabolism in offspring., BBSRC (BB/M027252/1)

Intra-abdominal infections (IAIs) are common surgical emergencies and have been reported as major contributors to non-trauma deaths in hospitals worldwide. The cornerstones of effective treatment of IAIs include early recognition, adequate source control, appropriate antimicrobial therapy, and prompt physiologic stabilization using a critical care environment, combined with an optimal surgical approach. Together, the World Society of Emergency Surgery (WSES), the Global Alliance for Infections in Surgery (GAIS), the Surgical Infection Society-Europe (SIS-E), the World Surgical Infection Society (WSIS), and the American Association for the Surgery of Trauma (AAST) have jointly completed an international multi-society document in order to facilitate clinical management of patients with IAIs worldwide building evidence-based clinical pathways for the most common IAIs. An extensive non-systematic review was conducted using the PubMed and MEDLINE databases, limited to the English language. The resulting information was shared by an international task force from 46 countries with different clinical backgrounds. The aim of the document is to promote global standards of care in IAIs providing guidance to clinicians by describing reasonable approaches to the management of IAIs.

NOD mice represent a unique strain that recapitulates some aspects of progressive MS when subjected to experimental autoimmune encephalomyelitis (EAE). It is unknown, however, whether a proneness to demyelination and/or defect in remyelination contribute to disease progression in NOD mice. Answering to this question might help deciphering the molecular and cellular events underpinning disease evolution in progressive MS. Here, we compared the cuprizone-dependent demyelination and remyelination responses, as well as their functional correlates, in NOD, C57BL/6, and SJL mice typically adopted to model progressive, chronic or relapsing EAE. We report that demyelination occurred to a similar extent in the three mice strains, and that in none of them there was evidence of axonal degeneration during prolonged demyelination. Moreover, immunostaining for GFAP+ astrocytes, Iba1+ microglia, and NG2+ oligodendrocyte precursor cells similarly increased in the 3 mouse strains after cuprizone exposure. The mice underwent concomitant and complete remyelination 2 weeks after cuprizone withdrawal. On a functional level, NOD mice showed the earliest reduction of spontaneous motility and full recovery, but no impairment of motor skill. Conversely, C57BL/6 animals showed phasic reduction of both spontaneous motility and motor skill. Lastly, SJL mice presented the most severe neurological impairment with long-lasting reduction of spontaneous motility and motor skill. Overall, data suggest that the unique feature of EAE progression in NOD mice is not due to proneness to demyelination or intrinsic defects in myelin formation. Findings also unravel important functional differences in the response of the three mouse stains to cuprizone that can be harnessed to design and interpret future experiments.

Background and aims: Hepatocytes undergo profound metabolic rewiring when primed to proliferate during compensatory regeneration and in hepatocellular carcinoma (HCC). However, the metabolic control of these processes is not fully understood. In order to capture the metabolic signature of proliferating hepatocytes, we applied state-of-the-art systems biology approaches to models of liver regeneration, pharmacologically and genetically activated cell proliferation, and HCC.Approach and results: Integrating metabolomics, lipidomics, and transcriptomics, we link changes in the lipidome of proliferating hepatocytes to altered metabolic pathways including lipogenesis, fatty acid desaturation, and generation of phosphatidylcholine (PC). We confirm this altered lipid signature in human HCC and show a positive correlation of monounsaturated PC with hallmarks of cell proliferation and hepatic carcinogenesis.Conclusions: Overall, we demonstrate that specific lipid metabolic pathways are coherently altered when hepatocytes switch to proliferation. These represent a source of targets for the development of therapeutic strategies and prognostic biomarkers of HCC.

Environmental cues are essential in defining tumour malignancy, by promoting tumour initiation, progression and metastatic spreading. Stromal cells may metabolically cooperate or compete with cancer cells, playing a mandatory role in defining cancer metabolic plasticity, potentially dictating the final tumour outcome. Assessing shared nutrients between different tumoural or stromal compartments is essential to understand the impact of environmental nutrients on the metabolic plasticity of tumours. Here, we review analytical and computational approaches for studying the tumour metabolic microenvironment, the destiny of nutrients shared among tumour and stromal populations, as well as the molecular modules of these metabolic relationships.

Metabolic reprogramming as well as the flexible utilisation of fuel sources by tumour cells has been considered not only intrinsic to malignant cells but also sustained by resident and/or recruited stromal cells. The complexity of tumour-stroma cross-talk is experienced by neoplastic cells through profound changes in the own metabolic machinery. In such context, mitochondria are dynamic organelles that receive, orchestrate and exchange a multiplicity of stromal cues within the tumour cells to finely regulate key metabolic and signalling pathways, allowing malignant cells to adapt and thrive in an ever-changing environment. In this review, we focus on how tumour mitochondria are coached by stromal metabolic supply and how this re-education sustains tumour malignant traits.

