Abstract
Changes in nicotinamide adenine dinucleotide (NAD+) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD+ levels also affect DNA repair capacity as NAD+ is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD+ levels by processing NAD+ and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD+ content and NAD+-dependent DNA repair capacity. Reduced intracellular NAD+ levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD+ reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD+ or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD+ bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD+. A positive correlation between CD73 expression and intracellular NAD+ content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD+ when supplemented with NAD+ or NMN.
Highlights
The tumor microenvironment has the potential to provide critical metabolites to promote tumor cell growth and immune modulation[1] as well as support cellular metabolism via metabolic coupling[2] or metabolic plasticity[3,4]
One of the membrane-bound enzymes which has been suggested to participate in NAD+ precursor uptake and metabolism is CD73 (NT5E), an enzyme that is often upregulated in cancer cells[57,58,92,93]
We decided to investigate the effect of alterations in NAD+ status on DNA damage and efficiency of DNA repair mechanisms including the CD73 enzyme as a target which could affect global NAD+ pools in cancer
Summary
The tumor microenvironment has the potential to provide critical metabolites to promote tumor cell growth and immune modulation[1] as well as support cellular metabolism via metabolic coupling[2] or metabolic plasticity[3,4]. As NAD+ is a substrate for the DNA repair and DNA damage response signaling enzymes PARP1, PARP2 and PARP325, fluctuations in the cellular levels of NAD+ can influence DNA repair mechanisms[26], modulate chromatin structure[27,28], regulate transcription[29], affect telomere function[30] and impact cell death pathways[15]. The same inhibitors failed when tested in clinical trials[41,42,43,44,45] This may indicate that when deprived of NAM as the main NAD+ source, cancer cells have an ability to utilize other NAD+ biosynthesis pathways[46,47]. There is an increased risk of hypoxia-induced necrosis and necrotic cells can subsequently become a localized source of NAD+ precursors[52]
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