Abstract
Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with increasing incidence in developing countries
Panc-1 cells were cultured in DMEM with 10% fetal calf serum; AsPC-1 and BxPC-3 cells were cultured in RPMI 1640 with 10% fetal calf serum, Capan-1 cells were cultured in IMDM with 20% fetal calf serum; Mia PaCa-2 cells were cultured in DMEM/F12 with 10% fetal calf serum and 2.5% horse serum; All media used for routine passaging contained GlutaMAX and 25 mM glucose
Screening conditions for the identification of novel metabolic regulators Five different PDAC cell lines were characterized for the metabolic phenotype by measuring oxygen consumption rate (OCR) (Fig 1A) and extracellular acidification rate (ECAR) as an indicator of glycolysis, respectively, (Fig 1B) using the Seahorse XF Analyzer
Summary
Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with increasing incidence in developing countries. It is an aggressive and highly metastatic cancer with an overall 5-year survival rate of less than 5% [1]. The affymetrix expression data are not necessary to replicate the findings of your study. All the necessary information and data is already available in our manuscript
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