In this paper we present an investigation of parental-diet-driven metabolic programming in offspring using a novel computational network analysis tool. The impact of high paternal carbohydrate intake on offsprings’ phospholipid and triglyceride metabolism in F1 and F2 generations is described. Detailed lipid profiles were acquired from F1 neonate (3 weeks), F1 adult (16 weeks) and F2 neonate offspring in serum, liver, brain, heart and abdominal adipose tissues by MS and NMR. Using a purpose-built computational tool for analysing both phospholipid and fat metabolism as a network, we characterised the number, type and abundance of lipid variables in and between tissues (Lipid Traffic Analysis), finding a variety of reprogrammings associated with paternal diet. These results are important because they describe the long-term metabolic result of dietary intake by fathers. This analytical approach is important because it offers unparalleled insight into possible mechanisms for alterations in lipid metabolism throughout organisms., Furse et al. use a purpose-built computational tool called Lipid Traffic Analysis to determine the spatial distribution of lipids throughout an organism. They use it to show that high paternal carbohydrate intake influences lipid metabolism in offspring two generations hence.

Contains fulltext : 238976.pdf (Publisher’s version ) (Open Access) On January 2020, the WHO Director General declared that the outbreak constitutes a Public Health Emergency of International Concern. The world has faced a worldwide spread crisis and is still dealing with it. The present paper represents a white paper concerning the tough lessons we have learned from the COVID-19 pandemic. Thus, an international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making. With the present paper, international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making.

The tumour microenvironment (TME) is now recognised as a hallmark of cancer, since tumour:stroma crosstalk supports the key steps of tumour growth and progression. The dynamic co-evolution of the tumour and stromal compartments may alter the surrounding microenvironment, including the composition in metabolites and signalling mediators. A growing number of evidence reports the involvement of the endocannabinoid system (ECS) in cancer. ECS is composed by a complex network of ligands, receptors, and enzymes, which act in synergy and contribute to several physiological but also pathological processes. Several in vitro and in vivo evidence show that ECS deregulation in cancer cells affects proliferation, migration, invasion, apoptosis, and metastatic potential. Although it is still an evolving research, recent experimental evidence also suggests that ECS can modulate the functional behaviour of several components of the TME, above all the immune cells, endothelial cells and stromal components. However, the role of ECS in the tumour:stroma interplay remains unclear and research in this area is particularly intriguing. This review aims to shed light on the latest relevant findings of the tumour response to ECS modulation, encouraging a more in-depth analysis in this field. Novel discoveries could be promising for novel anti-tumour approaches, targeting the microenvironmental components and the supportive tumour:stroma crosstalk, thereby hindering tumour development.

BackgroundMetabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance.MethodsA survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines.ResultsmiR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines.ConclusionsWe disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30–90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT.

Objectives This study is aimed to report a case of SV-related gastritis and the results of a systematic review of SV infections in the literature. Methods Following a case presentation, we systematically searched different databases (MEDLINE, PubMed, Scopus, Web of Science, EMBASE) for the items “sarcina,” “ventriculi,” “clostridium” with AND/OR. Results A total of 55 articles reporting 65 cases of Sarcina Ventriculi were found. Thus, 66 patients, including our case, were reviewed. Median age was 51 years (IQR: 0-87 years). Females accounted for 51% of cases. 68% of patients had one or more comorbidities. SV was isolated in the gastrointestinal tract (88%), respiratory (5%), urine (4%), and bloodstream (3%) systems. Upper endoscopy was performed in 52 patients (79%). Biopsies were obtained in all 52 cases, and resulted normal in 23%. Surgery was warranted in 15 patients (23%), and specific antimicrobial therapy was delivered in 34 (52%) patients. Mortality was 14%. At follow-up, 88% of patients showed complete eradication of the SV infection. Conclusions Upper gastrointestinal biopsy positive for SV should prompt an evaluation of the clinical conditions, being the risk of gastric perforation not negligible. Antibiotic therapy may eradicate the infection and prevent complications. Emergency surgery is required in case of source control.

We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel strategy to treat Ewing sarcoma. The most potent compound 12 is suitable for progression to in vivo studies. The specific attributes of 12 included nanomolar inhibitory potency against both phosphoinositide-dependent kinase-1 (PDK1) and Aurora A (AurA) kinase, with acceptable in vitro ADME-Tox properties (cytotoxicity in 2 healthy and 14 hematological and solid cancer cell-lines; inhibition of PDE4C1, SIRT7, HDAC4, HDAC6, HDAC8, HDAC9, AurB, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and hERG). X-ray crystallography and docking studies led to the identification of the key AurA and PDK1/12 interactions. Finally, in vitro drug-intake kinetics and in vivo PK appear to indicate that these compounds are attractive lead-structures for the design and synthesis of PDK1/AurA dual-target molecules to further investigate the in vivo efficacy against Ewing Sarcoma.

