Abstract 4386: Cytoplasmic NAD+inhibits mRNA translation in ovarian cancers

Abstract

Abstract Introduction: NAD+ is a key metabolite involved in redox reactions and energy production, supporting important biological pathways, such as cell growth, proliferation, and metastasis. In addition, NAD+ regulates important cellular processes, such as gene transcription, by acting as a substrate for enzymes including PARPs and Sirtuins. PARP enzymes are the major consumers of NAD+; they transfer the ADP-ribose moiety from NAD+ to their substrate proteins. We recently showed that nuclear PARP activity is differentially regulated by compartmentalized synthesis of NAD+ by the nuclear and cytosolic NAD+ synthases, NMNAT-1 and NMNAT-2, respectively. While the role of the nuclear PARPs, PARP-1 and PARP-2, in catalyzing polyADP-ribosylation (PARylation) is well understood, little is known about the function of cytosolic PARPs, which catalyze monoADP-ribosylation (MARylation). Methods: We utilized several biochemical and genomic techniques to understand the role of MARylation in ovarian cancer. We performed cell fractionation and confocal microscopy assays to detect the localization of MARylation in OVCAR3 ovarian cancer cells. We also used a genetically encoded NAD+ biosensor to measure changes in nuclear and cytosolic compartment-specific NAD+ levels upon NMNAT-1 or NMNAT-2 depletion by shRNAs. In addition, we used puromycin incorporation assays to determine the amount of protein synthesis, and Polysome-sequencing to identify the changes in mRNA translation upon NMNAT-2 knockdown. Finally, we used Thioflavin T staining to measure protein aggregation and performed cell growth assays to demonstrate the functional effects of cytosolic NAD+ depletion on ovarian cancer phenotypes. Results: In this study, we observed that NMNAT2 expression is upregulated in ovarian cancer tissues and that high NMNAT2 expression correlates with poor progression-free survival in ovarian cancer patients. Since cytosolic PARPs predominantly catalyze MARylation of target proteins involved in mRNA regulation, we hypothesized that NMNAT-2 depletion restricts the NAD+ available for the cytosolic PARPs, and alters mRNA function. Indeed, our results show that NMNAT-2 knockdown inhibits MARylation of cytosolic proteins in ovarian cancer cells without affecting nuclear PARylation. Furthermore, NMNAT-2 knockdown results in a higher mRNA association with polysomes, increased mRNA translation, and a paradoxical reduction in cell growth. Intriguingly, we found that the increased load of protein synthesis upon NMNAT-2 knockdown leads to aggregation of proteins, resulting in proteotoxicity. Summary: Collectively, in this study, we identified a role for NMNAT-2 in the regulation of cytosolic MARylation and ovarian cancer phenotypes. We found that ovarian cancer cells depend on NMNAT-2 to maintain high levels of cytosolic NAD+, which regulate proteostasis by inhibiting protein synthesis. Supported by grants from the NIH/NIDDK and CPRIT to W.L.K. Citation Format: Sridevi Challa, Keun W. Ryu, Tulip Nandu, William L. Kraus. Cytoplasmic NAD+inhibits mRNA translation in ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4386.