PO-238 Dichloroacetate (DCA) treatment affects mitochondrial activity and stemness in pancreatic cancer (PC) cell lines

Abstract

IntroductionTargeting metabolism represents a new approach to treat cancer, expecially when conventional chemoterapy fails. In this study, we tested a metabolic approach to treat PC, investigating, in vitro and in vivo, its response to DCA treatment.Material and methodsTwo PC cell lines, BXPC3 and PANC1, were treated with DCA 4 and 10 mM for 24 hour. Cell viability and proliferation were assessed by MTS assay and xCELLigence, apoptosis and ROS by flow-cytometry; pPDHSer293/tot PDH, LC3B, DRP1, MFN1, MNF2, OPA1 and TOMM20 protein expression was evaluated by western blotting, lin28 gene expression by qPCR. The oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured by Seahorse Technology. Ultra-low attachment plates were used to form spheroids. In vivo, DCA was administered to BXPC3-luc tumor-bearing nude mice. After measuring bioluminescence signalling, the tumour masses were harvested, photographed and weighed.Results and discussionsDCA treatment reduced cell proliferation, decreasing cell survival with an increase in ROS production and apoptosis in both cell lines. Despite PDH activation by dephosphorylation, DCA did not restore bioenergetic profile but decreased OCR, a measure of oxidative phosphorylation efficiency. ECAR was not affected, suggesting that the glicolytic capacity was not modified by DCA treatment. These observations led us to explore mitophagy, whose activation was confirmed by LC3B protein overexpression and TOMM20 downregulation, and mitochondrial dynamics also altered following DCA treatment as shown by the downregulation of MFN1, MFN2, OPA1, key proteins of mitochondrial fusion. Interestingly, DCA was able to negatively affect the cancer stem cell (CSCs) fraction in both cell lines, reducing the expression of stemness genes such as Lin28 and inhibiting spheroid formation. When added to 3D cultures already formed, it was able to downregulate stemness genes expression, leading to a significant size reduction and affecting spheroid viability. Finally, DCA efficacy was confirmed in a xenograft pancreatic cancer mouse model in which DCA treatment displayed a significant retarded progression of PC, reducing diameter of the tumour mass.ConclusionOur data suggest that DCA is able to strongly affect PC cells metabolism counteracting mitochondrial activity. This effect is not related to PDH activity stimulation. In addition, the ability of DCA to hit CSCs offers a further rationale to candidate this drug for PC treatment, trying to reach a complete tumour eradication.