Endogenous ether lipids differentially promote tumor aggressiveness by regulating the SK3 channel

Abstract

SK3 channels are potassium channels found to promote tumor aggressiveness. We have previously demonstrated that SK3 is regulated by synthetic ether lipids, but the role of endogenous ether lipids is unknown. Here, we have studied the role of endogenous alkyl- and alkenyl-ether lipids on SK3 channels and on the biology of cancer cells. Experiments revealed that the suppression of alkylglycerone phosphate synthase or plasmanylethanolamine desaturase 1, which are key enzymes for alkyl- and alkenyl-ether-lipid synthesis, respectively, decreased SK3 expression by increasing micro RNA (miR)-499 and miR-208 expression, leading to a decrease in SK3-dependent calcium entry, cell migration, and matrix metalloproteinase 9-dependent cell adhesion and invasion. We identified several ether lipids that promoted SK3 expression and found a differential role of alkyl- and alkenyl-ether lipids on SK3 activity. The expressions of alkylglycerone phosphate synthase, SK3, and miR were associated in clinical samples emphasizing the clinical consistency of our observations. To our knowledge, this is the first report showing that ether lipids differentially control tumor aggressiveness by regulating an ion channel. This insight provides new possibilities for therapeutic interventions, offering clinicians an opportunity to manipulate ion channel dysfunction by adjusting the composition of ether lipids.

Keywords: SK3 channel; ether lipids; miRNA; potassium channels.

Fig. 1

AGPS promotes SK3 expression by regulating miRNA: evidence in human cancer cells and tissues. A–C: AGPS correlates with SK3 in invasive breast carcinoma and prostate adenocarcinoma. A: Scatter plot representing the Pearson’s correlation analysis between AGPS and KCNN3 mRNA (translating SK3 protein) quantified by RT-qPCR. Cohort 1, N = 50, r = 0.4, ∗P < 0.05. B: Representative figures of immunohistochemical staining for AGPS and SK3 in human breast cancer tissues (scale bar = 100 μm). Top: most tumors positive for SK3 were also positive for AGPS (23 on 28). Bottom: the majority of SK3 negative cases were also negative for AGPS (30 on 50). Cohort 2, N = 78, Chi2, ∗∗∗∗P = 0.0002. No correlation between AGPS staining and histoprognostic grade SBR or with the lymph node status was found (P = 0.36). C: Scatter plot representing the Pearson’s correlation of the mRNA expression of KCNN3 in function of the mRNA expression of AGPS in prostate adenocarcinoma. TGCA-PRAD dataset, N = 495, r = 0.4, ∗∗∗∗P < 0.0001. D: AGPS is critical for SK3 expression in cancer cells. Left: SK3 protein level studied in stain-free Western-blot is highly decreased 72 h after transfection with siAGPS#1 compared to siCTL in MDA-MB-435s cells. SK3 detection band area was normalized to the total protein signal of the lane and then relativized to the siCTL condition (N = 4). Right: KCNN3 mRNA level analyzed by RT-qPCR is decreased 72 h after transfection with siAGPS#1 compared to siCTL in MDA-MB-435s, A673, PC3, and C4-2 cells (median ± interquartile range, Mann–Whitney test, ∗∗∗∗P < 0.0001). The numbers in brackets indicate the number of independent experiments. E: AGPS knockdown does not affect KCNN3 promoter activity. KCNN3 promoter activity studied by reporter luciferase assay was performed 48 h (N = 4) and 72 h (N = 6) after siAGPS#1 transfection in MDA-MB-435s cells. Data are relativized to siCTL condition (median ± interquartile range, N = 6, Mann–Whitney test, not significant). F: AGPS knockdown increases the expression of putative miRNA–targeting KCNN3 mRNA. miR-499-5p (N = 7), miR-208-3p (N = 4), miR-218-3p (N = 3), and miR-135-5p (N = 3) were quantified by RT-qPCR 48 h after transfection with siAGPS#1 in MDA-MB-435s cells. Data are relativized to siCTL condition (median ± interquartile range, Mann–Whitney test, ∗∗∗∗P < 0.0001, ∗∗P < 0.01). G: miR-208-3p and miR-499-5p bind to 3′ UTR KCNN3 mRNA. Left: organization of endogenous KCNN3 mRNA, miRNAs, and GW482 (RNA-binding protein) within RNA-induced silencing complex (RISC) assessed by cross-linking immunoprecipitation in MDA-MB-435s cells. The binding of GW182, miR-499a-5p, or miR-208-3p to the 3′ UTR KCNN3 mRNA were detected. In opposite, the binding were not detected with miR-218-3p and miR-135-5p (N = 3, median ± interquartile range). Right: alignment of the sequences of miR-499-5p and miR-208-3p with their target site in the 3′ UTR of KCNN3 mRNA. The binding site is the same for both miRNAs and is highly conserved among mammals. H: miR-208-3p and miR-499-5p mimics downregulate KCNN3 mRNA. KCNN3 mRNA was quantified by RT-qPCR 48 h after transfection with miR-499-5p or miR-208-3p mimics and relativized to the mimic control condition in MDA-MB-435s cells. (N = 4, median ± interquartile range, Kruskal–Wallis test, P < 0.0001 and post hoc Dunn’s test (compared to control condition),∗∗∗∗P < 0.0001). I: AGPS and KCNN3 mRNA are inversely correlated with miR-208-3p and miR-499-5p in breast carcinoma. AGPS, KCNN3, miR-499-5p, and miR-208-3p were quantified by RT-qPCR. Graphics show Pearson’s correlation analysis. Cohort 3, N = 28, KCNN3 versus miR-208-3p+miR-499-5p: r = −0.40, ∗P < 0.05, AGPS versus miR-208-3p and miR-499-5p: r = 0.38, ∗P < 0.05. J: Schematic representation of SK3 regulation by AGPS. AGPS, alkylglycerone phosphate synthase; miRNA, micro RNA; qPCR, quantitiative polymerase chain reaction; RT-qPCR, reverse transcription quantitiative polymerase chain reaction; SBR, Scarff Bloom et Richardson; TCGA, The Cancer Genome Atlas; UTR, untranslated region.

