Identification of K_Ca3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines

<div><p>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 <i>KRAS</i> as well as loss-of-function mutations in <i>p53</i>. 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 <i>KCNN4</i> gene, which encodes for the Ca²⁺-sensitive K⁺ channel K_Ca3.1. 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 K_Ca3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of K_Ca3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.</p></div

K_Ca3.1 inhibition modulates oxygen consumption profile in a subset of PDAC cell lines.

<p><b>(A)</b> Seahorse XF Mito Stress test was performed to measure mitochondrial function upon various concentrations of rac-16 treatment of Mia PaCa-2, <b>(B)</b>, Panc-1, <b>(C)</b> BxPC-3, and <b>(D)</b> Capan-1 cells. n = 3. Oligo is oligomycin A, FCCP is carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, A/R is a mix of antimycin A and rotenone. n = 6.</p

K_Ca3.1 inhibition affects proliferation.

<p><b>(A)</b> xCELLigence proliferation assay on Mia PaCa-2, <b>(B)</b> Panc-1 <b>(C)</b> Capan-1 and <b>(D)</b> BxPC-3 cells grown in the presence of 11 mM glucose or galactose as the main energy source and treated with increasing concentrations of rac-16, n = 5 for each condition. The slope of the exponential phase of the growth curve was calculated in the complementary software. The relative growth was calculated as the ratio between the slopes of DMSO-and rac-16 treated cells in the appropriate media. <b>(E)</b> Viability assay of Mia PaCa-2 cells transfected with non-targeted siRNA or individual siRNAs against KCNN4. Assay was conducted 72 hours post-transfection, n = 3. ANOVA Bonferroni test was used to compare each of the rac-16 treatments vs DMSO; ns, not significant, p>0.05,*, p-value is 0.01 to 0.05; **, p-value < 0.01.</p

Metabolism of PDAC cell lines.

<p><b>(A)</b> Measurement of OCR, <b>(B)</b> ECAR, <b>(C)</b> OCR/ECAR and <b>(D)</b> metabolic phenotyping were performed in Seahorse assay medium supplied with 11 mM Glucose, 2 mM Pyruvate and 2 mM Glutamine. 20 000 cells/well were seeded one day prior to the experiment. n = 12 for each cell line. Tukey’s Multiple Comparison ANOVA test was performed. All groups are significantly different unless indicated otherwise.</p

Transportome hits which downregulate oxygen consumption >10%.

<p>Transportome hits which downregulate oxygen consumption >10%.</p

Transportome hits which upregulate oxygen consumption >10%.

<p>Transportome hits which upregulate oxygen consumption >10%.</p

KCNN4 hit confirmation.

<p><b>(A)</b> OCR of Mia PaCa-2 cells transfected with individual siRNAs against KCNN4, 20 000 cells/well were re-seeded the night before the assay to avoid cell number-dependent changes. n = 6; <b>(B)</b> Upper panel: <i>KCNN4</i> TaqMan gene expression assay. <i>KCNN4</i> mRNA levels were normalized to HPRT. K_Ca3.1 Lower panel: Western Blot analysis of <i>KCNN4</i> protein expression, <i>HSP90</i> was used as loading control. Arrow indicates the KCNN4 protein band. <b>(C)</b> Oxygen consumption measurements of Mia PaCa-2, <b>(D)</b> Panc-1, <b>(E)</b> Capan-1 and <b>(F)</b> BxPC-3 cells treated with rac-16 (KCNN4 inhibitor) at different concentrations as indicated, 100 nM NS309 (KCNN4 activator) and high concentrations of extracellular KCl (60 mM), n = 5. Data are represented as the mean ± SD. ANOVA test with Bonferroni post-hoc was performed to compare NT siRNA and siRNA targeting KCNN4 mRNA; ns, not significant, p>0.05, *, p-value is 0.01 to 0.05; **, p-value is < 0.01.</p

Optimization of screening conditions in Mia PaCa-2 cells.

<p><b>(A)</b> Oxygen consumption measurements (OCR) of Mia PaCa-2 cells in 25mM glucose or 25mM galactose. The Complex I inhibitor rotenone (1 μM) was used as a positive control for the inhibition of OxPhos, n = 6; <b>(B)</b> Measurement of OCR in media supplied with different carbon sources: 25 mM Glucose, 2 mM Glutamine, 2 mM Na Pyruvate, n = 12; <b>(C)</b> CellTiter Blue fluorescence measurements of cell titrations cultured in different assay media for 2 hrs, n = 12; <b>(D)</b> OCR measurements of Mia PaCa-2 cell titrations in different assay media, n = 12; <b>(E)</b> Nuclei number count using Hoechst stain in Seahorse plate, n = 12. For A, t-test was performed, *, p-value is 0.01 to 0.05; **, p-value is 0.001 to 0.01; B, Tukey’s Multiple Comparison ANOVA Test was performed. Groups are significantly different unless indicated otherwise. For C and D, two-tailed t-test was performed, ns, not significant, p>0.05, *, p-value is 0.01 to 0.05; **, p-value is < 0.01.</p