Nicotinamide Phosphoribosyltransferase in Malignancy

Introduction

Nicotinamide adenine dinucleotide (NAD) was first discovered in 1904 by Sir Arther Harden who identified a low-molecular-weight compound in yeast termed cozymase that was required for sugar fermentation. In the 1930s, Warburg found this compound to be a hydride-accepting and donating molecule that played a role in multiple cellular reactions. ¹ The enzymatic activity responsible for NAD synthesis was identified in 1957 and designated as nicotinamide mononucleotide (NMN) pyrophosphorylase. ² In 1966, Gholson predicted that NAD is actively turned over within cells, and this prediction was confirmed when the half-life of cellular NAD was found to be 1.0 ± 0.3 hours within cultured cells.^3,4 The enzyme responsible for NAD synthesis was first cloned from activated peripheral human lymphocytes in 1994. It was initially identified as a secreted cytokine that synergized with interleukin-7 and stem cell factor to stimulate early stage B-cells, hence its designation as pre-B cell colony-enhancing factor (PBEF). ⁵ Later work confirmed a role for PBEF as a cytokine that is up-regulated in activated neutrophils and recombinant PBEF inhibits neutrophil apoptosis when placed in culture media. ⁶ In 2005, Fukuhara et al. ⁷ identified a visceral fat-secreted adipokine, corresponding to PBEF, with insulin-mimetic effects that was designated visfatin. The article was later retracted based on difficulty with data reproducibility. ⁸

Data demonstrating that PBEF played a role in intracellular NAD synthesis came in 2001, when Martin et al. ⁹ demonstrated that the Haemophilus ducreyi gene nadV, which has homology to mammalian Nampt/PBEF, is an NAD phosphoribosyltransferase that when expressed allowed H. ducreyi to grow in NAD free media. Later, Rongvaux et al. ¹⁰ demonstrated that murine PBEF was an intracellular nicotinamide phosphosribosyltransferase. The cloned murine Nampt gene was also able to confer growth in Actinobacillus pleuropneumoniae lacking nadV, indicating conservation of the NAD synthesis pathway between mammals and bacteria. ¹⁰ Based on this work, the enzyme was designated nicotinamide phosphosribosyltransferase (Nampt).^9,10

Nampt is now known to catalyze NAD synthesis by transferring the phosphoribosyl group of 5-phosporibosyl-1-pyrophosphate to nicotinamide (NAM), forming NMN. NAD synthesis is completed by NMN adenylyltransferase (Nmnat), which converts NMN into NAD. ¹⁰ Nampt’s catalytic activity is ~46-fold lower than Nmnat activity, so Nampt catalyses the rate-limiting step of NAD synthesis. Thus, even very small changes in Nampt, but not Nmnat levels, can profoundly affect NAD metabolism and NAD-dependent events.^10,11 The specific murine Nmnat isoform analyzed in this study was not identified, although the Nmnat had Km and Vmax values consistent with those previously reported for human Nmnat-1. ¹¹ Interestingly, there are 3 different Nmnat enzymes located in the nucleus, cytosol, and mitochondria (Nmnats1-3, respectively).^10,11 NAD is synthesized either de novo from precursors such as tryptophan, and nicotinic or quinolinic acids, or by the Nampt/Nmnat-catalyzed salvage pathway, with the later pathway being significantly faster, more efficient, and also the major NAD biosynthesis pathway in mammals (Figure 1).^1,11,12 Nampt is found both intracellularly in the cytoplasm (designated iNampt), nucleus, and possibly the mitochondria in most cell types, and extracellularly in the plasma (eNampt), which was previously designated as visfatin.^7,8,13,14

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Figure 1.

The NAD salvage pathway (modified from 11). NAD is consumed in many different reactions, including PARP1, CD38, and SirT1 activities.

