Evidence for a pro-oncogenic role of Sirtuin 6 in breast tumorigenesis

Background: Sirtuin 6 (SIRT6) is a member of the sirtuin family, NAD+-dependent deacetylases with key roles in cell metabolism, DNA repair and inflammation. High SIRT6 levels in breast tumors confer an adverse prognosis. However, the underlying mechanism for such observations have remained unclear in so far. Here I sought to define the effect of a heterozygous Sirt6 deletion on polyoma middle T antigen-induced mouse mammary tumorigenesis and to establish the biochemical and molecular effects of overexpressing vs. reducing SIRT6 in different breast cancer (BC) models. Methods: SIRT6 was overexpressed in either wild type or catalytically inactive (H133Y) form, or silenced in BC cell lines (MDA-MB-231 and MCF7), and we monitored oxygen consumption rate, mitochondrial complex I, III, IV, and ATP synthase activity, cell migration and invasion in Matrigel and in transwell assays, matrix metalloproteinase 9 (MMP9) expression and intracellular Ca2+ concentration ([Ca2+]i). In vivo, we monitored the growth of MDA-MB-231 xenografts in which SIRT6 was silenced vs. control tumors. In addition, we crossed Sirt6+/- mice with MMTV-PyMT+/- mice and comparatively monitored tumor latency and overall survival in MMTV-PyMT+/-;Sirt6+/- vs. MMTV-PyMT+/-;Sirt6+/+ mice. Results: In cultured BC cell lines, overexpression of a catalytically active SIRT6 (but not of the catalytically inactive isoform) boosted OXPHOS and the ATP/AMP ratio. Opposite effects were obtained by SIRT6 silencing. Modulating SIRT6 profoundly affected MMP9 expression and [Ca2+]i. Namely, in MDA-MB-231, SIRT6 overexpression increased, while SIRT6 silencing reduced MMP9 production. [Ca2+]i was increased in WT-SIRT6 overexpressing MDA-MB-231 and such an effect appeared to reflect Ca2+ freeing from its thapsigargin-sensitive stores. Consistent with these data, SIRT6-overexpressing MDA-MB-231 were more invasive than their control cells in vitro assays. In vivo, subcutaneous xenografts of SIRT6-silenced MDA-MB-231 cells were found to grow more slowly than the control tumors. MMTV-PyMT+/-;Sirt6+/- mice exhibited a markedly increased tumor latency and an increased overall survival as compared to the control MMTV-PyMT+/-;Sirt6+/+ animals. The metabolic features of the tumor masses isolated from MMTV-PyMT+/-;Sirt6+/- mice resembled those observed in BC cell lines with silenced SIRT6, showing decrease mitochondrial complexes activity and impaired energy status. The anticancer effects of Sirt6 heterozygous deletion did not reflect reduced glucose levels in Sirt6+/- mice, as the latters had normal blood glucose concentrations. Conclusions: Our data show that reducing Sirt6 levels has significant antitumor activity in in vivo BC models. SIRT6 enhances OXPHOS and energy status in BC cells. In addition, by virtue of its ability to enhance MMP9 expression and [Ca2+]i, SIRT6 could be a potential target for countering invasion and metastasis. Future studies should assess which molecular features predict the potential benefit of SIRT6 inhibition in BC and test the anticancer activity of SIRT6 inhibitors in BC models

The use of the orthotopic model to validate antivascular therapies for cancer

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First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT(+) hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT(+) hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn(F7/F7) mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT(+) hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355

Nicotinic acid phosphoribosyltransferase regulates cancer cell metabolism, susceptibility to NAMPT inhibitors and DNA repair.

In the last decade, substantial efforts have been made to identify NAD(+) biosynthesis inhibitors, specifically against nicotinamide phosphoribosyltransferase (NAMPT), as preclinical studies indicate their potential efficacy as cancer drugs. However, the clinical activity of NAMPT inhibitors has proven limited, suggesting that alternative NAD(+) production routes exploited by tumors confer resistance. Here, we show the gene encoding nicotinic acid phosphoribosyltransferase (NAPRT), a second NAD(+)-producing enzyme, is amplified and overexpressed in a subset of common types of cancer, including ovarian cancer, where NAPRT expression correlates with a BRCAness gene expression signature. Both NAPRT and NAMPT increased intracellular NAD(+) levels. NAPRT silencing reduced energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitized cells to NAMPT inhibitors both in vitro and in vivo; similar results were obtained with the NAPRT inhibitor 2-hydroxynicotinic acid. Reducing NAPRT levels in a BRCA2-deficient cancer cell line exacerbated DNA damage in response to chemotherapeutics. In conclusion, NAPRT-dependent NAD(+) biosynthesis contributes to cell metabolism and to the DNA repair process in a subset of tumors. This knowledge could be used to increase the efficacy of NAMPT inhibitors and chemotherapy. Cancer Res; 77(14); 3857-69. ©2017 AACR

Use of Attribute Driven Incremental Discretization and Logic Learning Machine to build a prognostic classifier for neuroblastoma patients

Cancer patient's outcome is written, in part, in the gene expression profile of the tumor. We previously identified a 62-probe sets signature (NB-hypo) to identify tissue hypoxia in neuroblastoma tumors and showed that NB-hypo stratified neuroblastoma patients in good and poor outcome 1. It was important to develop a prognostic classifier to cluster patients into risk groups benefiting of defined therapeutic approaches. Novel classification and data discretization approaches can be instrumental for the generation of accurate predictors and robust tools for clinical decision support. We explored the application to gene expression data of Rulex, a novel software suite including the Attribute Driven Incremental Discretization technique for transforming continuous variables into simplified discrete ones and the Logic Learning Machine model for intelligible rule generation. We applied Rulex components to the problem of predicting the outcome of neuroblastoma patients on the bases of 62 probe sets NB-hypo gene expression signature. The resulting classifier consisted in 9 rules utilizing mainly two conditions of the relative expression of 11 probe sets. These rules were very effective predictors, as shown in an independent validation set, demonstrating the validity of the LLM algorithm applied to microarray data and patients' classification. The LLM performed as efficiently as Prediction Analysis of Microarray and Support Vector Machine, and outperformed other learning algorithms such as C4.5. Rulex carried out a feature selection by selecting a new signature (NB-hypo-II) of 11 probe sets that turned out to be the most relevant in predicting outcome among the 62 of the NB-hypo signature. Rules are easily interpretable as they involve only few conditions. Our findings provided evidence that the application of Rulex to the expression values of NB-hypo signature created a set of accurate, high quality, consistent and interpretable rules for the prediction of neuroblastoma patients' outcome. We identified the Rulex weighted classification as a flexible tool that can support clinical decisions. For these reasons, we consider Rulex to be a useful tool for cancer classification from microarray gene expression dat

Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: Endorsement of macrophage polarization

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secre- tion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mech- anism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macro- phage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infil- trating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs

SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer