Metabolic regulation of the plasma membrane calcium pump in pancreatic ductal adenocarcinoma.

  • Andrew James

    Student thesis: Phd


    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer withpoor prognosis and limited treatment options. Since many patients present withmetastatic disease and are thus ineligible for surgical resection, PDAC is almostubiquitously fatal; new treatment options are therefore needed to combat thisdisease. A key hallmark of many cancers, including PDAC, is metabolicreprogramming and a shift towards a high glycolytic rate, known as the Warburgeffect. This allows cancer cells to generate ATP in the face of hypoxia and tomeet the increased metabolic requirements associated with rapid proliferation.We hypothesised that this shift towards glycolytic metabolism has importantimplications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since theplasma 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 thePMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were thereforeassessed. Moreover, the effects of numerous mechanistically distinct metabolicinhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated.Treatment with glycolytic inhibitors induced significant ATP depletion, PMCAinhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells,while mitochondrial inhibitors had no effect. Subsequently, these experimentswere repeated on PDAC cells cultured in media formulated to "switch" theirhighly glycolytic phenotype back to one more reliant on mitochondrialmetabolism. Culture in nominal glucose-free media supplemented with eithergalactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch inmetabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis weresignificantly decreased, and in the case of cells cultured in KIC, oxidativephosphorylation rate was preserved (assessed using Seahorse XF technology).Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolyticinhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and[Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These datademonstrate that in PDAC cells exhibiting a high rate of glycolysis,glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thusis critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all foundto associate with the plasma membrane in MIA PaCa-2 cells, possibly in atyrosine phosphorylation-dependent manner. To investigate whether thedynamic membrane-association of glycolytic enzymes provides a privilegedsupply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitorswas assessed on PMCA activity. However, while these inhibited PMCA activity,this occurred without accompanying global ATP depletion. These data indicatethat 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 therapeuticlocus. However, further research is required to determine whether membraneboundglycolytic enzymes regulate its activity.
    Date of Award1 Aug 2015
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorMartin Steward (Supervisor) & Jason Bruce (Supervisor)


    • PMCA
    • pancreatic cancer
    • Calcium overload
    • cancer metabolism
    • Metabolism
    • Warburg
    • Cell death
    • Calcium signalling
    • Calcium ATPase
    • ATP
    • Glycolysis

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