Fig. 2

AGPS drives SK3-dependent cell migration, invasion, and adhesion. A: The ability of SK3⁺ cells to migrate is significantly reduced unlike SK3^- cells upon AGPS knockdown. MDA-MB-435s cells containing a sh-control (SK3⁺ cell) or a sh-SK3 (SK3^- cell) were transfected with siAGPS#1 and seeded in migration inserts after 48 h. Results are relative to siCTL. Representative pictures of migration inserts are shown with nuclei staining in white color. (Scale bar = 30 μm, N = 3, median ± interquartile range, Mann–Whitney test, ∗∗∗P < 0.001, ns = not significant). B: Constitutive calcium entry in SK3⁺ is significantly reduced unlike SK3^- cells upon AGPS knockdown. MDA-MB-435s cells containing a sh-control (SK3⁺ cell) or a sh-SK3 (SK3^- cell) were transfected with siAGPS#1 during 72 h hours before the measurement of constitutive calcium entry by using Fura-2-AM probe. Results presented as histograms are relative to siCTL. Representative recordings of fluorescence are shown. (N = 3, median ± interquartile range, Mann–Whitney test, ∗∗P < 0.01, ns = not significant). C and D: Cell invasion and adhesion are reduced upon AGPS or SK3 knockdown. C: MDA-MB-435s cells were transfected with siCTL (N = 4), siKCNN3#1 (N = 4), siAGPS#1 (N = 3) 2 days before cell invasion assays (24 h). Data are relativized to siCTL condition (median ± interquartile range, Kruskal–Wallis test, P < 0.0001 and post hoc Dunn’s test, compared to siCTL condition, ∗∗P < 0.01, ∗∗∗P < 0.001). Representative pictures of cells after cell adhesion assays. The scale bar represents 60 μm, and nuclei staining are in white color. D: MDA-MB-435s cancer cells were transfected with siCTL, siKCNN3#1, or siAGPS#1 3 days before cell adhesion assay (2 h). P < 0.001. Data are relativized to siCTL condition (N = 3, median ± interquartile range, Kruskal–Wallis test, P < 0.0001, and post hoc Dunn’s test, compared to siCTL condition, ∗∗∗P < 0.001. E and F: Cell invasion is slightly reduced and cell adhesion is unmodified by pharmacological inhibitors of SK3 currents. E: MDA-MB-435s cells were pretreated daily during 2 days then during invasion assay (24 h) with apamin (100 nM) or ohmline (1 μM), two inhibitors of SK3 currents. Data are relativized to control condition (median ± interquartile range, Kruskal–Wallis test, P < 0.0001 and post hoc Dunn’s test, compared to siCTL condition, ∗∗P < 0.01. F: MDA-MB-435s cells were pretreated daily during 3 days then during adhesion assay (2 h) with apamin (100 nM) or ohmline (1 μM), two inhibitors of SK3 currents. Data are relativized to control condition (N = 4, median ± interquartile range, Kruskal–Wallis test, P = 0.05. Representative pictures of cells after cell adhesion assays. The scale bar represents 60 μm; and nuclei staining are in white color. AGPS, alkylglycerone phosphate synthase.

