Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3)

Abstract

High glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells. PDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADP-ribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging. PFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2. PFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.