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NAMPT overexpression in prostate cancer and its contribution to tumor cell survival and stress response

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in regenerating nicotinamide adenine dinucleotide (NAD+) from nicotinamide in mammals. NAMPT has crucial roles for many cellular functions by regulating NAD+-dependent SIRT1 deacetylase. However, roles of NAMPT in cancer are poorly defined. In this study, we show that NAMPT is prominently overexpressed in human prostate cancer cells along with SIRT1. Elevation of NAMPT expression occurs early for the prostate neoplasia. Inhibition of NAMPT significantly suppresses cell growth in culture, soft agar colony formation, cell invasion and growth of xenografted prostate cancer cells in mice. NAMPT knockdown sensitizes prostate cancer cells to oxidative stress caused by H2O2 or chemotherapeutic treatment. Overexpression of NAMPT increases prostate cancer cell resistance to oxidative stress, which is partially blocked by SIRT1 knockdown. We demonstrate that in addition to modulating SIRT1 functions, the NAMPT inhibition reduces forkhead box, class ‘O’ (FOXO)3a protein expression and its downstream anti-oxidant genes catalase and manganese superoxide dismutase. Our results suggest important roles of concomitant upregulation of NAMPT and SIRT1 along with increased FOXO3a protein level for prostate carcinogenesis and their contribution to oxidative stress resistance of prostate cancer cells. These findings may have implications for exploring the NAMPT pathway for prostate cancer prevention and treatment.

Introduction

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for many enzymes and cellular functions. In mammals, NAD+ can be biosynthesized from nicotinamide (NAM), nicotinic acid or tryptophan, with NAM as the primary precursor (Garten et al., 2009). The rate limiting step for NAD+ biosynthesis from NAM is the transfer of a phosphoribosyl group to NAM, which is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT) (Revollo et al., 2004), also known as pre-B-cell colony enhancing factor (Samal et al., 1994). NAMPT has a crucial role for growth factor-induced myeloid differentiation (Skokowa et al., 2009), circadian clock (Nakahata et al., 2009; Ramsey et al., 2009), glucose-restriction-impaired skeletal myoblast differentiation (Fulco et al., 2008) and insulin secretion in pancreatic beta cells (Revollo et al., 2007). Overexpression of NAMPT extends lifespan of human vascular smooth muscle cells (van der Veer et al., 2007), resembling the effect of yeast NAD+ salvage pathway gene PNC1 for promoting longevity (Anderson et al., 2003).

Sirtuins are a family of protein deacetylases and adenosine diphosphate (ADP)-ribosyltransferases whose functions require NAD+ (Saunders and Verdin, 2007). Among them, SIRT1 has the highest homology to yeast Sir2 (silent information regulator 2) that extends lifespan under calorie restriction (Lin et al., 2000). SIRT1 is involved in regulation of a variety of cellular functions including survival, glucose homeostasis and fat metabolism. SIRT1 promotes mammalian cell survival under oxidative and genotoxic stresses through deacetylation of multiple substrates including p53 (Luo et al., 2001; Vaziri et al., 2001) and forkhead box, class ‘O’ (FOXO) transcriptional factors (Brunet et al., 2004; Daitoku et al., 2004; Motta et al., 2004). SIRT1 serves as a key physiological downstream effector for NAMPT in regulating cell survival, differentiation and circadian clock (van der Veer et al., 2007; Fulco et al., 2008; Nakahata et al., 2009; Ramsey et al., 2009; Skokowa et al., 2009).

SIRT1 is overexpressed in multiple primary solid tumors and hematopoietic malignancies (Bradbury et al., 2005; Huffman et al., 2007; Jang et al., 2008, 2009; Jung-Hynes et al., 2009; Nosho et al., 2009). Inhibition of SIRT1 suppresses growth and promotes apoptosis in human cancer cells (Luo et al., 2001; Vaziri et al., 2001; Chu et al., 2005; Ford et al., 2005; Ota et al., 2006; Kojima et al., 2008; Jung-Hynes et al., 2009), but has little impact on the survival of normal cells (Ford et al., 2005; Jung-Hynes et al., 2009). Consistently, loss of tumor suppressor gene p53 results in increase of SIRT1 expression (Nemoto et al., 2004), and we have shown that the epigenetically-regulated tumor suppressor HIC1 (hypermethylated in cancer 1) directly represses SIRT1 expression (Chen et al., 2005).

Increased expression of NAMPT was previously reported in colorectal cancer (Hufton et al., 1999; Van Beijnum et al., 2002). A specific small molecule inhibitor of NAMPT, FK866, inhibits cancer cell growth and induces apoptosis by NAD+ depletion (Hasmann and Schemainda, 2003). Administration of FK866 in vivo suppresses growth of murine renal cell carcinoma and mammary carcinoma in mice (Drevs et al., 2003; Muruganandham et al., 2005). Similarly, another NAMPT inhibitor, GMX1778, exhibits significant anti-tumor activity (Watson et al., 2009). However, precise roles of NAMPT in cancer are poorly understood. In this study, we demonstrated that NAMPT is overexpressed in numerous human cancer cells along with SIRT1, with prominent roles for prostate cancer. NAMPT promotes prostate cancer cell growth, survival and tumorigenesis in part by regulating SIRT1 functions. Surprisingly, we found that NAMPT also regulates FOXO3a protein expression for anti-oxidative stress response of prostate cancer cells, which appears independent of SIRT1 modulation.