Leukocyte infiltration plays an active role in controlling tumor development. In the early stages of carcinogenesis, T cells counteract tumor growth. However, in advanced stages, cancer cells and infiltrating stromal components interfere with the immune control and instruct immune cells to support, rather than counteract, tumor malignancy, via cell-cell contact or soluble mediators. In particular, metabolites are emerging as active players in driving immunosuppression. Here we demonstrate that in a prostate cancer model lactate released by glycolytic cancer-associated fibroblasts (CAFs) acts on CD4+ T cells, shaping T-cell polarization. In particular, CAFs exposure (i) reduces the percentage of the antitumoral Th1 subset, inducing a lactate-dependent, SIRT1-mediated deacetylation/degradation of T-bet transcription factor; (ii) increases Treg cells, driving naive T cells polarization, through a lactate-based NF-kB activation and FoxP3 expression. In turn, this metabolic-based CAF-immunomodulated environment exerts a pro-invasive effect on prostate cancer cells, by activating a previously unexplored miR21/TLR8 axis that sustains cancer malignancy.

Even though pharmacological approaches able to counteract age-dependent cognitive impairment have been highly investigated, drugs improving cognition and memory are still an unmet need. It has been hypothesized that sustaining energy dynamics within the aged hippocampus can boost memory storage by sustaining synaptic functioning and long term potentiation (LTP). Dexpramipexole (DEX) is the first-in-class compound able to sustain neuronal bioenergetics by interacting with mitochondrial F1Fo-ATP synthase. In the present study, for the first time we evaluated the effects of DEX on synaptic fatigue, LTP induction, learning and memory retention. We report that DEX improved LTP maintenance in CA1 neurons of acute hippocampal slices from aged but not young rats. However, we found no evidence that DEX counteracted two classic parameters of synaptic fatigue such as fEPSP reduction or the train area during the high frequency stimulation adopted to induce LTP. Interestingly, patch-clamp recordings in rat hippocampal neurons revealed that DEX dose-dependently inhibited (IC50 814 nM) the IA current, a rapidly-inactivating K+ current that negatively regulates neuronal excitability as well as cognition and memory processes. In keeping with this, DEX counteracted both scopolamine-induced spatial memory loss in rats challenged in Morris Water Maze test and memory retention in rats undergoing Novel Object Recognition. Overall, the present study discloses the ability of DEX to boost hippocampal synaptic plasticity, learning and memory. In light of the good safety profile of DEX in humans, our findings may have a realistic translational potential to treatment of cognitive disorders.

Summary Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development., Graphical Abstract, Highlights • This is a novel, robust and scalable protocol to differentiate hPSCs into BAs • The protocol recapitulates the different stages of human BAT development • hPSC-derived BAs show transcriptomic and functional features of bona fide human BAs • It represents a useful tool for temporal analysis of human BAs differentiation, Obese patients possess low amounts of BAT, so an efficient way to expand its mass and activation is necessary to treat obesity and complications. There is limited knowledge about human BAT physiology and accessing it is challenging. To overcome this, Carobbio and colleagues developed a new protocol to differentiate human PSCs into functional brown adipocytes, recapitulating human BAT developmental path.

Summary Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig−/−) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig−/− mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion., Graphical abstract, Highlights • The absence of Adig impairs adipogenesis in vitro • High-fat diet (HFD)-induced weight gain is ameliorated in Adig−/− mice • Fat-mass-adjusted leptin levels are reduced in Adig−/− mice • Leptin secretion is reduced in Adig−/− adipose explants, Alvarez-Guaita et al. show that Adipogenin (Adig) deficiency impairs adipogenesis in cultured cells. High-fat diet (HFD)-induced weight gain is ameliorated in Adig−/− mice, and fat-mass-adjusted leptin levels are lower. Leptin secretion from Adig−/− adipose explants is also reduced, suggesting that Adig influences leptin secretion.

Epithelial-to-mesenchymal transition (EMT) is involved in prostate cancer (PCa) metastatic progression, and its plasticity suggests epigenetic implications. Deregulation of DNA methyltransferases (DNMTs) and several microRNAs (miRNAs) plays a relevant role in EMT, but their interplay has not been clarified yet. In this study, we provide evidence that DNMT3A interaction with several miRNAs has a central role in an ex vivo EMT PCa model obtained via exposure of PC3 cells to conditioned media from cancer-associated fibroblasts. The analysis of the alterations of the miRNA profile shows that miR-200 family (miR-200a/200b/429, miR-200c/141), miR-205 and miR-203, known to modulate key EMT factors, are down-regulated and hyper-methylated at their promoters. DNMT3A (mainly isoform a) is recruited onto these miRNA promoters, coupled with the increase of H3K27me3/H3K9me3 and/or the decrease of H3K4me3/H3K36me3. Most interestingly, our results reveal the differential expression of two DNMT3A isoforms (a and b) during ex vivo EMT and a regulatory feedback loop between miR-429 and DNMT3A that can promote and sustain the transition towards a more mesenchymal phenotype. We demonstrate the ability of miR-429 to target DNMT3A 3′UTR and modulate the expression of EMT factors, in particular ZEB1. Survey of the PRAD-TCGA dataset shows that patients expressing an EMT-like signature are indeed characterized by down-regulation of the same miRNAs with a diffused hyper-methylation at miR-200c/141 and miR-200a/200b/429 promoters. Finally, we show that miR-1260a also targets DNMT3A, although it does not seem to be involved in EMT in PCa.