Yang et al. ¹⁵ found that cell survival following genotoxic stress was dependent on the mitochondrial, but not the nuclear or cytoplasmic NAD pools. Cell survival was increased with increasing iNampt expression, an event dependent on expression of the mitochondrial NAD⁺-dependent deacetylases SirT3 and SirT4, indicating that mitochondrial Nampt/NAD synthesis plays a vital role in cellular function and survival. However, other investigators have not found mitochondrial Nampt. For example, Nikiforov et al. ¹⁶ found no mitochondrial iNampt and instead found that the mitochondrial-specific Nmnat3 produced all the mitochondrial NAD from NMN imported from the cytosol. The reason for this discrepancy is unknown, but it may be due to using different cells, or different mitochondrial isolation and analysis techniques. Interestingly, approximately 70% of the intracellular NAD pool is mitochondrial. ¹

The functions of eNampt are presently poorly understood and there is conflicting data on eNampt function. For example, Revollo et al. ¹⁷ found that Nampt haplodeficient mice have reduced plasma NMN and eNampt and defects in glucose-stimulated insulin secretion in pancreatic β-cells. This defect is corrected by NMN administration. The authors concluded that eNampt-mediated systemic NAD synthesis is critical for normal β-cell function. However, Zhang et al. ¹⁴ found that eNampt promoted macrophage survival following endoplasmic reticulum stress, by stimulating interleukin-6 secretion and Stat3 activation. Interestingly, enzymatically inactive mutated eNampt was as biologically active as wild-type eNampt. Thus, eNampt exerts some functions independently of NAD synthesis. Last, low plasma eNampt levels correlate with hepatic mitochondrial dysfunction, thereby indicating that eNampt regulates some aspects of intracellular biochemistry. ¹⁸ An NAD-synthesizing role for eNampt also seems unlikely as other investigators have found eNampt to have very low catalytic activity under normal physiologic conditions, due to the extracellular space having very low adenosine-triphosphate concentrations.^19,20 eNampt is secreted by a nonclassical pathway in several different cell types, including differentiated adipocytes, macrophages, cardiomyocytes, and hepatocytes.^{21 -25} Interestingly, overexpression and secretion of eNampt in murine cardiomyocytes resulted in cardiac hypertrophy. Cardiomyocyte eNampt secretion was inhibited in cell culture by treatment with NAM or trichostatin, indicating that extracellular signals my partially regulate eNampt secretion. ²⁵

Nampt is found at 7q22 and spans 34.7 kb having 11 exons and 10 introns, giving a cDNA of 2,357 kb translated into a 491 amino acid, 52 kDa protein. Three predominant mRNA transcripts have been identified, comprising 2.0, 2.4, and 4.0 kb transcripts.^13,15 Nampt mRNA is found in all tissues, suggesting it has a vital and indispensible function.^10,26 The enzyme shows a high degree of evolutionary conservation, and enzymes with closely related sequences are found in prokaryotes, sponges, insects, and mammals.^10,27 Crystallographic studies show that Nampt is a dimeric class type II phosphoribosyltransferases where 2 identical Nampt subunits contribute to an enzymatic active site, thus converting NAM and 5-phosporibosy1-1-pyrophosphate into NMN by an A_ND_N mechanism.^27,28 iNampt is phosphorylated at histidine 247, resulting in a 160,000-fold increased enzymatic affinity for NAM. ²⁹ iNampt and eNampt undergo other posttranslational modifications, including acetylation and ubiquitization. The significance of these modifications is presently poorly understood. ¹¹ Presently all 3 of these names (PBEF, Visfatin, and Nampt) are used, although the Human Genome Organization Gene Nomenclature Committee approved the name of Nampt.

Nampt, NAD⁺, and Cancer

NAD is a cofactor that plays a central role in cellular electron transfer redox reactions, alternating between oxidized and reduced forms (NAD⁺ + e⁻ ⇔ NADH) and is a universal energy- and signal-carrying molecule. NAD functions in many cellular events, including transcriptional regulation, longevity and caloric-restriction responses, cell cycle progression, apoptosis, DNA repair, circadian rhythms, chromatin dynamics regulation, telomerase activity, intracellular calcium mobilization. It also regulates the histone deacetylases (SirT1-T7), CtBP, CD38, and the poly(ADP-ribose) polymerases that play a central role in the maintenance of organismal metabolic homeostasis and genomic stability.^{1,30 -36} Unlike many cellular redox reactions, several NAD-dependent signaling processes degrade NAD by transferring the ADP-ribose moiety onto a receptor with the concomitant release of NAM. Thus, constant NAD resynthesis is an absolute requirement for cell survival, especially for rapidly growing cells. ³³

