Landscape of Druggable Molecular Pathways Downstream of Genomic CDH1/Cadherin-1 Alterations in Gastric Cancer.

Loss of CDH1/Cadherin-1 is a common step towards the acquisition of an abnormal epithelial phenotype. In gastric cancer (GC), mutation and/or downregulation of CDH1/Cadherin-1 is recurrent in sporadic and hereditary diffuse GC type. To approach the molecular events downstream of CDH1/Cadherin-1 alterations and their relevance in gastric carcinogenesis, we queried public databases for genetic and DNA methylation data in search of molecular signatures with a still-uncertain role in the pathological mechanism of GC. In all GC subtypes, modulated genes correlating with CDH1/Cadherin-1 aberrations are associated with stem cell and epithelial-to-mesenchymal transition pathways. A higher level of genes upregulated in CDH1-mutated GC cases is associated with reduced overall survival. In the diffuse GC (DGC) subtype, genes downregulated in CDH1-mutated compared to cases with wild type CDH1/Cadherin-1 resulted in being strongly intertwined with the DREAM complex. The inverse correlation between hypermethylated CpGs and CDH1/Cadherin-1 transcription in diverse subtypes implies a common epigenetic program. We identified nonredundant protein-encoding isoforms of 22 genes among those differentially expressed in GC compared to normal stomach. These unique proteins represent potential agents involved in cell transformation and candidate therapeutic targets. Meanwhile, drug-induced and CDH1/Cadherin-1 mutation-related gene expression comparison predicts FIT, GR-127935 hydrochloride, amiodarone hydrochloride in GC and BRD-K55722623, BRD-K13169950, and AY 9944 in DGC as the most effective treatments, providing cues for the design of combined pharmacological treatments. By integrating genetic and epigenetic aspects with their expected functional outcome, we unveiled promising targets for combinatorial pharmacological treatments of GC.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

Nitric Oxide in Selective Cerebral Perfusion Could Enhance Neuroprotection During Aortic Arch Surgery.

BACKGROUND: Hypothermic circulatory arrest (HCA) in aortic arch surgery has a significant risk of neurological injury despite the newest protective techniques and strategies. Nitric oxide (NO) could exert a protective role, reduce infarct area and increase cerebral perfusion. This study aims to investigate the possible neuroprotective effects of NO administered in the oxygenator of selective antegrade cerebral perfusion (SCP) during HCA. METHODS: Thirty male SD adult rats (450-550 g) underwent cardiopulmonary bypass (CPB), cooling to 22°C body core temperature followed by 30 min of HCA. Rats were randomized to receive SCP or SCP added with NO (20 ppm) administered through the oxygenator (SCP-NO). All animals underwent CPB-assisted rewarming to a target temperature of 35°C in 60 min. At the end of the experiment, rats were sacrificed, and brain collected. Immunofluorescence analysis was performed in blind conditions. RESULTS: Neuroinflammation assessed by allograft inflammatory factor 1 or ionized calcium-binding adapter molecule 1 expression, a microglia activation marker was lower in SCP-NO compared to SCP (4.11 ± 0.59 vs. 6.02 ± 0.18%; p < 0.05). Oxidative stress measured by 8oxodG, was reduced in SCP-NO (0.37 ± 0.01 vs. 1.03 ± 0.16%; p < 0.05). Brain hypoxic area extent, analyzed by thiols oxidation was attenuated in SCP-NO (1.85 ± 0.10 vs. 2.74 ± 0.19%; p < 0.05). Furthermore, the apoptotic marker caspases 3 was significantly reduced in SCP-NO (10.64 ± 0.37 vs. 12.61 ± 0.88%; p < 0.05). CONCLUSIONS: Nitric oxide administration in the oxygenator during SCP and HCA improves neuroprotection by decreasing neuroinflammation, optimizing oxygen delivery by reducing oxidative stress and hypoxic areas, finally decreasing apoptosis.

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.

OBJECTIVES: Among the factors that could determine neurological outcome after hypothermic circulatory arrest (HCA) rewarming is rarely considered. The optimal rewarming rate is still unknown. The goal of this study was to investigate the effects of 2 different protocols for rewarming after HCA on neurological outcome in an experimental animal model. METHODS: Forty-four Sprague Dawley rats were cooled to 19 ± 1°C body core temperature by cardiopulmonary bypass (CPB). HCA was maintained for 60 min. Animals were randomized to receive slow (90 min) or fast (45 min) assisted rewarming with CPB to a target temperature of 35°C. After a total of 90 min of reperfusion in both groups, brain samples were collected and analysed immunohistochemically and with immunofluorescence. In 10 rats, magnetic resonance imaging was performed after 2 and after 24 h to investigate cerebral perfusion and cerebral oedema. RESULTS: Interleukin 6, chemokine (C-C motif) ligand 5, intercellular adhesion molecule 1 and tumour necrosis factor α in the hippocampus are significantly less expressed in the slow rewarming group, and microglia cells are significantly less activated in the slow rewarming group. Magnetic resonance imaging analysis demonstrated better cerebral perfusion and less water content in brains that underwent slow rewarming at 2 and 24 h. CONCLUSIONS: Slow rewarming after HCA might be superior to fast rewarming in neurological outcome. The present experimental study demonstrated reduction in the inflammatory response, reduction of inflammatory cell activation in the brain, enhancement of cerebral blood flow and reduction of cerebral oedema when slow rewarming was applied.

Methylation Dynamics of RASSF1A and Its Impact on Cancer.

