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Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells - PubMed

Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells

Sarah C Bowlby et al. PLoS One. 2012.

Abstract

Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+)), a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+) in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+)-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt) in maintaining de novo lipogenesis in prostate cancer (PCa) cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC) lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK) and phosphorylation of acetyl-CoA carboxylase (ACC). In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nampt regulates NAD+ levels and survival in PCa cells

(A) Nampt protein levels were determined by western blotting in PREC and PCa cells. (B) Nampt localization was also determined in the cytoplasmic and mitochondrial fractions of PCa cells by western blot, with COX-IV as a mitochondrial marker (C) Total NAD levels were measured in PCa cells after 48 hour treatment with vehicle (DMSO 0.1%), or FK866 (100 nM) in the absence or presence of NAD+ (100 µM) (*p<0.01). (D) Total NADP levels were measured in PC-3 cells after 48 hour treatment with vehicle (DMSO 0.1%), or FK866 (100 nM) in the absence or presence of NAD+ (100 µM) (*p<0.01). (E) The effect of FK866 on survival was assessed by trypan blue exclusion in PREC, LNCaP, and PC-3 cells after 48 hours of treatment. (F) Clonogenic survival of PC-3 and DU145 cells was measured following a dose-response of FK866, or 100 nM FK866 plus NAD+ (100 µM) for 24 hours (*p<0.01). (G) Expression of Naprt1 and β-actin were determined by western blot. The ability of NMN and Na to protect cells from FK866 was determined by trypan blue exclusion in LNCaP cells (48 hour treatment) and clonogenic survival in PC-3 and DU145 cells (24 hour treatment).

Figure 2
Figure 2. Nampt activity is required for fatty acid synthesis in cancer cells

(A) PCa cells were treated with FK866 (100 nM) in the absence or presence of NAD+, NMN, or Na for 48 hours and fatty acid synthesis was determined by the incorporation of 14C-acetate into the lipid (*p<0.0001). Expression of ACC, AceCS1, FASN, and β-actin was determined by western blot. (B) Fatty acid synthesis was measured in Snb-19 glioblastoma cells, Src-transformed 3T3 fibroblasts, and MCF-7 breast cancer cells as in (A) (*p<0.0001). (C) PC-3 cells were transfected with scrambled or Nampt-targeting siRNA (100 nM) and fatty acid synthesis was assayed 5 days post-transfection (*p<0.0001). (D) PC-3 cells were treated as indicated and the fatty acid profile was determined by GC-MS.

Figure 3
Figure 3. Nampt activity is required for phosphatidylcholine (PC) synthesis in cancer cells

(A) PCa cells were treated for 48 hours with vehicle, or FK866 (100 nM) in the absence or presence of NAD+ (100 µM) and lipid synthesis was assessed by 14C-choline incorporation (*p<0.0001). (B) PC-3 cells were transfected with scrambled or Nampt-targeting siRNA (100 nM) and lipid synthesis was assayed 5 days post-transfection (*p<0.0001). (C) PC-3 cells were treated as indicated and the PC profile was determined by mass spectrometry (*p<0.05).

Figure 4
Figure 4. Nampt inhibition does not affect the phosphatidylserine (PS), phosphatidylethanolamine (PE), or phosphatidylinositol (PI) lipid classes.

(A–C) PC-3 cells were treated with vehicle, FK866 (100 nM) in the absence or presence of NMN (500 µM) for 48 hours and the PS, PE, and PI profiles were determined by mass spectrometry (*p<0.05).

Figure 5
Figure 5. Inhibition of Nampt activates AMPK signaling

(A) Prostate tumor cells were treated with vehicle, FK866 (10 nM or 100 nM), or FK866 (100 nM) plus NAD+, Na or NMN. After 48 hours, ATP levels were measured by luminescence and normalized to DNA content (*p<0.0001). (B) The levels of pACC and ACC were determined in cells treated with vehicle (0.1%), or FK866 (100 nM) in the absence or presence of NAD+ for 48 hours (100 µM). (C) PC-3 cells were treated with vehicle, FK866 (100 nM), Compound C (CC, 10 µM), or the combination of both (FK+CC) for 48 hours and pACC, ACC, pAMPK, and AMPK levels were determined by western blot. (D) PC-3 cells were treated with vehicle, FK866, Compound C, or the combination of both for 48 hours and fatty acid synthesis was measured (*p<0.0001, #p = 0.0007). Cell killing was also determined by trypan blue exclusion (*p<0.0001).

Figure 6
Figure 6. Sirtuin activity is required for lipogenesis in prostate cancer cells

(A) PCa cells were treated as indicated for 48 hours and global protein acetylation was measured by western blot. (B) PCa cells were treated with DMSO (0.1%) or Sirtinol (100 µM) for 48 hours and 14C-acetate and -choline incorporation into lipid were measured (*p<0.0001). (C) PC-3 cells were treated with scrambled, SIRT1, or SIRT3 targeting siRNA (100 nM each) and fatty acid synthesis was measured. The expression levels of SIRT1, SIRT3, and β-actin were determined by western blot. (D) PC-3 and LNCaP cells were transfected with scrambled (200 nM) or the combination of SIRT1.2 and SIRT3.2 targeting siRNA (100 nM each). The incorporation of 14C-acetate and -choline into lipid were determined 5 days after transfection (*p = 0.0002). The levels of SIRT1, SIRT3, and β-actin were determined by western blot.

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