SIRT6 promotes metastasis and relapse in HER2-positive breast cancer.

The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

Single-cell proteo-genomic reveals a comprehensive map of centrosome-associated spliceosome components.

Ribonucleoprotein (RNP) condensates are crucial for controlling RNA metabolism and splicing events in animal cells. We used spatial proteomics and transcriptomic to elucidate RNP interaction networks at the centrosome, the main microtubule-organizing center in animal cells. We found a number of cell-type specific centrosome-associated spliceosome interactions localized in subcellular structures involved in nuclear division and ciliogenesis. A component of the nuclear spliceosome BUD31 was validated as an interactor of the centriolar satellite protein OFD1. Analysis of normal and disease cohorts identified the cholangiocarcinoma as target of centrosome-associated spliceosome alterations. Multiplexed single-cell fluorescent microscopy for the centriole linker CEP250 and spliceosome components including BCAS2, BUD31, SRSF2 and DHX35 recapitulated bioinformatic predictions on the centrosome-associated spliceosome components tissue-type specific composition. Collectively, centrosomes and cilia act as anchor for cell-type specific spliceosome components, and provide a helpful reference for explore cytoplasmic condensates functions in defining cell identity and in the origin of rare diseases.

Repurposing pentamidine using hyaluronic acid-based nanocarriers for skeletal muscle treatment in myotonic dystrophy.

In a context of drug repurposing, pentamidine (PTM), an FDA-approved antiparasitic drug, has been proposed to reverse the splicing defects associated in myotonic dystrophy type 1 (DM1). However, clinical use of PTM is hinder by substantial toxicity, leading to find alternative delivery strategies. In this work we proposed hyaluronic acid-based nanoparticles as a novel encapsulation strategy to efficiently deliver PTM to skeletal muscles cells. In vitro studies on C2C12 myoblasts and myotubes showed an efficient nanoparticles' internalization with minimal toxicity. More interestingly, our findings evidenced for the first time the endosomal escape of hyaluronic acid-based nanocarriers. Ex vivo studies showed an efficient nanoparticles' internalization within skeletal muscle fibers. Finally, the therapeutic efficacy of PTM-loaded nanosystems to reduce the number of nuclear foci has been demonstrated in a novel DM1 in vitro model. So far, current data demonstrated the potency of hyaluronic acid-based nanosystems as efficient nanocarrier for delivering PTM into skeletal muscle and mitigate DM1 pathology.

Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study.

Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.

The transcriptional profile of adipose-derived stromal cells (ASC) mirrors the whitening of adipose tissue with age.

Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

Low Ozone Concentrations Differentially Affect the Structural and Functional Features of Non-Activated and Activated Fibroblasts In Vitro.

Oxygen-ozone (O2-O3) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O3 concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O2-O3 gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O3 stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-ß1 secretion, while in LPS-activated fibroblasts, O3 stimulated only antioxidant response and cytokines secretion. Therefore, the low O3 concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated.

Interferon regulatory factor 7 impairs cellular metabolism in aging adipose-derived stromal cells.

Dysregulated immunity and widespread metabolic dysfunctions are the most relevant hallmarks of the passing of time over the course of adult life, and their combination at midlife is strongly related to increased vulnerability to diseases; however, the causal connection between them remains largely unclear. By combining multi-omics and functional analyses of adipose-derived stromal cells established from young (1 month) and midlife (12 months) mice, we show that an increase in expression of interferon regulatory factor 7 (IRF7) during adult life drives major metabolic changes, which include impaired mitochondrial function, altered amino acid biogenesis and reduced expression of genes involved in branched-chain amino acid (BCAA) degradation. Our results draw a new paradigm of aging as the 'sterile' activation of a cell-autonomous pathway of self-defense and identify a crucial mediator of this pathway, IRF7, as driver of metabolic dysfunction with age.

Ozone Activates the Nrf2 Pathway and Improves Preservation of Explanted Adipose Tissue In Vitro.

In clinical practice, administration of low ozone (O3) dosages is a complementary therapy for many diseases, due to the capability of O3 to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O3 concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O3 treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O3 treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O3 concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O3 as an adjuvant treatment to improve fat engraftment.