Results

NAMPT is overexpressed in human prostate cancer

The deacetylation and ADP-ribosyltransferation by sirtuins produce NAM that is known as a natural inhibitor of sirtuins (Bitterman et al., 2002). The accumulation of high concentration of intracellular NAM may thus impair functions of SIRT1 and other sirtuin enzymes, and preventing accumulation of intracellular NAM would be critical for maintaining active SIRT1 functions. NAM reduction can be achieved through NAM methylation by NAM N-methyltransferase that targets it for excretion (Williams and Ramsden, 2005), or by recycling NAM back to NAD+ by the salvage pathway (Figure 1a). We hypothesized that enzymes responsible for maintenance of cellular NAM level, NAM N-methyltransferase and rate-limiting NAD+ salvage pathway enzyme NAMPT, might also be upregulated in cancer cells in order to maintain active SIRT1 function. We screened human cancer cell lines by western blotting and found that SIRT1 was overexpressed in a variety of cancer cell lines along with increased NAMPT expression (Figure 1b); in contrast, NAM N-methyltransferase expression was hardly detectable in cancer cell lines except a CML cell line, KCL-22 (Supplementary Figure 1), indicating that cancer cells efficiently recycle NAM to replenish NAD+ supply.

Figure 1
figure1

NAMPT expression in human cancers. (a) The NAD biosynthesis pathways in mammalian cells. Npt, Nampt, Nmnat and Nnmt are nicotinic acid phosphoribosyltransferase, nicotinamide phosphoribosyltransferase, nicotinamide/nicotinic acid mononucleotide adenylyltransferase and nicotinamide N-methyltransferase, respectively. (b) Western blots of NAMPT and SIRT1 for normal human tissues and cancer cell line lysates. (c) NAMPT immunohistochemistry of normal human prostate tissues and prostate cancers from tissue arrays. NAMPT-positive cells were stained brown. (d) Statistics of prostate tissue array analysis. Statistical significance was calculated using two-tailed χ2 and Fisher tests. BPH, benign prostate hyperplasia; NaMN, nicotinic acid mononucleotide; NB, normal breast; NC, normal colon; NMN, nicotinamide mononucleotide; NMNAM, N-methyl nicotinamide; NL, normal lung; NP, normal prostate; NPM, normal peripheral blood mononuclear cells; PCA, prostate carcinoma.

We next examined NAMPT expression in normal human tissues and cancers by immunohistochemistry of tissue arrays. The specificity of the NAMPT antibody was validated as detailed in the methods and Supplementary Figure 2. NAMPT was expressed in certain normal tissues with the highest level in liver, pancreas, adrenal gland and muscle (Supplementary Figure 3A), consistent with previous reports (Samal et al., 1994; Fukuhara et al., 2005). We found that NAMPT expression was upregulated in many cancer types including prostate, colon, brain and lung cancer, as well as lymphoma when compared with their normal counterparts, with prostate exhibiting the most consistent and striking difference between normal tissue and cancer in arrays obtained from different sources (Figure 1c and Supplementary Figure 3B). NAMPT was only weakly detectable in one out of six normal prostate cases, each with three sections of peripheral or transitional zone studied; however, NAMPT was strongly expressed in 39 out of 41 (95%) prostate carcinomas (P=7.9 × 10−5, Figure 1d). Although no prostate intraepithelial neoplasia had been included in these arrays, we did find that NAMPT expression was moderately elevated in all three cases of benign prostate hyperplasia on the arrays (P=0.048). Together, these data suggest that NAMPT is overexpressed in prostate cancer, which likely occurs early and increases towards late stages of the malignancy.

NAMPT regulates NAD+ metabolism for prostate cancer cell growth and survival

To determine whether high levels of NAMPT in prostate cancer cells are functionally important, we first examined effects of NAMPT inhibition in prostate cancer cell lines PC3 and LNCaP with NAMPT inhibitor FK866. We found that FK866 inhibited the growth of both PC3 and LNCaP cells in a dose-dependant manner, and 50 nM FK866 dramatically suppressed cell growth after overnight treatment (Figure 2a). The drug treatment depleted cellular NAD+ content (Figure 2b). Using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay coupled with flow cytometry, we found that NAMPT inhibition by 50 nM FK866 induced dramatic apoptosis in both cell lines, with LNCaP cells being more sensitive to the treatment (Figure 2c).

