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.

Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content.

Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit+. Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGFß, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL-6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

Small molecule-induced epigenomic reprogramming of APL blasts leading to antiviral-like response and c-MYC downregulation.

Acute promyelocytic leukemia (APL) is an aggressive subtype of acute myeloid leukemia (AML) in which the PML/RARα fusion protein exerts oncogenic activities by recruiting repressive complexes to the promoter of specific target genes. Other epigenetic perturbations, as alterations of histone H3 lysine 9 trimethylation (H3K9me3), have been frequently found in AMLs and are associated with leukemogenesis and leukemia progression. Here, we characterized the epigenomic effects of maltonis, a novel maltol-derived molecule, in APL cells. We demonstrate that maltonis treatments induce a profound remodulation of the histone code, reducing global H3K9me3 signal and modulating other histone post-translational modifications. Transcriptomic and epigenomic analyses revealed that maltonis exposure induces changes of genes expression associated with a genomic redistribution of histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 acetylation (H3K27ac). Upregulation of interferon alpha and gamma response and downregulation of c-MYC target genes, in function of c-MYC reduced expression (monitored in all the hematopoietic neoplasms tested), represent the most significant modulated pathways. These data demonstrate the ability of maltonis to epigenetically reprogram the gene expression profile of APL cells, inducing an intriguing antiviral-like response, concomitantly with the downregulation of c-MYC-related pathways, thus making it an attractive candidate for antileukemic therapy.

The early-life stress induced by oxytocin inhibition in p53 knockout mouse dams increases adulthood tumorigenesis in first and second generations.

BACKGROUND: Early-life stress due to poor parental care has been suggested to increase cancer risk, though, so far, no experimental evidence established a link between defective parental behavior and spontaneous tumorigenesis in progeny. Essential maternal behavior is regulated, in particular, by the oxytocin (OT) hormonal circuit, which in turn responds to stimuli from the offspring and impinges on the central nervous systems. METHODS: By providing L-368,899 OT receptor (OTR) inhibitor to lactating mothers, we set up a model of defective maternal care in p53 knockout mice. RESULTS: The progeny of these dams showed, later in life, higher cortisol levels, shortened life span and increased tumorigenic potential of bone marrow cells (BMC). Notably, these phenotypes were transmitted to the following generation. CONCLUSIONS: Therefore, the inhibition of OT function in mothers is a novel paradigm of early-life stress that is inherited across generations and increases cancer risk in tumor-prone mice.

Time makes histone H3 modifications drift in mouse liver.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Histone H3 Lysine 4 and 27 Trimethylation Landscape of Human Alzheimer's Disease.

BACKGROUND: Epigenetic remodeling is emerging as a critical process for both the onset and progression of Alzheimer's disease (AD), the most common form of neurodegenerative dementia. However, it is not clear to what extent the distribution of histone modifications is involved in AD. METHODS: To investigate histone H3 modifications in AD, we compared the genome-wide distributions of H3K4me3 and H3K27me3 in entorhinal cortices from severe sporadic AD patients and from age-matched healthy individuals of both sexes. RESULTS: AD samples were characterized by typical average levels and distributions of the H3K4me3 and H3K27me3 signals. However, AD patients showed a lower H3K4me3 and higher H3K27me3 signal, particularly in males. Interestingly, the genomic sites found differentially trimethylated at the H3K4 between healthy and AD samples involve promoter regions of genes belonging to AD-related pathways such as glutamate receptor signaling. CONCLUSIONS: The signatures of H3K4me3 and H3K27me3 identified in AD patients validate the role of epigenetic chromatin remodeling in neurodegenerative disease and shed light on the genomic adaptive mechanisms involved in AD.

Hematopoietic progenitor cell liabilities and alarmins S100A8/A9-related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults.