ACSL3-PAI-1 signaling axis mediates tumor-stroma cross-talk promoting pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked fibrosis and low immunogenicity, features that are linked to treatment resistance and poor clinical outcomes. Therefore, understanding how PDAC regulates the desmoplastic and immune stromal components is of great clinical importance. We found that acyl-CoA synthetase long-chain 3 (ACSL3) is up-regulated in PDAC and correlates with increased fibrosis. Our in vivo results show that Acsl3 knockout hinders PDAC progression, markedly reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells, and increases cytotoxic T cell infiltration. This effect is, at least in part, due to decreased plasminogen activator inhibitor-1 (PAI-1) secretion from tumor cells. Accordingly, PAI-1 expression in PDAC positively correlates with markers of fibrosis and immunosuppression and predicts poor patient survival. We found that PAI-1 pharmacological inhibition strongly enhances chemo- and immunotherapeutic response against PDAC, increasing survival of mice. Thus, our results unveil ACSL3-PAI-1 signaling as a requirement for PDAC progression with druggable attributes.

The ACSL3-LPIAT1 signaling drives prostaglandin synthesis in non-small cell lung cancer.

Enhanced prostaglandin production promotes the development and progression of cancer. Prostaglandins are generated from arachidonic acid (AA) by the action of cyclooxygenase (COX) isoenzymes. However, how cancer cells are able to maintain an elevated supply of AA for prostaglandin production remains unclear. Here, by using lung cancer cell lines and clinically relevant KrasG12D-driven mouse models, we show that the long-chain acyl-CoA synthetase (ACSL3) channels AA into phosphatidylinositols to provide the lysophosphatidylinositol-acyltransferase 1 (LPIAT1) with a pool of AA to sustain high prostaglandin synthesis. LPIAT1 knockdown suppresses proliferation and anchorage-independent growth of lung cancer cell lines, and hinders in vivo tumorigenesis. In primary human lung tumors, the expression of LPIAT1 is elevated compared with healthy tissue, and predicts poor patient survival. This study uncovers the ACSL3-LPIAT1 axis as a requirement for the sustained prostaglandin synthesis in lung cancer with potential therapeutic value.

RAS as Supporting Actor in Breast Cancer.

Oncogenic activation of RAS isoforms leads tumor initiation and progression in many types of cancers and is gaining increasing interest as target for novel therapeutic strategies. In sharp contrast with other types of cancer, the importance of RAS in breast tumorigenesis has long been undermined by the low frequency of its oncogenic mutation in human breast lesions. Nevertheless, a wealth of studies over the last years have revealed how the engagement of RAS function might be mandatory downstream varied oncogenic alterations for the progression, metastatic dissemination, and therapy resistance in breast cancers. We review herein the major studies over the last three decades which have explored the controversial role of RAS proteins and their mutation status in breast tumorigenesis and have contributed to reveal their role as supporting actors, instead of as primary cause, in breast cancer.

The Role of Nrf2 in the Antioxidant Cellular Response to Medical Ozone Exposure.

Ozone (O3) is a natural, highly unstable atmospheric gas that rapidly decomposes to oxygen. Although not being a radical molecule, O3 is a very strong oxidant and therefore it is potentially toxic for living organisms. However, scientific evidence proved that the effects of O3 exposure are dose-dependent: high dosages stimulate severe oxidative stress resulting in inflammatory response and tissue injury, whereas low O3 concentrations induce a moderate oxidative eustress activating antioxidant pathways. These properties make O3 a powerful medical tool, which can be used as either a disinfectant or an adjuvant agent in the therapy of numerous diseases. In this paper, the cellular mechanisms involved in the antioxidant response to O3 exposure will be reviewed with special reference to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its role in the efficacy of ozone therapy.

Light Entrains Diurnal Changes in Insulin Sensitivity of Skeletal Muscle via Ventromedial Hypothalamic Neurons.

Loss of synchrony between geophysical time and insulin action predisposes to metabolic diseases. Yet the brain and peripheral pathways linking proper insulin effect to diurnal changes in light-dark and feeding-fasting inputs are poorly understood. Here, we show that the insulin sensitivity of several metabolically relevant tissues fluctuates during the 24 h period. For example, in mice, the insulin sensitivity of skeletal muscle, liver, and adipose tissue is lowest during the light period. Mechanistically, by performing loss- and gain-of-light-action and food-restriction experiments, we demonstrate that SIRT1 in steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamic nucleus (VMH) convey photic inputs to entrain the biochemical and metabolic action of insulin in skeletal muscle. These findings uncover a critical light-SF1-neuron-skeletal-muscle axis that acts to finely tune diurnal changes in insulin sensitivity and reveal a light regulatory mechanism of skeletal muscle function.

Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study.

