Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2+

Abstract

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca 2+ /ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca 2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.

PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner

“…Consistent with this view, we and others have demonstrated the relevance of PEPCK-M in cell growth and chemoresistance in several cancer cell models in vitro and in vivo [ 6 – 9 ]. However, the way PEPCK-M fluxes interact with tumor cell metabolism to promote survival are yet to be identified, and is the focus of the present work.…”

“…Our data is consistent with this view, and with previous observations that increased proline metabolism correlated with higher ROS production at the PRODH/POX node, and a blockade in TCA cycle flux [ 15 ], a general mechanism to cope with hypoxic stress [ 31 ]. These events identify a metabolic disturbance that bear on the health status of the cells with reduced PEPCK-M activity (i.e., sh1-PCK2), albeit with minimal effects on cell viability and apoptosis in the absence of nutrient stress [ 5 , 6 , 9 ].…”

“…Moreover, the synthesis of proline is activated by c-Myc [ 14 ], and PEPCK-M might influence this pathway through its regulation of calcium fluxes by modulating PEP levels and SERCA activity in cancer cells [ 6 , 33 ], that in turn triggers the CAMKIIγ-dependent phosphorylation and stabilization of c-Myc. Interestingly, PEPCK-M can impact on metabolism by regulating the PEP levels even in the presence of excess glucose in HCT116 cells [ 6 ]. However, in the present study using HeLa cells, we have not been able to measure specific flux of carbons from glutamine into serine and glycine in these conditions, even with extended incubation times.…”

“…However, the way PEPCK-M fluxes interact with tumor cell metabolism to promote survival are yet to be identified, and is the focus of the present work. On this, we have recently shown that PEPCK-M activity, and glucose availability, modulates cytosolic calcium signaling by upkeeping the pool of PEP, an inhibitor of SERCA, even in the presence of glucose in two models of colon carcinoma, HCT116 and SW480 [ 6 ]. Calcium then signals downstream effectors such as NFAT and c-Myc with implications on tumor cell biology.…”

The Two-Way Relationship Between Calcium and Metabolism in Cancer

“…Recently, the regulation of Ca 2+ homeostasis by PEP has been described in two human colon carcinoma cell lines, in which increased cytosolic Ca 2+ due to SERCA modulation stabilizes the master regulator of glycolytic metabolism, the Myc protooncogene protein, ultimately supporting cancer cell proliferation (Moreno-Felici et al, 2019).…”