Drugs able to efficiently counteract progression of multiple sclerosis (MS) are still an unmet need. Several lines of evidence indicate that histone deacetylase inhibitors (HDACi) are clinically-available epigenetic drugs that might be repurposed for immunosuppression in MS therapy. Here, we studied the effects of HDACi on disease evolution in myelin oligodendrocyte glycoprotein (MOG)-immunized NOD mice, an experimental model of progressive experimental autoimmune encephalomyelitis (PEAE). To obtain data of potential clinical relevance, the HDACi panobinostat, givinostat and entinostat were administered orally adopting a daily treatment protocol after disease onset. We report that the 3 drugs efficiently reduced in vitro lymphocyte proliferation in a dose-dependent manner. Notably, however, none of the drugs delayed evolution of PEAE or reduced lethality in NOD mice. In striking contrast with this, however, the lymphocyte proliferation response to MOG as well as Th1 and Th17 spinal cord infiltrates were significantly lower in animals exposed to the HDACi compared to those receiving vehicle. When put into a clinical context, for the first time data cast doubt on the relevance of HDACi to treatment of progressive MS (PMS). Also, our findings further indicate that, akin to PMS, neuropathogensis of PEAE in NOD mice becomes independent from autoimmunity, thereby corroborating the relevance of this model to experimental PMS research.

It is well acknowledged that tumors display extensive cellular and molecular heterogeneity. Despite of cell-intrinsic (genetic and epigenetic) changes, tumor cells evolve into high-grade malignancies, along with the tumor microenvironment representing a landscape of cellular and structural components. Malignant cells experience a dynamic and complex scenario of communications with nonmalignant stromal cells, recruited and/or corrupted in loco by tumor-derived soluble factors, cytokines, metabolites, and matrix-remodeling agents. Together with acidity and hypoxia, microenvironmental signals favor tumor progression and exacerbate malignant phenotype, from the tumor initiation to the metastatic spreading.

Summary Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers., Graphical Abstract, Highlights • ETR cells show high miR-23b-3p that reduces SLC6A14 and amino acids upload • ETR cells promote autophagy and aspartate and glutamate import via SLC1A2 • Aspartate and glutamate fuel anabolic and catabolic pathways in ETR breast cancers • Targeting amino acid metabolic reprogramming is effective in ETR cells, Bacci et al. find that endocrine-resistant ER+ breast cancers are characterized by enhanced miR-23b-3p, autophagy activation, and import of aspartate and glutamate that fuel catabolic and anabolic pathways, which are essential for their aggressive features. The molecular players involved in this metabolic scenario are of clinical significance and have prognostic and predictive value.

Increasing brown adipose tissue (BAT) mass and activation has been proposed as a potential therapeutic strategy to treat obesity and associated cardiometabolic complications. Given that obese and diabetic patients possess low amounts of BAT, an efficient way to expand their BAT mass would be necessary if BAT is to be useful. Currently, there is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Moreover, to have access to human BAT is challenging, given its low volume and being anatomically dispersed. These constrain makes detailed mechanistic studies related to BAT development and function in humans virtually impossible. To overcome these limitations, we have developed a human-relevant new protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs). Unique to this protocol is that it is chemically-defined to recapitulate a physiological step-by-step developmental path of BAT that captures transient paraxial mesoderm and BAT progenitor states, on its way to reaching the adipocyte stage finally. These hPSC-derived BAs express brown adipocyte and thermogenic markers, are insulin sensitive, and respond to β-adrenergic stimuli. This new protocol is a scalable tool to study human BAs during development.

While the consequences of poor maternal diet on the offspring’s cardio-metabolic health have been studied in detail, the role of the father’s diet on the health of his offspring is poorly understood. We used a known mouse model to establish the impact of an isocaloric paternal low-protein high-carbohydrate diet on the offspring’s lipid metabolism. Detailed lipid profiles were acquired from F1 neonate (3 weeks), F1 adult (16 weeks) and F2 neonate male and female offspring, in serum, liver, brain, heart and abdominal adipose tissues by Mass Spectrometry and Nuclear Magnetic Resonance. Using a purpose-built computational tool for analysing lipid metabolism as a network, we characterised the number, type and abundance of lipid variables in and between tissues (Lipid Traffic Analysis), finding a variety of alterations associated with paternal diet. These elucidate a mechanism for the defective physiological behaviour of systems at risk of cardio-metabolic disease.

