Abstract
BackgroundHigh 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.MethodsPDAC 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.ResultsPFKFB3 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.ConclusionsPFKFB3 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.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and with a 5-year survival rate of < 3%, it has one of the worst survival rates of all cancers [1, 2]
PFKFB3 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and predicts poor prognosis Expression of PFKFB1–4 and PFKP in tumour tissue versus healthy pancreatic epithelia was assessed by interrogating the Oncomine database and array dataset: Badea Pancreas [35]
PFK15 reduces cell proliferation and induces cell death in PDAC MIA PaCa-2 cells Glycolytic Adenosine triphosphate (ATP) is required for Plasma membrane calcium ATPase (PMCA) function; inhibition of glycolysis using iodoacetate (IAA) and bromopyruvate (BrPy) causes cytotoxic calcium overload and cell death in PDAC cells [9, 10]
Summary
Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and with a 5-year survival rate of < 3%, it has one of the worst survival rates of all cancers [1, 2]. After identifying that a glycolytic ATP supply to PMCAs is critical for PDAC cell survival, the logical step was to identify key glycolytic enzymes that are upregulated in PDAC and contribute to this phenotype. Though cancer cells exhibit a high rate of glycolysis, PKM2 has a lower catalytic activity than PKM1; this produces a bottleneck in glycolysis which allows the build-up of glycolytic intermediates that can be utilised for anabolic processes, at the expense of ATP production [14,15,16,17]. 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
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