Figure 2
figure2

Effects of NAMPT inhibition by FK866 on prostate cancer cells. (a) XTT cell proliferation assay of PC3 and LNCaP cells treated with FK866. After over-night seeding in triplicate, cells were treated with FK866 with concentrations indicated and analyzed by XTT proliferation assay. (b) Cellular NAD+ level upon FK866 treatment for 48 h. NAD+ level in LNCaP cells dropped below the detectable level after 50 nM FK866. (c) TUNEL assay of apoptosis in PC3 (left) and LNCaP (right) cells treated with 50 nM FK866. One half million cells were seeded overnight and treated with FK866 or mock for days indicated. Cells were collected and fixed for TUNEL labeling and analyzed by flow cytometry. (d) Nicotinic acid (NA) rescue. LNCaP cells were treated with 50 nM FK866 without or with NA at various concentrations indicated. Viable cells were counted 3 days after treatment. (e) NAD+ levels in LNCaP cells treated with FK866, NA or combination.

We then knocked down NAMPT using a lentiviral small hairpin RNA vector to determine the gene specific effects of NAMPT inhibition. Effective gene knockdown was achieved in both PC3 and LNCaP cells (Figure 3a), which resulted in significant reduction of cellular NAD+ level and induced apoptosis (Figures 3b and c). We enriched NAMPT knockdown cells by fluorescence-activated cell sorting for expression of green fluorescent protein from the vector and used these cells for analysis of cell growth. We found that NAMPT knockdown suppressed growth of both PC3 and LNCaP cells, as measured by sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay or cell counting (Figures 3d and e), consistent with FK866 treatment.

Figure 3
figure3

Effects of NAMPT stable knockdown on prostate cancer cell proliferation and survival. (a) Stable knockdown of NAMPT in PC3 and LNCaP cells with lentiviral shNAMPT vectors. Mock knockdown was carried out with a scrambled shRNA. The efficiency of knockdown was analyzed four days after viral transduction. (b) Effect of NAMPT knockdown on cellular NAD+ level. NAD+ was measured 5 days after transduction of PC3 cells. (c) NAMPT knockdown increased the spontaneous apoptosis of PC3 cells measured by TUNEL assay. Cells were analyzed 4 days after transduction. (d) Growth of LNCaP (left) and PC3 (right) cells after NAMPT knockdown analyzed by XTT assay. A total of 4000 FACS-sorted GFP positive cells were seeded in each well in triplicate in a 96-well plate for the assay. (e) Growth of LNCaP (left) and PC3 (right) cells after NAMPT knockdown analyzed by viable cell counts with the method of trypan-blue dye exclusion. GFP positive cells, 1 × 105 per well, were seeded in duplicate in a 6-well plate for the assay.

The effect of NAMPT inhibition can be rescued by repletion of NAD+ through biosynthesis from nicotinic acid (Hasmann and Schemainda, 2003). Consistently, we found that supply of nicotinic acid at as low as 10 μM fully reversed the inhibitory effect of FK866 on cell growth (Figure 2d), in association with restoration of cellular NAD+ contents (Figure 2e). Together, these data indicate that NAMPT is critical for regulating NAD+ metabolism for the growth and survival of both androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells.

NAMPT knockdown suppresses tumor phenotypes of prostate cancer cells

In addition to suppression of cell growth, we found that NAMPT knockdown significantly impaired the ability of soft agar colony formation for both PC3 and LNCaP cells (Figure 4a). Using an invasion chamber assay, we found that NAMPT knockdown reduced PC3 migration through extracellular matrix (Figure 4b). To determine if NAMPT has a role in prostate cancer growth in vivo, mock or NAMPT knockdown PC3 cells were inoculated into immunodeficient mice. The xenografted prostate tumors in NAMPT knockdown group grew significantly slower than those in mock knockdown group (P<0.01 after the first 8 days of growth, Figure 4c). When tumors were harvested at the end of the study, all 10 mice in the control group had grown tumors; in contrast, 4 out of 11 mice in the NAMPT knockdown group did not have tumors and the rest had significantly smaller tumors than those in the control group (P=0.006, Figure 4d). These results indicate that NAMPT knockdown inhibits prostate tumorigenesis in vitro and in vivo.

Figure 4
figure4

NAMPT knockdown suppressed tumor phenotypes of prostate cancer cells. (a) Suppression of anchorage-independent cell growth. After 3 days of mock or NAMPT shRNA transduction, LNCaP (left) and PC3 (right) cells were FACS sorted. GFP positive cells were seeded in triplicate with 500 cells/well in soft agar. Colonies were scored after 20 days of incubation. (b) Suppression of tumor invasion in vitro. At 3 days after transduction, mock or NAMPT knockdown PC3 cells were seeded in the upper migration chambers. Invasive cells in the lower chamber were quantified using a fluorometric assay. (c) Suppression of in vivo tumor growth. Mock or NAMPT shRNA knockdown PC3 cells were xenografted into NOD-SCID mice, 10 mice in the mock group and 11 mice in shNAMPT group. Tumor volume was measured at days indicated. (d) Left, weight of PC3 cell xenografted tumors harvested at the end of experiment from NOD-SCID shown in (c). Right, images of dissected tumors.

