Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD⁺ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD⁺ consuming enzymes. We recently demonstrated that enhancement of NAD⁺/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD⁺ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD⁺ levels promote tumor cell dissemination, we here asked whether inhibition of NAD⁺ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD⁺ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD⁺ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD⁺ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.     

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

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.     

Identification of small-molecule urea derivatives as novel NAMPT inhibitors via pharmacophore-based virtual screening

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-prophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD⁺) synthesis. Recently, intracellular NAD⁺ has received substantial attention due to the recent discovery that several enzymes including poly(ADP-ribose) polymerases (PARPs), mono(ADP-ribose) transferases (ARTs), and sirtuins (SIRTs), use NAD⁺ as a substrate, suggesting that intracellular NAD⁺ level may regulate cytokine production, metabolism, and aging through these enzymes. NAMPT is found to be upregulated in various types of cancer, and given its importance in the NAD⁺ salvage pathway, NAMPT is considered as an attractive target for the development of new cancer therapies. In this study, the reported NAMPT inhibitors bearing amide, cyanoguanidine, and urea scaffolds were used to generate pharmacophore models and pharmacophore-based virtual screening studies were performed against ZINC database. Following the filtering steps, ten hits were identified and evaluated for their in vitro NAMPT inhibitory effects. Compounds GF4 (NAMPT IC₅₀ = 2.15 ± 0.22 μM) and GF8 (NAMPT IC₅₀ = 7.31 ± 1.59 μM) were identified as new urea-typed inhibitors of NAMPT which also displayed cytotoxic activities against human HepG2 hepatocellular carcinoma cell line with IC₅₀ values of 15.20 ± 1.28 and 24.28 ± 6.74 μM, respectively.     

Nicotinamide Phosphoribosyltransferase Promotes Epithelial-to-Mesenchymal Transition as a Soluble Factor Independent of Its Enzymatic Activity

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.     

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116R^FK866. In this study, we investigated gene mutations in parental HCT116 and HCT116R^FK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD⁺ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116R^FK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.     

NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD⁺ level. NAMPT is a rate-limiting enzyme for NAD⁺ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD⁺ production to maintain mitochondrial NAD⁺ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD⁺ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD⁺ content to promote cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression rescued the mitochondrial membrane potential and content, suggesting that maintained mitochondrial content and integrity by NAMPT overexpression might be one of the key mechanisms to maintain mitochondrial NAD⁺ level and subsequently dictate cell survival under oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and membrane potential.     

Exogenous NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γ-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP), which can potentially increase the level of intracellular NAD⁺, on the level of γ-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NADP in different doses immediately after IR causes an increased level of γ-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that there could be a relationship between intracellular NAD⁺ content and DNA damage response in vivo.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD⁺) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD⁺ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD⁺ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD⁺, mediates skin inflammation through parthanatos cell death.

A study of chronic skin inflammation reveals that hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation via parthanatos cell death, identifying NAMPT, PARP1 and AIFM1 as novel therapeutic targets for psoriasis.

Highlights

• Nicotinamide phosphoribosyltransferase (NAMPT) inhibition alleviates inflammation in zebrafish and human organotypic 3D skin models of psoriasis.
• NADPH oxidase–derived reactive oxygen species (ROS) mediate keratinocyte DNA damage and poly(ADP-ribose) polymerase 1 (PARP1) overactivation.
• Inhibition of parthanatos cell death phenocopies the effects of NAMPT inhibition in zebrafish and human psoriasis models.
• NAMPT and poly(ADP-ribose) (PAR) metabolism is altered in psoriasis patients.

     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,in Human Cancer Cells: METABOLIC BASIS AND POTENTIAL CLINICAL IMPLICATIONS*

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD⁺, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD⁺ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.     

NAMPT reduction‐induced NAD+ insufficiency contributes to the compromised oocyte quality from obese mice

Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD⁺) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD⁺ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity‐associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD⁺) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD⁺ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.Subject terms: Drug development, Cancer metabolism

     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.