(528f) Metabolomics Reveals That Inhibition of Nucleotide Synthesis Underlies Senescence of Human Mammary Epithelial Cells

Cellular senescence in mammalian cells causes an irreversible cell cycle arrest. The senescent phenotype is thought to protect against cancer and can be induced by diverse stimuli including the extensive shortening of telomeric DNA that occurs after ~50 cell divisions. This intrinsic cellular mechanism is known as replicative senescence. To develop a detailed molecular understanding of replicative senescence, we used primary human mammary epithelial cells (HMEC) and quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. In cells undergoing replicative senescence, quantitative metabolomics revealed an accumulation of nucleosides and a depletion of deoxyribonucleoside triphosphates. To gain insight into the metabolic fluxes that underlie these changes in metabolite pool size, we cultured cells with stable isotope-labeled nutrients including [U-13C]-glucose, [1,2-13C]-glucose and [U-13C]-glutamine. Stable isotope tracing showed a consistent blockage of both glucose- and glutamine-derived biomass into nucleotide synthesis pathways in senescent cells. Notably, metabolic fluxes remained unchanged for other cell essential pathways including the TCA cycle. To test whether cellular immortalization would reverse these observations, we expressed telomerase in HMEC. In addition to preventing senescence, telomerase expression restored metabolic flux from glucose into nucleotide synthesis pathways. Finally, we tested the role of nucleotide synthesis in young, proliferating cells. Treatment of proliferating HMEC with an inhibitor of ribonucleotide reductase (RNR), the enzyme that catalyzes the formation of deoxyribonucleoside diphosphates from nucleoside diphosphates, induced senescence with concomitant decreased nucleotide pool size and metabolic flux from glucose. Taken together, our results suggest that downregulation of nucleotide synthesis plays a causative role in establishment of replicative senescence in human mammary epithelial cells. This work will enable future studies into how cancer cells avoid senescence and development of therapeutics targeting pathways that allow tumor cells to escape from senescence.