NAMPT is crucial for prostate cancer cell response to oxidative and chemotherapeutic stresses

We investigated if NAMPT regulates prostate cancer cell responses to oxidative stress and chemotherapeutic agents. After treatment of mock and NAMPT knockdown PC3 cells with H2O2 for 24 h, we found a significantly higher rate of cell death in NAMPT knockdown cells than in mock knockdown (Figure 5a). Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed that these NAMPT knockdown cells underwent rapid apoptosis upon the treatment (Figure 5b). Knockdown of NAMPT in LNCaP cells also sensitized them to oxidative stress as in PC3 cells (Figure 5c). Similar effects were observed using a second set of NAMPT small hairpin RNA previously described (Yang et al., 2007) (Supplementary Figure 4). To further support findings for NAMPT knockdown, we cloned full-length human NAMPT complementary DNA and generated a NAMPT overexpression lentiviral vector. When overexpressed in LNCaP cells that have a relatively lower level of NAMPT expression than PC3 cells as shown in Figure 1b, NAMPT protected LNCaP cells from oxidative stress (Figure 5e).

Figure 5
figure5

NAMPT knockdown sensitized prostate cancer cells to oxidative stress and chemotherapeutic agents. (a) Effects of NAMPT knockdown on the sensitivity of PC3 cells to H2O2. One half million FACS sorted PC3 cells with NAMPT knockdown were seeded in duplicate in a 6-well plate overnight. Cells were then treated with H2O2 at concentrations indicated for 24 h. Viable cells were counted by trypan-blue dye exclusion. (b) TUNEL assay of apoptosis. NAMPT knockdown PC3 cells seeded on chamber slides were treated with 200 μM H2O2 for 24 h before TUNEL labeling. Apoptotic cells were shown in gold to red as a result of merging TUNEL label TMR red with nuclear DAPI counter stain. (c) NAMPT knockdown sensitized LNCaP cells to H2O2 treatment. Cells were treated as in (a). (d) NAMPT knockdown sensitized PC3 cells to PEITC treatment. After overnight seeding, cells were treated with PEITC for two days, and viable cells were counted. (e) Effects of NAMPT overexpression on LNCaP cells. LNCaP cells were transduced with NAMPT expressing or empty lentiviral vector (mock), sorted for GFP expression, and used for H2O2 treatment with 2 × 105 cells seeded per well. The level of NAMPT overexpression was shown in the left panel.

To determine if NAMPT inhibition sensitizes prostate cancer cells to chemotherapeutic agents, we treated mock and NAMPT knockdown cells with paclitaxel, etoposide or phenylethyl isothiocyanate (PEITC). We found that NAMPT knockdown sensitized PC3 cells to these chemotherapeutic agents with the most dramatic effect on PEITC (Figure 5d and Supplementary Figure 5). PEITC induces cancer cell apoptosis by disabling glutathione antioxidant system, causing severe accumulation of cellular reactive oxygen species and oxidative mitochondrial damage (Trachootham et al., 2006; Xiao et al., 2006). The significant effect of NAMPT knockdown on PEITC killing of prostate cancer cells resembles the effect of H2O2 treatment described above. Together, these data suggest that NAMPT enhances prostate cancer-cell survival under oxidative and chemotherapeutic stresses.

SIRT1 is a key downstream target of NAMPT for prostate cancer cell growth and survival

SIRT1 has a prominent role for mammalian cell survival under stress and is a key downstream effector for NAMPT in numerous physiological settings as described above. SIRT1 is overexpressed in primary human prostate cancer and mouse models of prostate cancer (Huffman et al., 2007; Jung-Hynes et al., 2009). However, roles of SIRT1 in prostate cancer remain controversial as two groups show that SIRT1 promotes prostate cancer cell growth and survival (Kojima et al., 2008; Jung-Hynes et al., 2009) whereas the other two groups show the opposite effect (Fu et al., 2006; Dai et al., 2007). To examine roles of SIRT1 in prostate cancer cells, we first treated LNCaP and PC3 cells with a sirtuin inhibitor, sirtinol (Grozinger et al., 2001). We found that sirtinol robustly inhibited cell growth and induced apoptosis (Figures 6a and b), similar to NAMPT inhibitor FK866. Using lentiviral small hairpin RNA, we knocked down SIRT1 and found that it did not obviously change cellular NAD+ level (Figure 6c). However, the knockdown reduced proliferation of LNCaP and PC3 cells, but to a less extent than NAMPT knockdown (compared Figure 6d versus Figure 3d). Similar to NAMPT knockdown, SIRT1 knockdown significantly inhibited soft agar colony formation of both LNCaP and PC3 cells (Figure 6e). SIRT1 knockdown also sensitized cells to H2O2 treatment (Figure 6f). H2O2 moderately reduced the cellular NAD+ level, but SIRT1 knockdown did not result in further reduction (Figure 6c). Our results are in-line with previous findings that SIRT1 is important for prostate cancer-cell survival and growth (Kojima et al., 2008; Jung-Hynes et al., 2009).