Summary Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis., Graphical Abstract, Highlights • IL-33 signaling promotes development of monocyte-derived red pulp macrophages (RPMs) • Il33−/− and Il1rl1−/− mice have decreased RPMs and splenic erythrophagocytosis • ERK activation is required for RPM development and is potentiated by hemin and IL-33 • GATA2 instructs RPM development and is aberrant in Il1rl1−/− RPM precursors, Splenic red pulp macrophages (RPMs) are critical for iron recycling. Here, Lu et al. implicate a role for IL-33 signaling in the regulation of GATA2, which controls the transition of monocytes to produce pre-RPMs that are competent to terminally differentiate into mature RPMs.

Neoplastic tissues are composed not only by tumor cells but also by several non-transformed stromal cells, such as cancer-associated fibroblasts, endothelial and immune cells, that actively participate to tumor progression. Starting from the very beginning of carcinogenesis, tumor cells, through the release of paracrine soluble factors and vesicles, i.e., exosomes, modify the behavior of the neighboring cells, so that they can give efficient support for cancer cell proliferation and spreading. A mandatory role in tumor progression has been recently acknowledged to metabolic deregulation. Beside undergoing a metabolic reprogramming coherent to their high proliferation rate, tumor cells also rewire the metabolic assets of their stromal cells, educating them to serve as nutrient donors. Hence, an alteration in the composition and in the flow rate of many nutrients within tumor microenvironment has been associated with malignancy progression. This review is focused on metabolic remodeling of the different cell populations within tumor microenvironment, dealing with reciprocal re-education through the symbiotic sharing of metabolites, behaving both as nutrients and as transcriptional regulators, describing their impact on tumor growth and metastasis.

Mitochondria play multifaceted roles in malignant tumor progression. Beyond their bioenergetic role, mitochondria are essential for providing malignant cells a higher plasticity to face the harsh environmental conditions. Cell-autonomous metabolic deregulation of cancer cells, or metabolic adaptation to microenvironmental cues (lack of nutrients, stromal supply, hypoxia, etc.), represent the triggering event of mitochondria overexploitation to orchestrate nutrient sensing and upload, signaling, and redox circuits. As readout of their higher function, mitochondria produce high amounts of reactive oxygen species (ROS) that are functional for multiple signaling networks underlying tumor proliferation, survival, and metastatic process. To compensate for the higher rate of mitochondrial ROS production, cancer cells have evolved adaptive mechanisms to increase their antioxidant systems and to address ROS activating pathways useful for the tumor cell adaptation to environmental changes. As these properties are critical for cancer progression, mitochondrial ROS have recently become an attractive target for anti-cancer therapies. We discuss how understanding of mitochondrial function in the tumor-specific generation of ROS will impact on the development of novel redox-based targeted therapeutic strategies.

Cancer progression is strictly dependent on the relationship between tumor cells and the surrounding stroma, which supports cancer malignancy promoting several crucial steps of tumor progression, including the execution of the epithelial to mesenchymal transition (EMT) associated with enhancement in cell invasion, resistance to both anoikis and chemotherapeutic treatments. Recently it has been highlighted the central role of microRNAs (miRNAs) as regulators of tumor progression. Notably, in several tumors a strong deregulation of miRNAs is observed, supporting proliferation, invasion, and metabolic reprogramming of tumor cells. Here we demonstrated that cancer-associated fibroblasts induce a downregulation of miR-1247 in prostate cancer (PCa) cells. We proved that miR-1247 repression is functional for the achievement of EMT and increased cell invasion as well as stemness traits. These phenomena contribute to promote the metastatic potential of PCa cells as demonstrated by increased lung colonization in in vivo experiments. Moreover, as a consequence of miR-1247 downregulation, we observed a correlated increased expression level of neuropilin-1, a miR-1247 target involved as a coreceptor in the epidermal growth factor receptor signaling. Taken together, our data highlight miR-1247 as a potential target for molecular therapies aimed to block the progression and diffusion of PCa.

There is growing evidence to suggest that bone marrow-derived mesenchymal stem cells (BM-MSCs) are key players in tumour stroma. Here, we investigated the cross-talk between BM-MSCs and osteosarcoma (OS) cells. We revealed a strong tropism of BM-MSCs towards these tumour cells and identified monocyte chemoattractant protein (MCP)-1, growth-regulated oncogene (GRO)-α and transforming growth factor (TGF)-β1 as pivotal factors for BM-MSC chemotaxis. Once in contact with OS cells, BM-MSCs trans-differentiate into cancer-associated fibroblasts, further increasing MCP-1, GRO-α, interleukin (IL)-6 and IL-8 levels in the tumour microenvironment. These cytokines promote mesenchymal to amoeboid transition (MAT), driven by activation of the small GTPase RhoA, in OS cells, as illustrated by the in vitro assay and live imaging. The outcome is a significant increase of aggressiveness in OS cells in terms of motility, invasiveness and transendothelial migration. In keeping with their enhanced transendothelial migration abilities, OS cells stimulated by BM-MSCs also sustain migration, invasion and formation of the in vitro capillary network of endothelial cells. Thus, BM-MSC recruitment to the OS site and the consequent cytokine-induced MAT are crucial events in OS malignancy.

