Abstract A33: CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+
Authors: Chmielewski Jeffrey P. , Wheeler Frances , Cramer Scott , Lihong Shi , Sirintrapun Joseph , Kridel Steven J. 
Published: May 2016

Abstract
In order to maintain high rates of proliferation, tumor cells must alter their metabolic machinery in favor of increased macromolecule synthesis and energy production. A key factor in tumor cell metabolism is Nicotinamide Adenine Dinucleotide, NAD+. NAD+ is used as a cofactor for catabolic reactions (NAD(H)), or after conversion to NADP(H), for anabolic reactions. In addition, NAD+ is utilized as part of the catalytic mechanism for several classes of enzymes including the sirtuins, PARPs, and CD38. Given the juxtaposition of NAD+ to cell metabolism and other critical cellular process we hypothesize a balance between synthesis and consumption of NAD+ is a primary metabolic determinant in cancer. In prostate cancer, the primary pathway by which NAD+ is synthesized is via the salvage pathway, which recycles nicotinamide into Nicotinamide Mononucleotide (NMN) via the rate-limiting enzyme Nicotinamide Phosphoribosyltransferase (Nampt). We determined the metabolic consequences of Nampt blockade in prostate cancer and glioblastoma (GBM) cell lines using the pharmacological inhibitor FK866. As expected, treatment with FK866 decreases the levels of NAD+/NADP+ and the NAD+ precursor nicotinamide. In relation to glycolysis, there were increases in metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, which requires NAD+ as a cofactor. Specifically, glucose is increased 1.6 fold in PC3 cells (p≤0.05), glucose-6-phosphate is decreased 1.7 fold SNB19s (p≤0.05), and both lines have decreased fructose-1-phosphate levels between 6 and11 fold (p≤0.05), respectively. To compliment these changes we measured the effects of Nampt inhibition on glycolysis in real time using a Seahorse XF extracellular flux analyzer. Nampt inhibition lowers basal glycolysis 40% (p&lt;0.01) in prostate cancer (PCa) cells and glycolytic capacity 46% (p&lt;0.01) and 64% (p&lt;0.01) in PCa and glioblastoma (GBM) cells, respectively. Reactions requiring NAD+ as a cofactor in the TCA cycle were similarly perturbed. Specifically, in both cell lines fumarate and malate were increased between 1.5 and 2 fold (p&lt;0.05) resulting from reduced malic enzyme activity. Corresponding to altered TCA cycle metabolites there was a 45% decrease in spare mitochondrial respiratory capacity (p&lt;0.01) in PCa cells and a 90% decrease in GBM cells (p&lt;0.01), relative to vehicle. Together these data suggest blockage of NAD+ synthesis is sufficient to perturb the activity of NAD+ dependent metabolic enzymes and induce energetic stress. Of the three classes of NAD+ consuming enzymes, the cADP-ribose synthase CD38 is the primary NAD'ase in cells. Western blot analysis of normal prostate epithelial cells (PrEC) and several prostate tumor cell lines demonstrates expression of CD38 is nearly non-existent in PCa. In line with this, CD38 mRNA levels are decreased 2-fold across 31 independent PCa samples when compared to mRNA from 8 normal prostate samples (p=0.007). CD38 expression levels were also determined by immunohistochemistry (IHC) in primary PCa tissue micro-arrays (TMA). In a total of 91 samples: 37 benign, 17 prostatic intraepithelial neoplasia (PIN), and 37 Cancers, CD38 staining intensity was reduced 20% in PIN samples (p≤0.001) and 50% in cancer samples (p≤0.01). In these experiments, no change in Nampt protein expression, mRNA levels, or staining intensity was detected. Induction of CD38 expression in prostate cancer cell lines resulted in blocked cell proliferation. CD38 expression also reduced NAD levels by 54% and 91% after 48 and 96 hours, respectively (p≤0.01). Concomitant with decreased NAD levels, CD38 expression also reduced spare mitochondrial respiratory capacity 24% (p=0.026), mimicking the effects of Nampt inhibition. Fatty acid synthesis was similarly inhibited following CD38 expression. When taken together, these data suggest an interplay between Nampt and CD38 regulates total NAD+ levels, ultimately supporting the metabolic reprogramming associated with prostate cancer.
Citation Format: Jeffrey P. Chmielewski, Frances Wheeler, Scott Cramer, Shi Lihong, Joseph Sirintrapun, Steven J. Kridel. CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A33.