Figure 6
figure6

Effects of SIRT1 inhibition on prostate cancer cells. (a) XTT assay of PC3 and LNCaP cells treated with SIRT1 inhibitor sirtinol at concentrations and days indicated. A total of 2000 cells were seeded in each well in triplicate in 96-well plate for the assay. (b) TUNEL analysis of apoptosis in PC3 and LNCaP cells 2 days after the treatment with 50 μM sirtinol. (c) SIRT1 knockdown (top) and NAD+ levels (bottom). NAD+ was measured with or without 200 μM H2O2 treatment for 12 h. (d, e) Effects of SIRT1 knockdown on proliferation (d) and soft agar colony formation (e) of LNCaP (top panels) and PC3 (bottom panels) cells. (f) Oxidative stress response after SIRT1 knockdown in PC3 cells. SIRT1 knockdown cells were treated with H2O2 and analyzed as described for Figure 5a.

We next characterized SIRT1 functions downstream of NAMPT. We found that NAMPT knockdown reduced cellular sirtuin enzymatic activity in PC3 cells (Figure 7a). NAMPT inhibition by FK866 or gene knockdown induced acetylation of SIRT1 downstream effectors p53 and FOXO1 (Figure 7b), two cell survival regulators, indicating an important role of SIRT1 downstream of NAMPT. We found that overexpression of SIRT1 had only limited effect on protecting PC3 cells upon NAMPT knockdown (Supplementary Figure 6), which may be attributed to the inactiveness of the overexpressed SIRT1 upon blockage of NAD+ biosynthesis and simultaneous accumulation of NAM. In-line with this notion, double knockdown of SIRT1 and NAMPT did not further reduce cell survival compared with NAMPT knockdown alone (Supplementary Figure 7). Therefore, we examined effects of NAMPT overexpression in LNCaP cells followed by SIRT1 knockdown. We found that SIRT1 knockdown significantly blocked proliferation and survival advantage conferred by NAMPT overexpression over time with or without 100 μM H2O2 treatment (Figure 7c). Similar results, but to less extent, were observed in PC3 cells (Supplementary Figure 8). Together, these studies suggest that SIRT1 is an important downstream effector of NAMPT in regulating prostate cancer-cell survival and proliferation, in particular, when cells are under oxidative stress.

Figure 7
figure7

SIRT1 is an important downstream target of NAMPT in prostate cancer cells. (a) Measurement of sirtuin deacetylase activity in PC3 cells upon NAMPT knockdown. HDAC activity was blocked with 800 nM trichostatin A. (b) Effects of NAMPT inhibition on acetylation of SIRT1 substrates. LNCaP cells were analyzed after treatment with 50 nM FK866 for 48 h, or 5 days after NMAPT knockdown. (c) Proliferation of LNCaP cells co-transduced with empty expression vector(C) plus scrambled shRNA vector (C-Scr), empty expression vector plus shSIRT1 (C-shSIRT1), NAMPT overexpression vector(NAMPT) plus scrambled shRNA vector (NAMPT-Scr) or NAMPT overexpression vector plus shSIRT1 (NAMPT-shSIRT1). Top: no H2O2 treatment; Bottom: treatment with 100 μM H2O2.

NAMPT regulates FOXO3a expression for oxidative stress response in prostate cancer cells

To gain further insight of NAMPT regulating oxidative stress response of prostate cancer cells, we analyzed expression of anti-oxidant genes. We found that NAMPT knockdown significantly downregulated RNA levels of catalase (CAT) in both PC3 and LNCaP cells and manganese superoxide dismutase (SOD) in PC3 cells, whereas expression of several other anti-oxidant genes was unchanged (Figure 8a). Consistent with decreased CAT and SOD gene expression, the level of reactive oxygen species was significantly increased in PC3 cells upon NAMPT inhibition (Figure 8b).

Figure 8
figure8

NAMPT regulates FOXO3a expression for oxidative stress response in prostate cancer. (a) Expression of anti-oxidant genes. Total RNA extracted from scrambled (SCR) and shNAMPT transduced PC3 (left) and LNCaP (right) cells were analyzed by reverse transcription followed by real time PCR for different anti-oxidant genes using 18S rRNA as a control. Asterisk (*) indicates significant difference, P<0.01 by t-test. GST (glutathione S-transferase), GPX (glutathione peroxidase), SPS2 (selenophosphate synthetase 2), PRX3 (peroxiredoxin 3), and TRX1 (thioredoxin 1) had no significant change upon NAMPT inhibition. (b) Cellular ROS was measured in cultured PC3 cells treated with DMSO (left panel) and 10 nM FK866 (right panel) by flow cytometry. (c) FOXO3a protein level in PC3 cells 4 days after inducible SIRT1 knockdown by doxycycline (Dox). (d) FOXO3a protein level in PC3 cells 5 days after NAMPT knockdown. (e) FOXO3a protein levels in PC3 cells 3 days after treatment with DMSO, FK866 or Tenovin-6 at indicated concentrations.

