Cutting off the fuel supply to calcium pumps in pancreatic cancer

  • Daniel Richardson

Student thesis: Phd

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and carries with it one of the poorest prognoses of all cancers. Cancer metabolism has been studied as a potential area for therapy for a number of years, however, to-date no therapies directly targeting cancer metabolism are available. In the last decade, glycolytic fuelling of plasma membrane calcium ATPases (PMCAs) as critical for maintaining low intracellular calcium concentration [Ca2+]i was identified as a potential ‘Achilles heel’ of cancer cells. PMCAs are the primary method of Ca2+ efflux in PDAC cells and receive a ‘preferential’ glycolytic ATP supply in highly glycolytic PDAC cells. We hypothesised that key glycolytic enzymes, overexpressed in PDAC, are integral in fuelling PMCAs, such that inhibiting these enzymes would cut off the fuel supply to PMCAs, leading to cytotoxic Ca2+ overload and cell death. We hypothesised that these effects would be specific to cancer cells, as healthy tissues do not rely on functional coupling of glycolysis and PMCAs. We hypothesised therefore that specific inhibitors for key glycolytic enzymes in PDAC would present potential as a targeted therapy for PDAC and other cancers exhibiting the Warburg Effect. The present thesis identified pyruvate kinase M2 (PKM2) as a key glycolytic enzyme that plays an important role in the fuelling of PMCAs in PDAC cells. PKM2 is overexpressed in PDAC where it correlates with poor survival. Inhibiting PKM2 with shikonin reduces cell proliferation and migration and induces cell death. This is due to ATP depletion, resulting in decreased PMCA activity and cytotoxic Ca2+ overload and cell death in PDAC cells. We hypothesized that functional coupling of glycolysis and PMCAs was mediated by co-localization and found that both PKM2 can be identified in plasma membrane fractions. Knockdown of PKM2 was shown to reduce PMCA activity, further suggesting that PKM2 is required for fuelling [Ca2+]i homeostasis. PKM2 knockdown also protected against shikonin induced cell death. Phosphofructokinase-fructose-bisphoshatase-3 (PFKFB3) was also identified as playing a role in glycolytic fuelling of PMCAs in PDAC. PFKFB3 is overexpressed in PDAC, where it correlates with poor survival. Inhibition of PFKFB3 with PFK15 caused reduced PDAC cell proliferation and induced cell death. This was shown to be at least in-part due to inhibition of PMCA activity, leading to cytotoxic Ca2+ overload and cell death. A pool of PFKFB3 was also identified at the plasma membrane, further suggesting that co-localization of glycolytic enzymes with PMCAs at the plasma membrane contributes to a preferential ATP supply to PMCAs, maintaining low [Ca2+]i. Lastly we hypothesized that the metabolic requirements of PDAC cells change throughout the cell cycle, such that particular points in the cell cycle exist where cells are more vulnerable to PKM2 inhibition. Inhibition of CDK4/6 using palbociclib and CDK1 using RO-3306 arrests cells in G1/S and G2/M respectively. G1/S and G2/M arrested cells were assessed for ATP levels, metabolic phenotype and the distribution of low activity dimeric and high activity tetrameric PKM2 and no significant differences were observed. Despite this G2/M arrested cells were sensitised to shikonin treatment, suggesting an important role for PKM2 in G2/M. The present thesis provides further evidence that glycolytic fuelling of PMCAs presents a potential therapeutic strategy for PDAC and highlights the potential for targeting cells in G2/M to increase the efficacy of PKM2 inhibitors.
Date of Award31 Dec 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorKaye Williams (Supervisor) & Jason Bruce (Supervisor)

Keywords

  • PMCA
  • Cell Cycle
  • Calcium Homeostasis
  • Metabolism
  • Warburg Effect
  • Cancer
  • Glycolysis

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