The aim of this post-mortem ultrastructural and immunohistochemical study is to explore the characteristics of acute myocardial ischemia in the context of sudden death, using the combination of two different methods, both more insightful than ordinary histology. Transmission electron microscope and immunohistochemistry, in addition to the traditional histology, were applied to study human heart specimens collected during forensic autopsies. The whole series was sub-grouped into cases (n=17) and controls (n=10). The control group consisted of unnatural death with a short agonal period (immediately lethal injuries). Heart samples of the two cohorts of subjects were prepared for electron microscopy. On the other hand, each specimen, formalin fixed and paraffin embedded, was stained with haematoxylin and eosin and immunoreacted with the following primary antibodies: antiFibronectin, antiConnexin-43, anti npCx43 (dephosphorylated form of Connexin43), antiZonula occludens-1. Immunopositivity of each marker in the myocardium was semi-quantitatively graded. Electron microscopy revealed a number of interesting differences between acute myocardial ischemia and controls, regarding the morphology of nucleus, mitochondria and intercellular junctions. By immunohistochemistry, fibronectin was found to be markedly increased in the extracellular matrix of the acute myocardial ischemia cases, with a remarkable difference in respect of controls. Connexin 43 staining disclosed a slightly increase in the cytoplasm of acute myocardial ischemia cases with respect to the controls, whereas no relevant differences were seen between cases and controls at intercellular junctions. Dephosphorylated form of Cx43 showed an evident difference of staining in cases compared to controls and overall this difference more evident in the cytoplasm. Zonula occludens 1, described as an important marker for functional modification of cardiac muscle fibers, resulted negative or very weak in the vast majority of both cases and controls. The present study attempts to simultaneously apply electron microscopy and immunohistochemistry, in order to figure out the morphological changes that might lead to pathological processes underlying the sudden, unexpected death due to acute myocardial ischemia, and consequently to find useful diagnostic markers of very early ischemic injury. Both methods showed significant differences between acute myocardial ischemia and controls, regarding, overall nuclei, mitochondria, and intercellular junctions.Â.

Mild ozonisation activates antioxidant cell response by the Keap1/Nrf2 dependent pathway.

Treatment with low-dose ozone is successfully exploited as an adjuvant therapy in the treatment of several disorders. Although the list of medical applications of ozone therapy is increasing, molecular mechanisms underlying its beneficial effects are still partially known. Clinical and experimental evidence suggests that the therapeutic effects of ozone treatment may rely on its capability to mount a beneficial antioxidant response through activation of the nuclear factor erythroid-derived-like 2 (Nrf2) pathway. However, a conclusive mechanistic demonstration is still lacking. Here, we bridge this gap of knowledge by providing evidence that treatment with a low concentration of ozone in cultured cells promotes nuclear translocation of Nrf2 at the chromatin sites of active transcription and increases the expression of antioxidant response element (ARE)-driven genes. Importantly, we show that ozone-induced ARE activation can be reverted by the ectopic expression of the Nrf2 specific inhibitor Kelch-like ECH associated protein (Keap1), thus proving the role of the Nrf2 pathway in the antioxidant response induced by mild ozonisation.

Theranostic Role of 32P-ATP as Radiopharmaceutical for the Induction of Massive Cell Death within Avascular Tumor Core.

Drug inaccessibility to vast areas of the tumor parenchyma is amongst the major hurdles for conventional therapies. Treatment efficacy rapidly decreases with distance from vessels and most of the tumor cells survive therapy. Also, between subsequent cycles of treatment, spared cancer cells replace those killed near the vessels, improving their access to nutrients, boosting their proliferation rate, and thus enabling tumor repopulation. Because of their property of "acting at a distance," radioisotopes are believed to overcome the physical barrier of vascular inaccessibility. Methods A novel molecular imaging tool called Cerenkov Luminescence Imaging (CLI) was employed for the detection of Cerenkov radiation emitted by beta particles, allowing in vivo tracking of beta-emitters. More precisely we investigated using a xenograft model of colon carcinoma the potential use of 32P-ATP as a novel theranostic radiopharmaceutical for tracing tumor lesions while simultaneously hampering their growth. Results Our analyses demonstrated that 32P-ATP injected into tumor-bearing mice reaches tumor lesions and persists for days and weeks within the tumor parenchyma. Also, the high-penetrating beta particles of 32P-ATP exert a "cross-fire" effect that induces massive cell death throughout the entire tumor parenchyma including core regions. Conclusion Our findings suggest 32P-ATP treatment as a potential approach to complement conventional therapies that fail to reach the tumor core and to prevent tumor repopulation.