FOXO3a is a direct transcriptional activator of CAT and SOD promoters (Kops et al., 2002; Nemoto and Finkel, 2002), and is highly expressed in prostate cancer cell lines (Modur et al., 2002). Whereas nuclear localization of FOXO3a is seen in normal prostate cells, increasing cytoplasmic accumulation of FOXO3a occurs in more advanced prostate cancer (Shukla et al., 2009). SIRT1 interacts with and deacetylates FOXO3a, which increases or decreases expression of different sets of FOXO3a target gene in response to oxidative stress (Brunet et al., 2004; Motta et al., 2004). SIRT1 can bind to FOXO3a promoter and presumably repress its transcription in PC3 and LNCaP cells (Kikuno et al., 2008). However, we found that SIRT1 knockdown did not affect FOXO3a protein expression (Figure 8c). In contrast, NAMPT knockdown significantly reduced FOXO3a expression in PC3 cells (Figure 8d). Similar effect was seen with treatment of NAMPT inhibitor FK866, but not SIRT1 inhibitor tenovin-6 (Lain et al., 2008) (Figure 8e). Interestingly, NAMPT inhibition did not change FOXO3a RNA level, but FOXO3a acetylation increased slightly (Supplementary Figure 9), indicating likely the involvement of NAD+-dependent posttranslational regulation of FOXO3a protein stability. Our results suggest that although SIRT1 is an important downstream effector of NAMPT for prostate cancer-cell survival under oxidative stress, NAMPT may also mediate a novel stress response pathway by regulating FOXO3a protein level, which does not require SIRT1.

Intersection of NAMPT with other NAD+-dependent cellular functions

NAMPT-regulated NAD+ metabolism is expected to impact many cellular pathways. Poly ADP-ribose polymerases (PARPs) are among the major cellular NAD+ consumers and have key roles in regulating cell survival (Schreiber et al., 2006). In response to DNA damage, PARP-1 use NAD+ to catalyze polymerization of ADP-ribose on acceptor proteins involved in damage repair. PARP inhibitors have been explored to kill cancer cells bearing defective DNA damage repair (Gartner et al., 2010). To examine if NAMPT inhibition could have additive or synergistic effect with PARP inhibition, we treated PC3 cells with a potent PARP inhibitor AZD2281. AZD2218 alone did not inhibit cell growth; surprisingly, when combined with FK866, AZD2281 antagonized the cell killing effect of FK866 in the absence or presence of H2O2, even though it did not restore cell growth (Figure 9a and Supplementary Figure 10). The effect of AZD2281 was not a result of restoration of cellular NAD+ level (Figure 9b), and is thus distinct from nicotinic acid rescue shown in Figure 2. PARP1-mediated synthesis of poly ADP-ribose in response to DNA damage can trigger release of apoptosis inducing factor from mitochondria to induce apoptosis, and PAPR1 inhibition may block apoptosis in certain cells (Yu et al., 2006). However, the antagonizing effect of AZD2281 occurs without PARP1 activation and there is insufficient cellular NAD+ for poly ADP-ribose reaction, suggesting the likely involvement of additional mechanisms for cell survival.

Figure 9
figure9

Intersection of NAMPT inhibition with other NAD+-dependent processes. (a, b) PC3 cells were treated with 20 nM FK866, 1 μM AZD2281 or combination of the two for 72 h. Viable cells (a) and NAD+ levels (b) were analyzed. (c) Glucose concentrations in PC3 cell culture medium after treatment with FK866 at indicated concentrations for 24 and 48 h. Cells continued consuming glucose after 24 h, and was not altered by the drug. (d) ATP levels in PC3 cells after FK866 treatment for 24 and 48 h.

Finally, we examined the impact of NAMPT inhibition on cellular energy production given that NAD+ is essential for glycolysis and oxidative phosphorylation. We found that treatment of FK866 did not result in significant change of glucose intake (Figure 9c), and cellular ATP levels were reduced only after 48 h of the drug treatment (Figure 9d). The change of ATP levels was not FK866-concentration dependent, and occurred later than XTT reading change that occurred after 24 h of treatment (Figure 2a). Therefore, the change of energy production may not be a driving event for growth suppression and survival of prostate cancer cells upon NAMPT inhibition.

Discussion

NAMPT is a critical rate-limiting enzyme of the NAD+ salvage pathway for numerous cellular functions including regulation of the SIRT1, but roles of NAMPT in cancer are poorly understood. Our study now demonstrates that NAMPT is upregulated in various cancer cells to efficiently recycle NAM to replenish NAD+ supply. NAMPT has a prominent role in prostate cancer, and its expression is upregulated early during prostate carcinogenesis and further increased towards late stages. NAMPT may promote prostate cancer-cell survival and growth through multiple cellular processes; among them, SIRT1 and FOXO3a are important downstream effectors for NAMPT functions.

Our work shows that SIRT1 promotes prostate cancer cell growth and survival, consistent with its overexpression in primary human and mouse prostate cancers (Huffman et al., 2007; Jung-Hynes et al., 2009). Our results are in-line with findings from other groups (Kojima et al., 2008; Jung-Hynes et al., 2009). Our study provides further evidence that in addition to increased SIRT1 expression during prostate carcinogenesis, concomitant increase of NAMPT for NAM recycling and replenishing NAD+ to maintain SIRT1 enzymatic activity is also required for prostate cancer-cell survival and growth, shedding novel insight on coordination of these two genes for tumorigenesis.

