Alessia Grozio

The NAD+-dependent histone deacetylase SIRT6 promotes cytokine production and migration in pancreatic cancer cells by regulating Ca2+ responses

Background: Cytokine secretion has unwanted consequences in malignant and in inflammatory disorders. The deacetylase SIRT6 has pro-inflammatory activity, but the underlying mechanisms and its biological significance remain unclear. Results: SIRT6 enhances cytokine secretion and cell motility in pancreatic cancer cells by activating Ca2+ signaling. Conclusion: SIRT6 promotes Ca2+-dependent responses. Significance: SIRT6 inhibitors may help combat malignant and inflammatory disorders. Cytokine secretion by cancer cells contributes to cancer-induced symptoms and angiogenesis. Studies show that the sirtuin SIRT6 promotes inflammation by enhancing TNF expression. Here, we aimed to determine whether SIRT6 is involved in conferring an inflammatory phenotype to cancer cells and to define the mechanisms linking SIRT6 to inflammation. We show that SIRT6 enhances the expression of pro-inflammatory cyto-/chemokines, such as IL8 and TNF, and promotes cell migration in pancreatic cancer cells by enhancing Ca2+ responses. Via its enzymatic activity, SIRT6 increases the intracellular levels of ADP-ribose, an activator of the Ca2+ channel TRPM2. In turn, TRPM2 and Ca2+ are shown to be involved in SIRT6-induced TNF and IL8 expression. SIRT6 increases the nuclear levels of the Ca2+-dependent transcription factor, nuclear factor of activated T cells (NFAT), and cyclosporin A, a calcineurin inhibitor that reduces NFAT activity, reduces TNF and IL8 expression in SIRT6-overexpressing cells. These results implicate a role for SIRT6 in the synthesis of Ca2+-mobilizing second messengers, in the regulation of Ca2+-dependent transcription factors, and in the expression of pro-inflammatory, pro-angiogenic, and chemotactic cytokines. SIRT6 inhibition may help combat cancer-induced inflammation, angiogenesis, and metastasis.

LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38+/LANCL2+ HeLa transfected with a chimeric G-protein (Gαq/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a Gi. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts

The plant hormone abscisic acid (ABA) is released from glucose‐challenged human pancreatic β cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2‐ to 9‐fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP‐1 stimulated ABA release from an insulinoma cell line and from human islets, by ~10‐ and 2‐fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3‐L1 cells differentiated to adipocytes, by increasing GLUT‐4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.—Bruzzone, S., Ameri, P., Briatore, L., Mannino, E., Basile, G., Andraghetti, G., Grozio, A., Magnone, M., Guida, L., Scarfì, S., Salis, A., Damonte, G., Sturla, L., Nencioni, A., Fenoglio, D., Fiory, F., Miele, C., Beguinot, F., Ruvolo, V., Bormioli, M., Colombo, G., Maggi, D., Murialdo, G., Cordera, R., De Flora, A., Zocchi, E. The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts. FASEB J. 26, 1251‐1260 (2012). www.fasebj.org

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells

Background: NAMPT inhibitors showed antitumor activity in preclinical cancer models, but no tumor remission occurred in clinical studies. Results: Cells treated with a NAMPT inhibitor are rescued by low NAD+e or NAD+ precursors, depending on CD38 and CD73 expression. Conclusion: CD73 enables, whereas CD38 impairs, extracellular NMN utilization by cells for NAD+ biosynthesis. Significance: Combining CD73 and NAMPT inhibition could represent a new anti-cancer strategy. NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.

Rejuvenating sirtuins: the rise of a new family of cancer drug targets.

Sirtuins are a family of NAD+-dependent enzymes that was proposed to control organismal life span about a decade ago. While such role of sirtuins is now debated, mounting evidence involves these enzymes in numerous physiological processes and disease conditions, including metabolism, nutritional behavior, circadian rhythm, but also inflammation and cancer. SIRT1, SIRT2, SIRT3, SIRT6, and SIRT7 have all been linked to carcinogenesis either as tumor suppressor or as cancer promoting proteins. Here, we review the biological rationale for the search of sirtuin inhibitors and activators for treating cancer and the experimental approaches to their identification.

Discovery of novel and selective SIRT6 inhibitors.

SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.

Binding of abscisic acid to human LANCL2

The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor.

Nicotinamide phosphoribosyltransferase promotes epithelial-to-mesenchymal transition as a soluble factor independent of its enzymatic activity

Background: Nicotinamide phosphoribosyltransferase (NAMPT) acts both as an enzyme in the production of the coenzyme NAD+ and as a secreted cytokine. Results: In breast cancer cells, NAMPT induces the epithelial-to-mesenchymal transition, a process that underlies metastasis, as a secreted protein independent of its enzymatic activity. Conclusion: Secreted NAMPT promotes epithelial-to-mesenchymal transition. Significance: Extracellular NAMPT neutralization may be of therapeutic value. Boosting NAD+ biosynthesis with NAD+ intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD+ biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFβ signaling pathway via increased TGFβ1 production. These findings have implications for the design of therapeutic strategies exploiting NAD+ biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV‐B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV‐B of human granulocytes and keratinocytes, the cells involved in UV‐triggered skin inflammation. The intracellular ABA concentration increased in UV‐B‐exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV‐B‐induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE2), and tumor necrosis factor‐α (TNF‐α) by UV‐B irradiated keratinocytes. Lanthionine synthetase C‐like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV‐B‐triggered release of PGE2, TNF‐α, and NO and ROS production. These results indicate that UV‐B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation. J. Cell. Physiol. 227: 2502–2510, 2012.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics

The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6s biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.