Abstract
BackgroundPancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus.MethodsPDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA.ResultsThe PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death.ConclusionsCutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options
pyruvate kinase-M2 (PKM2) inhibitor shikonin reduces PDAC cell proliferation To investigate the importance of PKM2 on cancer hallmark responses, we first tested the effect of PKM2 modulators on Mia PaCa-2 cell growth/viability using a sulforhodamine B (SRB) colorimetric assay and tetrazolium-based cell counting kit (CCK8) assay
The current study demonstrates that PKM2 overexpression in human PDAC is associated with poor survival
Summary
Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. RESULTS: The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PDAC cells undergo a switch from mitochondrial to glycolytic metabolism (Warburg effect7,8), which facilitates numerous cancer hallmarks, including cell proliferation, invasion and resistance to apoptosis.[9] Our previous studies show that this increased glycolytic rate is important for fuelling the ATP-dependent plasma membrane calcium pump (PMCA), as inhibition of glycolytic ATP production in PDAC cells causes cytotoxic Ca2+ overload and cell death.[10,11] This dependency of the PMCA on glycolytic ATP in PDAC represents a potential therapeutic avenue, and understanding the underlying molecular mechanisms may reveal novel therapeutic targets against which novel drugs can be designed
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