Fig. 3

A new nonpore function for SK3 promotes cell invasion and adhesion through AGPS/SK3/MMP9 axis. A: MMP9 expression is under the control of AGPS and SK3 expressions. MMP9 mRNA was quantified by RT-qPCR 72 h after transfection with siCTL, siAGPS#1, siKCNN3#1, or siKCNN3#2, and relativized to siCTL condition (median ± interquartile range, Kruskal–Wallis test, P < 0.0001, and post hoc Dunn’s test, compared to control condition: ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, and ∗∗P < 0.01). The numbers in brackets indicate the number of independent experiments. B: MMP9 expression is upregulated by the overexpression of wild-type SK3 as well as mutated pore (H425V) SK3 construct. MMP9 mRNA was quantified by RT-qPCR 72 h after transfection of MDA-MB-435s cells with 0.25 μg control (CTL), human wild-type SK3 (hSK3wt), or human SK3np (hSK3np) plasmids. (N = 3, median ± interquartile range, Kruskal–Wallis test for the experiment with MDA-MB-435s cells, ∗∗∗P < 0.001,∗∗∗∗P < 0.0001). C: MMP9 expression is unmodified by a treatment with SK3 inhibitors. Cells are treated daily during 3 days with apamin (100 nM) or ohmline (1 μM) and MMP9 mRNA was quantified by RT-qPCR and relativised to control condition. (N = 3, median ± interquartile range, Kruskal–Wallis test for the experiment with MDA-MB-435s cells: P > 0.05, Mann–Whitney test for the experiment with A-673 cells, ∗P < 0.05). D: Cell invasion and adhesion are under control of MMP9 expression. MDA-MB-435s cells were transfected with siCTL or siMMP9. Invasion assay was performed for 24 h, 48 h post transfection. Adhesion assay was performed after 72h h of transfection. Data are relativized to siCTL condition (median ± interquartile range, Mann–Whitney test, ∗∗∗P < 0.001). The numbers in brackets indicate the number of independent experiments. E and F: Effect of mutated pore SK3 construct overexpression on cell migration and adhesion. MDA-MB-435s cells were transfected with 0.25 μg control (CTL) or human SK3np (hSK3np) plasmids. E: Adhesion assay was performed after 72 h by seeding the transfected cells in plates coated with electrodes to measure cell index every 4 min for 2 h. Left: representative graphic showing the median and the interquartile range for one experiment. Right: after 2 h, the relative cell index was significantly increased in the hSK3mut condition (median ± interquartile range, Mann–Whitney test, ∗∗P < 0.01). F: Cell migration assay was performed for 24 h, 48 h post transfection (median ± interquartile range, Mann–Whitney test, ∗∗∗P < 0.001). The numbers in brackets indicate the number of independent experiments. AGPS, alkylglycerone phosphate synthase; MMP, matrix metalloproteinase; qPCR, quantitiative polymerase chain reaction; RT-qPCR, reverse transcription quantitiative polymerase chain reaction.