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [3H]NAD+ and [14C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5′-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5′-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1–mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G0/G1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells.

Background and purpose Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury. Experimental approach The effects of dexpramipexole on bioenergetics, Ca2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging. Key results Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro. Conclusion and implications Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke. Linked articles This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Mitochondrial encephalopathies are fatal, infantile neurodegenerative disorders caused by a deficit of mitochondrial functioning, for which there is urgent need to identify efficacious pharmacological treatments. Recent evidence shows that rapamycin administered both intraperitoneally or in the diet delays disease onset and enhances survival in the Ndufs4 null mouse model of mitochondrial encephalopathy. To delineate the clinical translatability of rapamycin in treatment of mitochondrial encephalopathy, we evaluated the drug's effects on disease evolution and mitochondrial parameters adopting treatment paradigms with fixed daily, oral doses starting at symptom onset in Ndufs4 knockout mice. Molecular mechanisms responsible for the pharmacodynamic effects of rapamycin were also evaluated. We found that rapamycin did not affect disease development at clinically-relevant doses (0.5 mg kg-1). Conversely, an oral dose previously adopted for intraperitoneal administration (8 mg kg-1) delayed development of neurological symptoms and increased median survival by 25%. Neurological improvement and lifespan were not further increased when the dose raised to 20 mg kg-1. Notably, rapamycin at 8 mg kg-1 did not affect the reduced expression of respiratory complex subunits, as well as mitochondrial number and mtDNA content. This treatment regimen however significantly ameliorated architecture of mitochondria cristae in motor cortex and cerebellum. However, reduction of mTOR activity by rapamycin was not consistently found within the brain of knockout mice. Overall, data show the ability of rapamycin to improve ultrastructure of dysfunctional mitochondria and corroborate its therapeutic potential in mitochondrial disorders. The non-clinical standard doses required, however, raise concerns about its rapid and safe clinical transferability.

Cancer-associated fibroblasts (CAFs) are the major cellular stromal component of many solid tumors. In prostate cancer (PCa), CAFs establish a metabolic symbiosis with PCa cells, contributing to cancer aggressiveness through lactate shuttle. In this study, we report that lactate uptake alters the NAD+/NADH ratio in the cancer cells, which culminates with SIRT1-dependent PGC-1α activation and subsequent enhancement of mitochondrial mass and activity. The high exploitation of mitochondria results in tricarboxylic acid cycle deregulation, accumulation of oncometabolites and in the altered expression of mitochondrial complexes, responsible for superoxide generation. Additionally, cancer cells hijack CAF-derived functional mitochondria through the formation of cellular bridges, a phenomenon that we observed in both in vitro and in vivo PCa models. Our work reveals a crucial function of tumor mitochondria as the energy sensors and transducers of CAF-dependent metabolic reprogramming and underscores the reliance of PCa cells on CAF catabolic activity and mitochondria trading.

Enteroendocrine cells (EECs) produce hormones such as glucagon-like peptide 1 and peptide YY that regulate food absorption, insulin secretion, and appetite. Based on the success of glucagon-like peptide 1–based therapies for type 2 diabetes and obesity, EECs are themselves the focus of drug discovery programs to enhance gut hormone secretion. The aim of this study was to identify the transcriptome and peptidome of human EECs and to provide a cross-species comparison between humans and mice. By RNA sequencing of human EECs purified by flow cytometry after cell fixation and staining, we present a first transcriptomic analysis of human EEC populations and demonstrate a strong correlation with murine counterparts. RNA sequencing was deep enough to enable identification of low-abundance transcripts such as G-protein–coupled receptors and ion channels, revealing expression in human EECs of G-protein–coupled receptors previously found to play roles in postprandial nutrient detection. With liquid chromatography–tandem mass spectrometry, we profiled the gradients of peptide hormones along the human and mouse gut, including their sequences and posttranslational modifications. The transcriptomic and peptidomic profiles of human and mouse EECs and cross-species comparison will be valuable tools for drug discovery programs and for understanding human metabolism and the endocrine impacts of bariatric surgery.

