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Title: Metabolic regulation of the plasma membrane calcium pump in pancreatic ductal adenocarcinoma
Author: James, Andrew
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with poor prognosis and limited treatment options. Since many patients present with metastatic disease and are thus ineligible for surgical resection, PDAC is almost ubiquitously fatal; new treatment options are therefore needed to combat this disease. A key hallmark of many cancers, including PDAC, is metabolic reprogramming and a shift towards a high glycolytic rate, known as the Warburg effect. This allows cancer cells to generate ATP in the face of hypoxia and to meet the increased metabolic requirements associated with rapid proliferation. We hypothesised that this shift towards glycolytic metabolism has important implications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining low [Ca2+]i and thus cell survival, is dependent on ATP to extrude cytosolic Ca2+. The relative contributions of mitochondrial vs glycolytic ATP in fuelling the PMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were therefore assessed. Moreover, the effects of numerous mechanistically distinct metabolic inhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated. Treatment with glycolytic inhibitors induced significant ATP depletion, PMCA inhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells, while mitochondrial inhibitors had no effect. Subsequently, these experiments were repeated on PDAC cells cultured in media formulated to "switch" their highly glycolytic phenotype back to one more reliant on mitochondrial metabolism. Culture in nominal glucose-free media supplemented with either galactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch in metabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis were significantly decreased, and in the case of cells cultured in KIC, oxidative phosphorylation rate was preserved (assessed using Seahorse XF technology). Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolytic inhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and [Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These data demonstrate that in PDAC cells exhibiting a high rate of glycolysis, glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thus is critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all found to associate with the plasma membrane in MIA PaCa-2 cells, possibly in a tyrosine phosphorylation-dependent manner. To investigate whether the dynamic membrane-association of glycolytic enzymes provides a privileged supply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitors was assessed on PMCA activity. However, while these inhibited PMCA activity, this occurred without accompanying global ATP depletion. These data indicate that glycolytic ATP is critical for the regulation of [Ca2+]i by the PMCA in PDAC, and that the glycolytic regulation of the PMCA may be an important therapeutic locus. However, further research is required to determine whether membrane-bound glycolytic enzymes regulate its activity.
Supervisor: Steward, Martin ; Bruce, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: PMCA ; cancer metabolism ; Calcium overload ; Warburg ; pancreatic cancer ; Calcium ATPase ; Glycolysis ; Cell death ; Calcium signalling ; ATP ; Metabolism