SIRT6 Suppresses Cancer Stem-like Capacity in Tumors with PI3K Activation Independently of Its Deacetylase Activity.

Cancer stem cells (CSCs) have high tumorigenic capacity. Here, we show that stem-like traits of specific human cancer cells are reduced by overexpression of the histone deacetylase sirtuin 6 (SIRT6). SIRT6-sensitive cancer cells bear mutations that activate phosphatidylinositol-3-kinase (PI3K) signaling, and overexpression of SIRT6 reduces growth, progression, and grade of breast cancer in a mouse model with PI3K activation. Tumor metabolomic and transcriptomic analyses reveal that SIRT6 overexpression dampens PI3K signaling and stem-like characteristics and causes metabolic rearrangements in this cancer model. Ablation of a PI3K activating mutation in otherwise isogenic cancer cells is sufficient to convert SIRT6-sensitive into SIRT6-insensitive cells. SIRT6 overexpression suppresses PI3K signaling at the transcriptional level and antagonizes tumor sphere formation independent of its histone deacetylase activity. Our data identify SIRT6 as a putative molecular target that hinders stemness of tumors with PI3K activation.

Enhanced insulin sensitivity in skeletal muscle and liver by physiological overexpression of SIRT6.

OBJECTIVE: Available treatment for obesity and type 2 diabetes mellitus (T2DM) is suboptimal. Thus, identifying novel molecular target(s) exerting protective effects against these metabolic imbalances is of enormous medical significance. Sirt6 loss- and gain-of-function studies have generated confounding data regarding the role of this sirtuin on energy and glucose homeostasis, leaving unclear whether activation or inhibition of SIRT6 may be beneficial for the treatment of obesity and/or T2DM. METHODS: To address these issues, we developed and studied a novel mouse model designed to produce eutopic and physiological overexpression of SIRT6 (Sirt6BAC mice). These mutants and their controls underwent several metabolic analyses. These include whole-blood reverse phase high-performance liquid chromatography assay, glucose and pyruvate tolerance tests, hyperinsulinemic-euglycemic clamp assays, and assessment of basal and insulin-induced level of phosphorylated AKT (p-AKT)/AKT in gastrocnemius muscle. RESULTS: Sirt6BAC mice physiologically overexpress functionally competent SIRT6 protein. While Sirt6BAC mice have normal body weight and adiposity, they are protected from developing high-caloric-diet (HCD)-induced hyperglycemia and glucose intolerance. Also, Sirt6BAC mice display increased circulating level of the polyamine spermidine. The ability of insulin to suppress endogenous glucose production was significantly enhanced in Sirt6BAC mice compared to wild-type controls. Insulin-stimulated glucose uptake was increased in Sirt6BAC mice in both gastrocnemius and soleus muscle, but not in brain, interscapular brown adipose, or epididymal adipose tissue. Insulin-induced p-AKT/AKT ratio was increased in gastrocnemius muscle of Sirt6BAC mice compared to wild-type controls. CONCLUSIONS: Our data indicate that moderate, physiological overexpression of SIRT6 enhances insulin sensitivity in skeletal muscle and liver, engendering protective actions against diet-induced T2DM. Hence, the present study provides support for the anti-T2DM effect of SIRT6 and suggests SIRT6 as a putative molecular target for anti-T2DM treatment.

Diet-induced unresolved ER stress hinders KRAS-driven lung tumorigenesis.

Dietary effects on tumor biology can be exploited to unravel cancer vulnerabilities. Here, we present surprising evidence for anti-proliferative action of high-calorie-diet (HCD) feeding on KRAS-driven lung tumors. Tumors of mice that commenced HCD feeding before tumor onset displayed defective unfolded protein response (UPR) and unresolved endoplasmic reticulum (ER) stress. Unresolved ER stress and reduced proliferation are reversed by chemical chaperone treatment. Whole-genome transcriptional analyses revealed FKBP10 as one of the most downregulated chaperones in tumors of the HCD-pre-tumor-onset group. FKBP10 downregulation dampens tumor growth in vitro and in vivo. Providing translational value to these results, we report that FKBP10 is expressed in human KRAS-positive and -negative lung cancers, but not in healthy parenchyma. Collectively, our data shed light on an unexpected anti-tumor action of HCD imposed before tumor onset and identify FKBP10 as a putative therapeutic target to selectively hinder lung cancer.