Background:The bisphosphonate Zoledronic acid (ZA) is a potent osteoclast inhibitor currently used in the clinic to reduce osteoporosis and cancer-induced osteolysis. Moreover, ZA exerts an anti-tumor effect in several tumors. Despite this evidence, the relevance of ZA in prostate cancer (PCa) is not completely understood.Objective:To investigate the effect of ZA administration on the invasive properties of PC3 cells, which are characterised by RhoA-dependent amoeboid motility.Methods:The effect of ZA administration on the in vitro invasive properties of PC3 cells was evaluated by cell migration in 3D collagen matrices, immunofluorescence and Boyden assays or transendothelial migration. Lung retention and colonization assays were performed to assess the efficacy of ZA administration in vivo.Results:PC3 cells are characterised by RhoA-dependent amoeboid motility. We now report a clear inhibition of in vitro PC3 cell invasion and RhoA activity upon ZA treatment. Moreover, to confirm a specific role of ZA in the inhibition of amoeboid motility of PC3 cells, we demonstrate that ZA interferes only partially with PC3 cells showing a mesenchymal phenotype due to both treatment with conditioned medium of cancer associated fibroblasts or to the acquisition of chemoresistance. Furthermore, we demonstrate that ZA impairs adhesion to endothelial cells and the trans-endothelial cell migration, two essential properties characterising amoeboid motility and PC3 metastatic dissemination. In vivo experiments prove the ability of ZA to inhibit the metastatic process of PC3 cells as shown by the decrease in lung colonization.Conclusion:This study demonstrates that ZA inhibits Rho-dependent amoeboid motility of PC3 cells, thus suggesting ZA as a potential therapy to impede the metastatic dissemination of PC3 cells.

Human melanomas contain a population of tumor-initiating cells that are able to maintain the growth of the tumor. We previously showed that the embryonic transcription factor SOX2 is essential for self-renewal and tumorigenicity of human melanoma-initiating cells. However, targeting a transcription factor is still challenging. Gentian violet (GV) is a cationic triphenylmethane dye with potent antifungal and antibacterial activity. Recently, a combination therapy of imiquimod and GV has shown an inhibitory effect against melanoma metastases. Whether and how GV affects melanoma cells remains unknown. Here we show that GV represses melanoma stem cell self-renewal through inhibition of SOX2. Mechanistically, GV hinders EGFR activation and inhibits the signal transducer and activator of transcription-3 [(STAT3)/SOX2] axis. Importantly, we show that GV treatment decreases STAT3 phosphorylation at residue tyrosine 705, thus preventing the translocation of STAT3 into the nucleus and its binding to SOX2 promoter. In addition, GV affects melanoma cell growth by promoting mitochondrial apoptosis and G2 cell cycle arrest. This study shows that in melanoma, GV affects both the stem cell and the tumor bulk compartments, suggesting the potential use of GV in treating human melanoma alone or in combination with targeted therapy and/or immunotherapy.

Tumor stromal cells can supply appropriate signals that may develop aggressive phenotypes of carcinoma cells and establish a complex scenario which culminates in metastasis. Recent works proposed that bone marrow-derived mesenchymal stem cells (MSC) are recruited to primary tumors. However, the exact functions of these cells in the tumor microenvironment are not well characterized, as it is reported that MSC can either promote or inhibit tumor progression. In the present study, we aim at investigating the signaling molecules which regulate the interplay between MSC, prostate carcinoma (PCa) cells and two important cellular types constituting the tumor-associated stroma, macrophages and fibroblasts, during their progression toward malignancy. We identified TGF-β1 as a crucial molecule able to attract MSC recruitment both to PCa cells as well as to tumor stroma components. Moreover, PCa- and tumor stroma-secreted TGF-β1 is important to induce MSC transdifferentiation into carcinoma-associated fibroblast (CAF)-like cells. Consequently, the CAF-like phenotype acquired by MSC is central to promote tumor progression related effects. Thus, tumor-educated MSC enhance PCa invasiveness compared to nonactivated MSC. Additionally, differing from normal MSC, CAF-like MSC perform vascular mimicry and recruit monocytes, which can be further polarized to M2 macrophages within the PCa environment. Our findings indicate a prominent role for TGF-β1 in MSC mobilization and activation strengthened by the fact that the blockade of TGF-β1 signaling impairs MSC promotion of PCa progression.

Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength in E. coli. Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases.

The presence of lactate in human tumours has been long neglected, confined to the role of a waste product derived from glycolysis and as a biomarker of malignancy. More recently, lactate has been rediscovered as signalling molecule that plays important roles in the regulation of the metabolic pathways, the immune response, and cell-to-cell communication within the tumour microenvironment. This review examines recent discoveries about the functional role of lactate in shaping the behaviour and the phenotype of tumour and tumour-associated cells, and describes potential clinical approaches to target lactate transport and metabolism in tumours.