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types.

High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy.

Oligodendrocyte loss can lead to cognitive and motor deficits. Current remyelinating therapeutic strategies imply either modulation of endogenous oligodendrocyte precursors or transplantation of in vitro expanded oligodendrocytes. Cell therapy, however, still lacks identification of an adequate source of oligodendrocyte present in adulthood and able to efficiently produce transplantable cells. Recently, a neural stem cell-like population has been identified in meninges. We developed a protocol to obtain high yield of oligodendrocyte lineage cells from one single biopsy of adult rat meningeal tissue. From 1 cm2 of adult rat spinal cord meninges, we efficiently expanded a homogenous culture of 10 millions of meningeal-derived oligodendrocyte lineage cells in a short period of time (approximately 4 weeks). Meningeal-derived oligodendrocyte lineage cells show typical mature oligodendrocyte morphology and express specific oligodendrocyte markers, such as galactosylceramidase and myelin basic protein. Moreover, when transplanted in a chemically demyelinated spinal cord model, meningeal-derived oligodendrocyte lineage cells display in vivo-remyelinating potential. This oligodendrocyte lineage cell population derives from an accessible and adult source, being therefore a promising candidate for autologous cell therapy of demyelinating diseases. In addition, the described method to differentiate meningeal-derived neural stem cells into oligodendrocyte lineage cells may represent a valid in vitro model to dissect oligodendrocyte differentiation and to screen for drugs capable to promote oligodendrocyte regeneration.

New neurons in adult brain: distribution, molecular mechanisms and therapies.

"Are new neurons added in the adult mammalian brain?" "Do neural stem cells activate following CNS diseases?" "How can we modulate their activation to promote recovery?" Recent findings in the field provide novel insights for addressing these questions from a new perspective. In this review, we will summarize the current knowledge about adult neurogenesis and neural stem cell niches in healthy and pathological conditions. We will first overview the milestones that have led to the discovery of the classical ventricular and hippocampal neural stem cell niches. In adult brain, new neurons originate from proliferating neural precursors located in the subventricular zone of the lateral ventricles and in the subgranular zone of the hippocampus. However, recent findings suggest that new neuronal cells can be added to the adult brain by direct differentiation (e.g., without cell proliferation) from either quiescent neural precursors or non-neuronal cells undergoing conversion or reprogramming to neuronal fate. Accordingly, in this review we will also address critical aspects of the newly described mechanisms of quiescence and direct conversion as well as the more canonical activation of the neurogenic niches and neuroblast reservoirs in pathological conditions. Finally, we will outline the critical elements involved in neural progenitor proliferation, neuroblast migration and differentiation and discuss their potential as targets for the development of novel therapeutic drugs for neurodegenerative diseases.

Characterization and Expression of Sphingosine 1-Phosphate Receptors in Human and Rat Heart.

Aim: Sphingosine 1-phosphate (S1P), sphingolipid derivatives are known anti-inflammatory, anti-apoptotic, and anti-oxidant agent. S1P have been demonstrated to have a role in the cardiovascular system. The purpose of this study was to understand the precise expression and distribution of S1P receptors (S1PRs) in human and rat cardiovascular tissues to know the significance and possible implementation of our experimental studies in rat models. Methods and Results: In this study, we investigated the localization of S1PRs in human heart samples from cardiac surgery department, University of Verona Hospital and rat samples. Immunohistochemical investigation of paraffin-embedded sections illustrated diffused staining of the myocardial samples from human and rat. The signals of the human heart were similar to those of the rat heart in all chambers of the heart. The immunohistochemical expression levels correlated well with the results of RT-PCR-based analysis and western blotting. We confirmed by all techniques that S1PR1 expressed strongly as compared to S1PR3, and are uniformly distributed in all chambers of the heart with no significant difference in human and rat myocardial tissue. S1PR2 expression was significantly weak while S1PR4 and S1PR5 were not detectable in RT-PCR results in both human and rat heart. Conclusion: These results indicate that experimental studies using S1PR agonists on rat models are more likely to have a potential for translation into clinical studies, and second important information revealed by this study is, S1P receptor agonist can be used for cardioprotection in global ischemia-reperfusion injury.

Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy.

Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis.

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

Meninges harbor cells expressing neural precursor markers during development and adulthood.

Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Comparative study of immune regulatory properties of stem cells derived from different tissues.

Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ectomesenchymal SCs, leptomeningeal SCs, and three different c-Kit-positive SC types, that is, amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti-apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs.

Assembly and trafficking of human small conductance Ca2+-activated K+ channel SK3 are governed by different molecular domains.

Intracellular trafficking is an important event in the control of type and number of ion channels expressed on the cell surface. In this study, we have identified molecular domains involved in assembly and trafficking of the human small conductance Ca2+-activated K+ channel SK3. Deletion of the N-terminus, the C-terminus, or the calmodulin-binding domain (CaMBD) led to retention of SK3 channels in the endoplasmic reticulum. Presence of the CaMBD allowed trafficking to the Golgi complex, and sequences downstream were required for efficient transport to the plasma membrane, suggesting several steps in the control of SK3 forward trafficking. Co-immunoprecipitation studies demonstrated that SK3 subunits lacking the N-terminus, the CaMBD, or the distal C-terminus, but not the entire C-terminus, were able to oligomerize with wild-type SK3 subunits. Thus, these two C-terminal regions of SK3 seem to contribute to assembly and trafficking of channels whereas the N-terminus is necessary for trafficking but not sufficient for oligomerization.