Several different human malignant tumors have been demonstrated to overexpress iNampt including colorectal, ovarian, breast, gastric, prostrate, well-differentiated thyroid, and endometrial carcinomas, and myeloma, melanoma, and astrocytomas. Increased iNampt expression also occurs in malignant lymphomas, including diffuse large B-cell lymphoma, follicular B-cell lymphoma, Hodgkin’s lymphoma, and peripheral T-cell lymphoma (Table 1).^37-55 eNampt also increases the growth fraction of the hepatocellular carcinoma HepG2 cell line in vitro, suggesting a role for it in hepatocellular carcinoma. ⁵³ Many of these studies have found interesting aspects of Nampt expression in cancer. Olesen et al. ⁵⁵ found higher iNampt expression in aggressive malignant lymphomas. Huang et al. ⁵⁶ found that iNampt expression increases cellular stromal cell-derived factor-1 levels in colon cancer cells, promoting colorectal carcinoma progression. Although most studies documented increased iNampt levels between benign and malignant tissue, several correlated iNampt expression with specific changes in tumor behavior. For example, Long et al. ⁴⁴ found iNampt was expressed 13 times higher in gastric cancer than in benign gastric tissue. Higher iNampt expression correlated with deeper tumor invasion, lymph node metastases, a higher clinical TMN stage, and a reduced patient survival. Similarly, increased iNampt correlates with increased tumor growth, metastases, cellular dedifferentiation, and the presence of a vertical growth phase in melanoma. Higher iNampt expression also confers a worse prognosis in endometrial adenocarcinoma and astrocytomas.^{47 -49,52} Wang et al. ⁴⁵ found that elevated iNampt expression in early prostate cancer and inhibition of iNampt suppressed cell growth in culture, cell invasion, and the growth of xenografted prostate cancer cells in mice. Last, several researchers found that iNampt expression confers resistance to chemotherapeutic agents, including fluorouracil, doxorubicin, paclitaxel, etoposide, and phenylethyl isothiocyanate.^40,41,43,45 Interestingly, in one case of signet ring cell gastric carcinoma following exposure to the Chernobyl nuclear accident, iNampt expression was very low in the malignant cells, which may suggest that iNampt expression may be different between radiation-induced and sporadic gastric cancers. ⁵⁷

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Table 1.

Nampt Overexpression in Several Human Malignancies ^a .

Tumor Type	Experimental Method(s)	Finding(s)	References
Colorectal carcinoma*	Suppression subtractive hybridization and immunohistochemistry	Six-fold iNampt overexpression compared to benign tissue	37, 38
Ovarian serous adenocarcinoma*	Tissue microarray	iNampt overexpressed	39
Breast cancer*	cDNA microarray, quantitative RT-PCR, immunohistochemistry, chromatin immunoprecipitation	iNampt overexpressed in doxorubicin resistant tumors, increased by hypoxia, promotes cell proliferation	40-42
Gastric cancer*	RT-PCR, Western blot	Overexpression, higher iNampt expression with deeper tumor invasion and in metastases	43, 44
Prostate cancer*	Western blot, immunohistochemistry	iNampt overexpressed, higher expression increases H2O2 and chemotherapeutic agent resistance, and FOXO3a expression	45, 46
Endometrial adenocarcinoma*	Tissue microarray, immunohistochemistry	iNampt overexpressed, expression is an independent overall survival predictor, higher expression with greater endometrial epithelial atypia	47
Melanoma*	Immunohistochemistry	iNampt overexpressed, higher levels in metastases and vertical growth phase melanoma, eNampt increases tumor growth	48, 49
Myeloma	Enzyme-linked immunosorbent assay (ELISA)	Overexpressed, inhibiting iNampt lowered SirT1 and PARP-1 activities, t(11;14) causes gains at 7q22 where Nampt is located	50, 51
Astrocytomas*	cDNA Microarray, RT-qPCR, ELISA	iNampt overexpressed, high expression with p53 correlates with poor survival, expression correlates with astrocytoma grade	52
HepG2 cells*	Western blot, cell proliferation assays, ELISA	eNampt increases HepG2 cell growth, eNampt is higher with advanced hepatocellular carcinoma stage	41, 53
Well-differentiated thyroid carcinomas*	Tissue microarray	Significant overexpression of iNampt and SirT3 in papillary and follicular thyroid carcinomas, PARP-1 overexpression seen in papillary thyroid carcinomas	54
Malignant lymphomas*	Immunohistochemistry on formalin-fixed, paraffin-embedded slides	Nampt expression is higher in more aggressive malignant lymphomas and is higher in Reed-Sternberg cells	52

a

“*” denotes malignancies with PARP-1 overexpression. PARP-1 overexpression is seen in diffuse large B-cell and follicular lymphomas. ⁵⁸