Background A wide variety of hemostats are available as adjunctive measures to improve hemostasis during surgical procedures if residual bleeding persists despite correct application of conventional methods for hemorrhage control. Some are considered active agents, since they contain fibrinogen and thrombin and actively participate at the end of the coagulation cascade to form a fibrin clot, whereas others to be effective require an intact coagulation system. The aim of this study is to provide an evidence-based approach to correctly select the available agents to help physicians to use the most appropriate hemostat according to the clinical setting, surgical problem and patient’s coagulation status. Methods The literature from 2000 to 2016 was systematically screened according to PRISMA [Preferred Reporting Items for Systematic Reviews and Meta-Analyses] protocol. Sixty-six articles were reviewed by a panel of experts to assign grade of recommendation (GoR) and level of evidence (LoE) using the GRADE [Grading of Recommendations Assessment, Development and Evaluation] system, and a national meeting was held. Results Fibrin adhesives, in liquid form (fibrin glues) or with stiff collagen fleece (fibrin patch) are effective in the presence of spontaneous or drug-induced coagulation disorders. Mechanical hemostats should be preferred in patients who have an intact coagulation system. Sealants are effective, irrespective of patient’s coagulation status, to improve control of residual oozing. Hemostatic dressings represent a valuable option in case of external hemorrhage at junctional sites or when tourniquets are impractical or ineffective. Conclusions Local hemostatic agents are dissimilar products with different indications. A knowledge of the properties of each single agent should be in the armamentarium of acute care surgeons in order to select the appropriate product in different clinical conditions.

// Elisa Giannoni 1 , Maria Letizia Taddei 1 , Andrea Morandi 1 , Giuseppina Comito 1 , Maura Calvani 1 , Francesca Bianchini 1 , Barbara Richichi 2 , Giovanni Raugei 1 , Nicholas Wong 3 , Damu Tang 3 , Paola Chiarugi 1 1 Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134, Florence, Italy 2 Department of Chemistry, University of Florence, 50019, Sesto Fiorentino, Italy 3 Division of Nephrology, Department of Medicine, McMaster University, L8N4A6, Hamilton, Ontario, Canada Correspondence to: Paola Chiarugi, e-mail: paola.chiarugi@unifi.it Keywords: pyruvate kinase M2, cancer associated fibroblasts, epithelial-mesenchymal transition, hypoxia inducible factor-1α, prostate cancer Received: March 05, 2015 Accepted: June 17, 2015 Published: June 27, 2015 ABSTRACT Cancer associated fibroblasts (CAFs) are key determinants of cancer progression. In prostate carcinoma (PCa), CAFs induce epithelial-mesenchymal transition (EMT) and metabolic reprogramming of PCa cells towards oxidative phosphorylation (OXPHOS), promoting tumor growth and metastatic dissemination. We herein establish a novel role for pyruvate kinase M2 (PKM2), an established effector of Warburg-like glycolytic behavior, in OXPHOS metabolism induced by CAFs. Indeed, CAFs promote PKM2 post-translational modifications, such as cysteine oxidation and Src-dependent tyrosine phosphorylation, allowing nuclear migration of PKM2 and the formation of a trimeric complex with hypoxia inducible factor-1α (HIF-1α) and the transcriptional repressor Differentially Expressed in Chondrocytes-1 (DEC1). DEC1 recruitment is mandatory for downregulating miR205 expression, thereby fostering EMT execution and metabolic switch toward OXPHOS. Furthermore, the analysis of a cohort of PCa patients reveals a significant positive correlation between PKM2 nuclear localization and cancer aggressiveness, thereby validating our in vitro observations. Crucially, in vitro and in vivo pharmacological targeting of PKM2 nuclear translocation using DASA-58, as well as metformin, impairs metastatic dissemination of PCa cells in SCID mice. Our study indicates that impairing the metabolic tumor:stroma interplay by targeting the PKM2/OXPHOS axis, may be a valuable novel therapeutic approach in aggressive prostate carcinoma.

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions.

Transcriptional deregulation emerges as a key pathogenetic mechanism in ALS pathogenesis, and non-class-specific histone deacetylase (HDACs) inhibitors proved of therapeutic efficacy in preclinical models of ALS. When tested in patients, however, these drugs failed, probably because of a lack of selectivity toward pathogenetic HDACs. Here, we studied the effects of MC1568, an inhibitor of Class-II HDACs which have been reported to contribute to ALS pathogenesis. We focused on transcriptional regulation of glutamate transporter EAAT2, whose reduced expression may contribute to motor neuron degeneration in ALS. We report that MC1568 highly increased EAAT2 transcripts in primary cultures of mouse glia, but these increases did not correlate with increased glutamate uptake capacity. Accordingly, we found that MC1568 augmented protein expression of EAAT2 together with its sumoylation, a post-translational modification typically altering protein function and localization. When tested in SOD1G93A mice, however, MC1568 fully restored the reduced spinal cord expression of EAAT2 and glutamate uptake up to control levels. A prolonged treatment with MC1568 (from onset to end stage) was unable to prolong survival of mice. Data reveal a key role of Class-II HDACs in expression and function of glutamate transporter, further corroborating preclinical and clinical evidence that the sole restoration of glutamate uptake is not of therapeutic relevance to ALS therapy.