Tumor cells require increased rates of cell metabolism to generate the macromolecules necessary to sustain proliferation. They rely heavily on NAD as a cofactor for multiple metabolic enzymes in anabolic and catabolic reactions. NAD also serves as a substrate for PARPs, sirtuins, and cyclic ADP-ribose synthases. Dysregulation of the cyclic ADP-ribose synthase CD38, the main NAD'ase in cells, is reported in multiple cancer types. This study demonstrates a novel connection between CD38, modulation of NAD, and tumor cell metabolism in prostate cancer. CD38 expression inversely correlates with prostate cancer progression. Expressing CD38 in prostate cancer cells lowered intracellular NAD, resulting in cell-cycle arrest and expression of p21 (CDKNA1). In parallel, CD38 diminishes glycolytic and mitochondrial metabolism, activates AMP-activated protein kinase (AMPK), and inhibits fatty acid and lipid synthesis. Pharmacologic inhibition of nicotinamide phosphoribosyltransferase (NAMPT) mimicked the metabolic consequences of CD38 expression, demonstrating similarity between CD38 expression and NAMPT inhibition. Modulation of NAD by CD38 also induces significant differential expression of the transcriptome, producing a gene expression signature indicative of a nonproliferative phenotype. Altogether, in the context of prostate cancer, the data establish a novel role for the CD38-NAD axis in the regulation of cell metabolism and development. This research establishes a mechanistic connection between CD38 and metabolic control. It also provides the foundation for the translation of agents that modulate NAD levels in cancer cells as therapeutics. .
The condensation state of chromosomes is a critical parameter in multiple processes within the cell. Failures in the maintenance of appropriate condensation states may lead to genomic instability, mis-expression of genes, and a number of disease states. During cell proliferation, replication of DNA represents an ongoing challenge for chromosome packaging as DNA must be unpackaged for replication and then faithfully repackaged. An integral member of the DNA replication machinery is the GINS complex which has been shown to stabilize the CMG complex which is required for processivity of the Mcm2–7 helicase complex during S phase. Through examination of the phenotypes associated with a null mutation in Psf2, a member of the evolutionarily conserved GINS complex, we find that Drosophila Psf2 likely has a role in establishing chromosome condensation and that the defects associated with this mis-condensation impact M phase progression, genomic stability, and transcriptional regulation.
Background: Chromatin remodeling is one of the most intriguing features of spermiogenesis, during which nuclei undergo drastic morphological changes leading to extensive chromatin compaction. Genetic and cytological accessibility make Drosophila melanogaster a powerful model to study this process. In fruit flies, paternal histones are largely replaced with sperm specific nuclear basic proteins in a highly coordinated manner. This remodeling is essential not only for sperm function but also for proper behavior of paternal chromosomes in the embryo. Our understanding of the changes associated with sperm chromatin and their role in embryonic chromosome behavior is incomplete, and will depend on the identification and characterization of additional components. One such newly identified gene, versager (vrs), is described here. Methods: Chromosome transmission was genetically monitored from vrs Z2566 males using chromosomespecific visible mutations. Recombination and deletion mapping was used to localize the mutation, and DNA sequencing was used to identify the causative lesion. Both in vivo RNAi expression knockdown and rescue by transgene expression of EGFP-tagged protein were used to verify the gene identity. The developmental expression pattern of Vrs was defined based on the EGFP signal in testis relative to RFP-tagged H2Av expression. Behavior of DAPI-stained chromosomes in early embryos from vrs Z2566 males was examined using confocal microscopy. Results: Genetic observations indicated that vrsZ2566 is required in males for high fidelity transmission of paternally derived chromosomes. DNA sequencing revealed that the vrs Z2566 mutation is a missense mutation in Celera predicted gene CG5538 and results in a D2V amino acid substitution. This residue was found to be conserved in Drosophila species and related Diptera. RNAi knockdown of vrs resulted in paternal-effect chromosome loss, and a vrs + -EGFP transgene fully rescued the mutant phenotype. Confocal microscopy of testis revealed nuclear localization of Vrs-EGFP specifically at the canoe stage of spermiogenesis, overlapping with the time of removal of H2Av-RFP at the histone-to-protamine transition. Examination of early stage embryos revealed micronuclei, isolated chromosomes and bridges indicative of chromosome loss events during the first three divisions. Approximately a quarter of later stage embryos arrested with an abnormal number of metaphase and fragmented nuclei. Conclusions: A novel sperm-specific paternal-effect gene, vrs, was identified that is expressed at the histone-to-protamine transition and is important for embryonic chromosome behavior and development. The histone-to-protamine transition may be a developmental period sensitive to perturbations that may lead to embryonic mitotic errors, aneuploidy and developmental arrest.
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<0.01) in prostate cancer (PCa) cells and glycolytic capacity 46% (p<0.01) and 64% (p<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<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<0.01) in PCa cells and a 90% decrease in GBM cells (p<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.
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