However, it should be noted that the role of SIRT1 in tumorigenesis is controversial. As described above, both tumor promoter and suppressor functions have been proposed for SIRT1 in prostate cancer. Recent mouse studies have not resolved such controversy. First, SIRT1 overexpression is shown to suppress intestinal polyp formation in Apcmin/+ mice (Firestein et al., 2008), suggesting a tumor suppressor function. In contrast, the SIRT1 homozygous knockout fails to increase intestinal polyp load in Apcmin/+ mice (Boily et al., 2009). Second, Oberdoerffer et al. (2008) show that SIRT1 overexpression or resveratrol treatment reduces thymic lymphoma in p53+/− background. However, noticeable increase of adenocarcinoma, leukemia and sarcoma occurs with SIRT1 overexpressing or resveratrol treated mice (Oberdoerffer et al., 2008), and it is unclear whether this is a tissue-dependent consequence of SIRT1 overexpression and non-specific drug effects. If SIRT1 overexpression were to mimic effects of calorie restriction, then the phenotypes observed by Oberdoerffer et al are different from calorie restriction that delays tumor formation but does not change tumor spectrum and incidence in p53+/− mice (Berrigan et al., 2002). Third, Wang et al. demonstrate that heterozygous loss of SIRT1 drastically accelerates tumorigenesis in p53+/− background (Wang et al., 2008). Surprisingly, in the study by Wang et al., the control p53+/− mice develop few tumors over 20 months of observation, which is in sharp contrast to numerous studies showing that p53+/− mice are highly susceptible to tumorigenesis within the similar time frame (Harvey et al., 1993; Venkatachalam et al., 1998; Cressman et al., 1999; Berrigan et al., 2002; Chen et al., 2004). It is unclear if the accelerated tumorigenesis observed by Wang et al. is an outcome of specific genetic background. Wang et al. also suggest that SIRT1 is a haploinsufficient tumor suppressor (Wang et al., 2008). However, no notable tumor formation has been reported in two aging studies using SIRT1−/− mice over 18 to 24 months (Boily et al., 2008; Li et al., 2008) and SIRT1 homozygous knockout does not increase intestinal tumors in Apcmin/+ mice (Boily et al., 2009). Therefore, more in vivo studies are needed for comprehensive understanding of roles of SIRT1 in cancer.

Our finding that SIRT1 is an important downstream effector of NAMPT for oxidative stress in prostate cancer cells is in agreement with the previous finding in normal human vascular smooth muscle cells (van der Veer et al., 2007). However, our study does not exclude the contribution of other sirtuins. Interestingly, mitochondrial SIRT3 and SIRT4, but not nuclear SIRT1, are essential for NAMPT-mediated cell survival in fibrosarcoma HT1080 and renal cancer HEK293 cell lines under genotoxic stress (Yang et al., 2007). This difference suggests potential influence of tumor cell types on the choice of survival genes. Future studies will further determine the roles of mitochondrial sirtuins for prostate cancer-cell survival.

Human cancers have altered metabolism and depend heavily on glycolysis of glucose, the Warburg's effect (Warburg, 1931). However, prostate cancer is metabolically distinct from other human cancers, and it does not significantly rely on glucose (Liu, 2006; Turkbey et al., 2009). Normal prostate glandular epithelial cells secrete large amount of citrate into the prostate fluid, which results in truncated Krebs cycle and low mitochondrial activity. In contrast, prostate cancer reverses this process and retains citrate for active mitochondrial biogenesis (Costello and Franklin, 2006). The active mitochondrial biogenesis accounts for the production of 90% intracellular reactive oxygen species (Balaban et al., 2005). Therefore, prostate cancer may evolve a strong anti-oxidant defense system. However, a key anti-oxidant gene for prostate epithelial cells, glutathione S-transferase Pi, is inactivated by promoter hypermethylation during prostate carcinogenesis (Lee et al., 1994) and molecular mechanisms of prostate cancer cells regulate oxidative stress are not fully understood. Our finding that NAMPT and SIRT1 inhibition significantly sensitizes prostate cancer cells to oxidative damaging agents (H2O2 and PEITC), suggests they may have important roles for regulating oxidative stress response of prostate cancer cells. Surprisingly, we find that inhibition of NAMPT, but not SIRT1, reduces FOXO3a expression, and thus anti-oxidant genes CAT and SOD. FOXO3a accumulation in cytoplasm becomes increasingly evident with prostate cancer progression (Shukla et al., 2009). Upon oxidative stress, FOXO3a translocates from cytoplasm to nucleus to activate anti-oxidant and survival genes (Brunet et al., 2004; Karger et al., 2009). We speculate that cytoplasmic FOXO3a may serve as a crucial anti-oxidant reservoir in prostate cancer and this FOXO3a stocking may be a NAD+ dependent process and regulated by NAMPT. FOXO3a protein can be degraded by AKT-triggered ubiquitination–proteasome pathway (Plas and Thompson, 2003) or by caspase-3-like proteases in hematopoietic cells (Charvet et al., 2003). Recently, it is shown that N-terminal acetylation of cellular proteins creates specific degradation signals targeted by ubiquitin-dependent proteasome pathway (Hwang et al., 2010). As the NAMPT inhibition reduces sirtuin deacetylase activity, it is possible that N-terminal acetylation of certain proteins may be increased for degradation. It remains to be determined if NAMPT may be involved in the above pathways for FOXO3a degradation in prostate cancer. Nonetheless, our study suggests that SIRT1 and FOXO3a have important roles for NAMPT-mediated survival and anti-oxidative stress response of prostate cancer cells.

