The Role of Plasma Membrane Calcium Pump during Pancreatic Cancer

UoM administered thesis: Phd

  • Authors:
  • Pishyaporn Sritangos

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a commonly diagnosed malignancy with one of the poorest patient survival prognoses that has barely improved over the past three decades, prompting the need to find novel therapeutic target and treatment strategies. Ongoing efforts have identified metabolic reprogramming and remodelling of Ca2+ signalling machinery as vulnerable targets which could be exploited therapeutically in multiple cancers, including PDAC. Evidence suggests that plasma membrane Ca2+ ATPases (PMCAs), shown to be the primary Ca2+ efflux machinery in PDAC, is functionally driven by glycolytic ATP to prevent cytotoxic Ca2+ overload in PDAC cells, which exhibit a highly glycolytic phenotype. It was hypothesized that plasma membrane (PM) associated glycolytic enzymes (GEs) are functionally coupled to ATP-consuming pumps, including the PMCAs. Glycolytic ATP fuels the pumps while the consumption of ATP prevents metabolite-induce inhibition of GEs, driving the glycolytic flux. Therefore, understanding the role of PMCAs in PDAC, its functional coupling with glycolytic ATP, and identification of putative PM-GEs binding protein responsible for this functional coupling, might provide a novel strategy for the therapeutic targeting of PDAC. The current thesis highlights the relevance of PMCA4 overexpression in patient-derived PDAC tumours and survival prognosis through datamining and also identifies MIAPaCa-2 cell line as a glycolysis-reliant PDAC model which predominantly overexpresses PMCA4 at both protein and mRNA level, representing the characteristics of patient-derived PDAC tumour. Knocking down PMCA4 expression using siRNA led to inhibited Ca2+ clearance, elevated resting intracellular Ca2+ ([Ca2+]i), inhibited cell migration and enhanced apoptotic cell death under Ca2+ stress; collectively suggesting that PMCA4 plays a critical role in Ca2+ homeostasis and contributes towards cancer phenotypes in PDAC. As PM-GEs and PMCA4 are reported to co-localise with caveolin-1 (Cav-1) enriched PM subdomains which may facilitate their functional coupling, PMCA and GEs co-localization with Cav-1 in MIAPaCa-2 cells was verified by sucrose gradient ultracentrifugation. Disruption of this Cav-1-enriched compartment, by cholesterol depletion, led to inhibited PMCA-mediated Ca2+ clearance which occurred independently of global ATP-depletion. This suggests that compartmental ATP may be important for fuelling PMCA activity. Although selective siRNA knockdown of Cav-1 resulted in modestly impaired PMCA activity which had no consequential effect on resting [Ca2+]i, nonetheless, this work provides the first evidence to suggest that Cav-1 expression could modulate PMCA4 activity in PDAC cells. Interestingly, neither PMCA4 nor Cav-1 siRNA knockdown had effect on basal metabolic phenotype as assessed by Agilent Seahorse XFe96 Mito stress and glycolytic stress tests. Lastly, we performed proximity-labelling bio-identification of pyruvate kinase M2 (PKM2), a key oncogenic GE, fused to a mutant biotin ligase BirA* and identified voltage-gated K+ channel subfamily H member 4 (KCNH4) as a novel putative PM-GEs binding protein potentially responsible for coupling glycolytic ATP to PMCA activity. However, further validation and experiments are required to determine whether these PM-GEs could be therapeutically targeted to inhibit oncogenic PMCA4 activity in PDAC.

Details

Original languageEnglish
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Award date1 Aug 2020