Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia

Abstract

Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.

PCNA has specific functions in regulation of metabolism in haematological cells

“…We also detect clear effects on bladder cancer metabolism by targeting PCNA with the APIM-peptide (Sogaard, Blindheim, et al, 2018); however, these effects differ from those detected in haematological cells. Tissue-or cell type specific roles of PCNA and a link to glucose metabolism has also been suggested previously (Naryzhny & Lee, 2010;Ohayon et al, 2016).…”

“…Thus, stress is mediating a switch in PCNA´s proteins partners which is important for the cellular stress response. Modulation of the immune reaction only in "activated" immune cells could be advantageous in the tumor microenvironment where pro- We are only beginning to gain knowledge about the non-canonical roles of PCNA, but recent results, including this study, have established that the cytosolic roles of PCNA are complex and vary in different cell types (Müller et al, 2013;Naryzhny & Lee, 2010;Ohayon et al, 2016;Olaisen et al, 2015;Rosental et al, 2011;Sogaard, Blindheim, et al, 2018;Sogaard, Moestue, et al, 2018;Sogaard et al, 2019;Witko-Sarsat et al, 2010;Yin et al, 2015). Experimental studies of multifunctional proteins are extremely challenging, and robust quantitative methodologies for measurements at the metabolome and protein level are needed.…”

“…Several lines of evidence support cytosolic or non-canonical roles for PCNA: I) Cytosolic PCNA is reported to regulate apoptosis by binding to procaspases in mature neutrophils (Witko-Sarsat et al, 2010), multiple myeloma (MM) cells (Müller et al, 2013), and neuroblastoma cells (Yin et al, 2015); II) Inhibition of the ability of APIM-containing proteins to interact with PCNA by treating cells with a cellpenetrating peptide containing the APIM sequence (APIM-peptide) strongly reduced the secretion of cytokines from human monocytes stimulated with Toll-like receptor ligands (Olaisen et al, 2015); III) Cytosolic PCNA in cancer cells inhibits activation of natural killer cells, thereby helping cancer cells to evade the immune system (Rosental et al, 2011); IV) Glycolytic enzymes associate with PCNA, and nuclear export of PCNA correlated with increased Warburg-effect in acute myeloid leukaemia (AML) cells (Naryzhny & Lee, 2010;Ohayon et al, 2016); V) Multiple proteins involved in PI3K/AKT and MAPK signaling were found in PCNA pull-downs (Olaisen et al, 2015), and targeting PCNA has been linked to downregulation of EGFR and PI3K/AKT pathways in bladder cancer cells and monocytes (Olaisen et al, 2015;Sogaard, Blindheim, et al, 2018); VI) Cytosolic PCNA participates in reactive oxygen species (ROS) regulation via interaction with a subunit of the NADPH oxidase complex, p47phox (Ohayon et al, 2019); VII) Combination treatments with APIM-peptide and receptor tyrosine kinase inhibitors against EGFR/HER2/VEGFR and cMET showed increased anticancer activity in absence of any DNA-damaging agent (Sogaard et al, 2019); and VIII) A functional conservation of PCNA as a scaffold protein in MAPK signaling has been shown between mammalians and yeast (Olaisen et al, 2018). These cytosolic scaffold functions of PCNA, as well as the verified APIM-PCNA interactions important in DNA damage repair and/or tolerance, are illustrated in Figure 1.…”

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils

“…Control IgG or rabbit polyclonal anti-PCNA Ab5 antibody (20 µg) was cross-linked to 50 µl of protein G Sepharose beads (Pierce) using 100 µl dimethyl pimelimidate (20 mM; Pierce) in crosslinking buffer (200 mM carbonate buffer, pH 9) for 30 min according to the manufacturer's instructions and as previously described (Borregaard et al, 1983;Ohayon et al, 2016). Proteins (200 µg) from human neutrophil cytosols were obtained by nitrogen cavitation to prevent granule rupture, preserving cytosolic proteins from artifactual proteolysis as previously described (Witko-Sarsat et al, 2010).…”

Dioxin Receptor Adjusts Liver Regeneration After Acute Toxic Injury and Protects Against Liver Carcinogenesis

“…2a ) . Interestingly, AhR −/− but not AhR +/+ livers had a significant amount of cytosolic PCNA protein at early stages of regeneration, probably resulting for a nuclear to cytoplasmic shuttling process that takes place to protect from apoptosis and to sustain cell survival and resistance to toxic agents 42 , 43 . Thus, the more efficient regeneration observed in AhR-null livers involved an earlier proliferative response.…”