Our observation that the NAMPT expression is elevated in benign prostate hyperplasia is also interesting. Chronic inflammation in the prostate gland, which produces reactive oxygen species, is an important etiological factor for prostate carcinogenesis (Nelson et al., 2003; De Marzo et al., 2007). Intriguingly, NAMPT is an inflammation-response gene, and its expression increases in multiple inflammatory tissues (Ognjanovic et al., 2001; Ye et al., 2005; Busso et al., 2008). That NAMPT expression is elevated early in prostate neoplasia suggests NAMPT may have a role in the etiology of prostate cancer. Inhibition of NAMPT could thus be chemoprotective for the prostate gland. It will be of great interest to further decipher roles of NAMPT-mediated pathways and sirtuins for biology of prostate carcinogenesis in future, which may have implication for prevention and treatment of prostate cancer.

Materials and methods

Immunohistochemistry analysis

Multi-tissue arrays were kindly provided by National Cancer Institute or purchased from Cybrdi, Inc. (Rockville, MD USA). We validated the specificity of an affinity purified rabbit NAMPT antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) by western blot analysis using normal human liver cell lysate and liver cancer cell line HepG2 as positive controls in which NAMPT is known to be expressed abundantly (Samal et al., 1994; Hasmann and Schemainda, 2003). To check antibody specificity for prostate tissue and tumor, we collected prostate cancer and normal prostate tissue from the same patient by histology-guided biopsy punch of frozen tissue blocks. The use of human tissues was approved by Institutional Review Board. Tissues were ground with RIPA buffer using a standard protocol and soluble fractions were used for western analysis. In all cases, the antibody detected a predominant band or one single band in the positive controls, several cancer cell lines, and primary prostate tissue and tumor (Supplementary Figure 2). The antibody specificity was further confirmed by NAMPT gene knockdown as described in the main text.

For tissue array immunohistochemistry, the embedded liver sections were used as a positive control (Supplementary Figure 3), and normal rabbit IgG was used as a negative staining control. Tissue slides were deparaffinized and rehydrated following standard methods. Antigen retrieval was achieved by incubating in 95 °C citrate buffer (10 mM sodium citrate) for 20 min followed by cooling for 20 min. For detecting NAMPT, tissue sections were incubated with an anti-NAMPT antibody (Bethyl; 1:200 dilution) overnight at 4 °C, followed by treatment with the Universal LSAB plus HRP Kit (Dako North America, Inc. Carpinteria, CA, USA) as per the manufacturer's instructions. Slides were then incubated with 0.05% (w/v) 3,3′-diaminobenzidine (Sigma-Aldrich, St Louis, MO, USA) for about 3 min, and counterstained with Mayer's hematoxylin (Sigma-Aldrich).

Tumor xenograft study

Animal studies were approved by the Institutional Animal Care and Use Committee. PC3 cells were transduced with scrambled (mock) or NAMPT lentiviral small hairpin RNA with multiplicity of infection of four. Three days after transduction, five million cells each suspended in 0.1 ml PBS were inoculated subcutaneously into the right flank of mice using a 27-gauge needle. Male non-obese diabetic severe combined immunodeficiency mice at 6 to 8 weeks of age were conditioned by γ-irradiation at the dose of 270 rads before tumor xenograft. Mice were ear-tagged and monitored for tumor volume. Tumor length (L) and width (W) were measured every 8 days with a caliper, and tumor volume were estimated for each time point from the formula, V=4/3 *π*[(L+W)/4]3. Growth curves for tumor volume over time for each tumor implant were plotted. Mice were euthanized after 40 days and tumors were harvested and weighed.

More Materials and methods are provided in Supplementary Information online.

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Acknowledgements

This study is supported by the start-up fund from City of Hope, career development award from STOPCANCER foundation and research scholar award from American Cancer Society (WYC). EA was supported by a training grant awarded to CSULB from California Institute of Regenerative Medicine.

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Wang, B., Hasan, M., Alvarado, E. et al. NAMPT overexpression in prostate cancer and its contribution to tumor cell survival and stress response. Oncogene 30, 907–921 (2011). https://doi.org/10.1038/onc.2010.468

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Keywords

  • prostate cancer
  • NAMPT
  • SIRT1
  • NAD
  • nicotinamide
  • Foxo3a

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