Treatment of Human Malignancies With iNampt Inhibitors

The overexpression of Nampt in several human malignancies, combined with its promotion of many aspects of the malignant phenotype,^{37 -54} suggests that Nampt inhibition may exert anticancer effects. Hasmann and Schemainda ¹⁰² found that the highly specific, noncompetitive Nampt inhibitor FK866 induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC₅₀ of ~1 nM. The mechanism involved a gradual NAD⁺ depletion, which could be partially reversed by adding NAM or nicotinic acid. Others researchers found that Nampt inhibition also causes ATP depletion, lowered PARP-1 and SirT1 activities, and eventual cell death.^{102 -105} Interestingly, due to their increased NAD and ATP catabolism, tumor cells are more sensitive to iNampt inhibition than are benign cells. ¹⁰⁶ Recently, several Nampt inhibitors have shown promise in treating several human malignancies and several are now in phase I and II clinical trials (Table 2).^{43,48,106 -110} Interestingly, Nampt inhibition by FK866 had little effect on cultured melanoma cells. ⁴⁸ Several of these studies employed a Nampt inhibitor with another chemotherapeutic agent to induce “synthetic lethality,” where the inhibition of 2 gene products causes cell death, while inhibition of either gene product alone does not significantly lower cell viability. ¹¹¹ For example, Bajrami et al. ⁷⁸ employed an olaparib (a PARP-1 inhibitor) sensitization screen to examine the effect of inhibiting different enzymes involved in NAD metabolism and their effect on triple-negative breast cancer cell growth in murine xenografts. Nampt was identified as a nonredundant modifier of the olaparib response. Based on this the authors concluded that Nampt/PARP-1 inhibitor combinations may have value in treating triple-negative breast cancer.

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Table 2.

Research Studies Demonstrating Nampt Inhibition May Have Some Clinical Efficacy in Treating Human Malignancies.

Tumor Type	Inhibitor	Finding(s)	Reference(s)
HepG2 liver carcinoma	FK866	FK866 induced delayed apoptosis by NAD depletion	103
Gastric carcinoma	FK866	FK866 increased fluorouracil sensitivity and decreased cell proliferation and VEGF, MMP2, MMP9, and NF-κB expression	107
Non–small cell lung cancer with epidermal growth factor receptor-gene mutation cell lines	FK866 and Nampt-siRNA transfection	Lung adenocarcinoma cell lines with EGFR mutations were more sensitive to the effects of Nampt inhibition	78
Triple negative breast cancer cell lines	FK866 and Olaparib (a PARP-1 inhibitor)	FK866 and Olaparib together suppressed cell line growth in xenografted mice more than either agent alone	70
Multiple hematopoietic cancer cells and hematopoietic progenitor cells	FK866 and WK175	FK866 inhibited malignant, but not benign, hematopoietic cell growth. FK866 also abrogated tumor growth in animal models of human leukemia	108-110
C6 glioblastoma cells	FK866	FK866 inhibited cell growth, depleted NAD, inhibited ERK activation, and induced G2/M cell-cycle arrest	111
Cultured melanoma cells	FK866	FK866 did not induce cell death or potentiate temozolomide or dacarbazine toxicities	46

Conclusion

Both iNampt and eNampt are overexpressed in several human malignancies, where increased expression of either form is often associated with malignant progression.^{37 -54,90 -92} Additionally, several early studies indicate that Nampt inhibition may have clinical efficacy in treating some human malignancies.^{43,70,78,107,108} Based on these data, Nampt plays an important role in carcinogenesis and possibly cancer treatment, especially as it relates to other proteins involved in NAD metabolism. There are many aspects of Nampt biology that are presently poorly understood and need further study. For example, Santidrian et al. ¹¹² found that a nonlethal reduction NAD⁺ levels by interfering with Nampt expression increased breast cancer metastases in an animal model, contradicting much of the data given above. Additionally, the same study revealed that mitochondrial complex I activity and the NAD⁺/NADH ratio regulated breast cancer progression. This last observation indicates that there are many aspects of NAD metabolism related to carcinogenesis and that Nampt activity in only one aspect of a complex NAD metabolome in cancer.