Frailty affects the physical, cognitive, and social domains exposing older adults to an increased risk of cardiovascular disease and death. The mechanisms linking frailty and cardiovascular outcomes are mostly unknown. Here, we studied the association of abundance (flow cytometry) and gene expression profile (RNAseq) of stem/progenitor cells (HSPCs) and molecular markers of inflammaging (ELISA) with the cardiorespiratory phenotype and prospective adverse events of individuals classified according to levels of frailty. Two cohorts of older adults were enrolled in the study. In a cohort of pre-frail 35 individuals (average age: 75 years), a physical frailty score above the median identified subjects with initial alterations in cardiorespiratory function. RNA sequencing revealed S100A8/A9 upregulation in HSPCs from the bone marrow (>10-fold) and peripheral blood (>200-fold) of individuals with greater physical frailty. Moreover higher frailty was associated with increased alarmins S100A8/A9 and inflammatory cytokines in peripheral blood. We then studied a cohort of 104 more frail individuals (average age: 81 years) with multidomain health deficits. Reduced levels of circulating HSPCs and increased S100A8/A9 concentrations were independently associated with the frailty index. Remarkably, low HSPCs and high S100A8/A9 simultaneously predicted major adverse cardiovascular events at 1-year follow-up after adjustment for age and frailty index. In conclusion, inflammaging characterized by alarmin and pro-inflammatory cytokines in pre-frail individuals is mirrored by the pauperization of HSPCs in frail older people with comorbidities. S100A8/A9 is upregulated within HSPCs, identifying a phenotype that associates with poor cardiovascular outcomes.

Dietary intake of cyanidin-3-glucoside induces a long-lasting cardioprotection from ischemia/reperfusion injury by altering the microbiota.

The anthocyanin class of flavonoids, including cyanidin-3-glucoside (C3G) present in berries, blood oranges and pigmented cereal crops, are food bioactives with antioxidant and anti-inflammatory action, capable to reduce myocardial ischemia/reperfusion (I/R) injury by unclear mechanism. Assessing the value of sporadic beneficial diet is critical for practical application. We aimed to determine whether and how the cardioptotective effect of dietary intake of anthocyanins persists. Gene expression, histology and resistance to I/R were investigated ex vivo in hearts from mice after a month beyond the cease of the C3G-enriched diet. Cardiac injury, oxidative stress and mitochondrial damage following I/R was effectively reduced in mice fed C3G-enriched diet, even after a month of wash out with standard diet. Cardioprotection was observed also in immune-deficient mice lacking mature B and T cells indicating the anti-inflammatory activity of C3G was not involved. Moreover, the transcription reprogramming induced by the C3G-enriched diets was rescued by the wash out treatment. Instead, we found C3G-enriched diet changed the microbiome and the transplantation of the fecal microbiota transferred the cardioprotection from mice fed C3G-enriched diet to mice fed standard diet. These findings established the effect of C3G dietary intake on gut microbiota determines long lasting cardioprotection.

The Histone H3 K4me3, K27me3, and K27ac Genome-Wide Distributions Are Differently Influenced by Sex in Brain Cortexes and Gastrocnemius of the Alzheimer's Disease PSAPP Mouse Model.

BACKGROUND: Women represent the majority of Alzheimer's disease patients and show typical symptoms. Genetic, hormonal, and behavioral mechanisms have been proposed to explain sex differences in dementia prevalence. However, whether sex differences exist in the epigenetic landscape of neuronal tissue during the progression of the disease is still unknown. METHODS: To investigate the differences of histone H3 modifications involved in transcription, we determined the genome-wide profiles of H3K4me3, H3K27ac, and H3K27me3 in brain cortexes of an Alzheimer mouse model (PSAPP). Gastrocnemius muscles were also tested since they are known to be different in the two sexes and are affected during the disease progression. RESULTS: Correlation analysis distinguished the samples based on sex for H3K4me3 and H3K27me3 but not for H3K27ac. The analysis of transcription starting sites (TSS) signal distribution, and analysis of bounding sites revealed that gastrocnemius is more influenced than brain by sex for the three histone modifications considered, exception made for H3K27me3 distribution on the X chromosome which showed sex-related differences in promoters belonging to behavior and cellular or neuronal spheres in mice cortexes. CONCLUSIONS: H3K4me3, H3K27ac, and H3K27me3 signals are slightly affected by sex in brain, with the exception of H3K27me3, while a higher number of differences can be found in gastrocnemius.

Comparative Analysis of Histone H3K4me3 Distribution in Mouse Liver in Different Diets Reveals the Epigenetic Efficacy of Cyanidin-3-O-glucoside Dietary Intake.

BACKGROUND: Different diets result in significantly different phenotypes through metabolic and genomic reprogramming. Epigenetic marks, identified in humans and mouse models through caloric restriction, a high-fat diet or the intake of specific bioactives, suggest that genomic reprogramming drives this metabolic reprogramming and mediates the effect of nutrition on health. Histone modifications encode the epigenetic signal, which adapts genome functions to environmental conditions, including diets, by tuning the structure and properties of chromatin. To date, the effect of different diets on the genome-wide distribution of critical histone marks has not been determined. METHODS: Using chromatin immunoprecipitation sequencing, we investigated the distribution of the trimethylation of lysine 4 of histone H3 in the liver of mice fed for one year with five different diets, including: chow containing yellow corn powder as an extra source of plant bioactives or specifically enriched with cyanidin-3-O-Glucoside, high-fat-enriched obesogenic diets, and caloric-restricted pro-longevity diets. CONCLUSIONS: Comparison of the resulting histone mark profiles revealed that functional food containing cyanidin determines a broad effect.

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Prolonged hypoxia delays aging and preserves functionality of human amniotic fluid stem cells.

Human amniotic fluid stem cells (hAFSCs) are an emerging tool in regenerative medicine because they have the ability to differentiate into various lineages and efficiently improve tissue regeneration with no risk of tumorigenesis. Although hAFSCs are easily isolated from the amniotic fluid, their expansion ex vivo is limited by a quick exhaustion which impairs replicative potential and differentiation capacity. In this study, we evaluate various aging features of hAFSCs cultured at different oxygen concentrations. We show that low oxygen (1% O2) extends stemness and proliferative features, and delays induction of senescence-associated markers. Hypoxic hAFSCs activate a metabolic shift and increase resistance to pro-apoptotic stimuli. Moreover, we observe that cells at low oxygen remain capable of osteogenesis for prolonged periods of time, suggesting a more youthful phenotype. Together, these data demonstrate that low oxygen concentrations might improve the generation of functional hAFSCs for therapeutic use by delaying the onset of cellular aging.

Comparison of the therapeutic effect of amniotic fluid stem cells and their exosomes on monoiodoacetate-induced animal model of osteoarthritis.

The cartilage tissue engineering associated with stem cell-related therapies is becoming very interesting since adult articular cartilage has limited intrinsic capacity for regeneration upon injury. Amniotic fluid stem cells (AFSC) have been shown to produce exosomes with growth factors and immunomodulating molecules that could stop tissue degradation and induce cartilage repair. Based on this state of the art, the main aim of this study was to explore the efficacy of the secreted exosomes, compared to their AFSC source, in MIA-induced animal model of osteoarthritis mimicking a chronic and degenerative process, where inflammation is also involved and lead to irreversible joint damage. Exosomes, obtained by the use of a commercial kit, prior to the injection in animal knee joints, were characterized for the presence of typical markers and HGF, TGFß, and IDO. Then, analyses were performed by histology, immunohistochemistry, and behavioral scoring up to 3 weeks after the treatment. Exosome-treated defects showed enhanced pain tolerance level and improved histological scores than the AFSC-treated defects. Indeed by 3 weeks, TGFß-rich exosome samples induced an almost complete restoration of cartilage with good surface regularity and with the characteristic of hyaline cartilage. Moreover, cells positive for resolving macrophage marker were more easily detectable into exosome-treated joints. Therefore, a modulating role for exosomes on macrophage polarization is conceivable, as demonstrated also by experiments performed on THP1 macrophages. In conclusion, this study demonstrates for the first time the efficacy of human AFSC exosomes in counteract cartilage damage, showing a positive correlation with their TGFß content.

Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging.

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age.The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence.

Amniotic fluid stem cell exosomes: Therapeutic perspective.

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158-167, 2018.

Development of a novel method for amniotic fluid stem cell storage.

BACKGROUND AIMS: Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. METHODS: We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. RESULTS: hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. CONCLUSIONS: This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.