Fig. 4

PEDS1, the key enzyme producing alkenyl-EL, is involved in the AGPS/SK3/MMP9 axis mediating cell migration, invasion, and adhesion. A: AGPS knockdown reduces all EL level in cancer cells. MDA-MB-435s cells were transfected with siCTL or siAGPS#1 during 96 h before lipid extraction. The heatmap represents the significantly altered molecular species of EL identified by UHPLC-MS in negative ion mode in the siCTL (left) and siAGPS (right) samples (see Supplemental Figs. S10 and S11 for the statistical tests performed for the selection, N = 7, Wilcoxon signed-rank test, ∗P < 0.05). Each column represents a different sample with siCTL and siAGPS samples working in pairs. Each line represents a different EL (identified on the right) and each colored square represents the Z-score of the EL (see material and methods for details on the Z-scores calculations). High Z-scores (red) indicate a higher quantity of EL in the sample and low Z-scores (blue) a lower quantity. B: Schematic representation of EL synthesis. The fatty acid of acyl dihydroxyacetone phosphate (acyl-DHAP) is replaced by a fatty alcohol produced by fatty acyl-CoA reductase 1 (FAR1) forming alkyl-DHAP in a reaction catalyzed by alkylglycerone phosphate synthase (AGPS). Alkyl-DHAP is catalyzed by several enzymes to form diverse alkyl-EL species. From alkyl-PE, plasmanylethanolamine desaturase 1 (PEDS1) forms alkenyl-PE and other enzymes enable the formation of other alkenyl-EL species. C–E: PEDS1 knockdown increases miRNAs targeting KCNN3 mRNA and reduces KCNN3 mRNA and SK3 expression. D: PEDS1 protein level studied in stain-free Western blot is highly decreased 72 h after transfection with siPEDS1 compared to siCTL in MDA-MB-435s cells. PEDS1 detection band area was normalized to the total protein signal of the lane and then relativized to the siCTL condition (N = 3). E: miR-499a-5p and miR-208-3p were quantified by RT-qPCR 48 h after transfection with siPEDS1 in MDA-MB-435s cells (N = 4). F: Left, KCNN3 mRNA was quantified by RT-qPCR 72 and 96 h after transfection with siPEDS1 in MDA-MB-435s cells (N = 3) (median ± interquartile range, Mann–Whitney test, ∗∗∗∗P < 0.001). Right: SK3 protein level was measured by stain-free Western blot 72 h after transfection with siPEDS1 compared to siCTL in MDA-MB-435s cells. SK3 detection band area was normalized to the total protein signal of the lane and then relativized to the siCTL condition (N = 3). F: PEDS1 knockdown reduces cancer cell migration, invasion, and adhesion. MDA-MB-435s cells were transfected with siCTL, siKCNN3#1, or siPEDS1 during 72 h before migration, invasion, and adhesion assays. Data are relativised to siCTL condition (N = 3–4, median ± interquartile range, Kruskal–Wallis test, P < 0.0001 and post hoc Dunn’s test, compared to control condition: ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, and ns = not significant). G: MMP9 expression is under the control of PEDS1 and SK3 expressions. MMP9 mRNA was quantified by RT-qPCR 72 h after transfection with siCTL, siKCNN3#1, or siPEDS1 and relativised to siCTL condition (N = 3, median ± interquartile range, Kruskal–Wallis test, P < 0.0001 and post hoc Dunn’s test, compared to control condition: ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns, not significant). H and I: PEDS1 knockdown also reduces the levels of all EL, possibly through the regulation of FAR1, the rate-limiting enzyme of EL synthesis. H: MDA-MB-435s cells were transfected with siCTL or siPEDS1 during 72 h before UHPLC-MS experiments in positive ion mode. Six independent experiments were performed. The fold changes plotted were calculated by relativizing the total abundances of alkyl-EL (left) and alkenyl-EL (right) in each siPEDS1 sample to their total abundance in the matching siCTL samples. (N = 6, median ± interquartile range, Mann–Whitney test, ∗∗P < 0.01). I: FAR1 protein level in MDA-MB-435s was measured by stain-free Western blot 72 h after transfection with siPEDS1 compared to siCTL. FAR1 detection band area was normalized to the total protein signal of the lane and then relativized to the siCTL condition (N = 3). DHAP, dihydroxyacetone phosphate; EL, ether lipid; miRNA, micro RNA; MMP, matrix metalloproteinase; qPCR, quantitiative polymerase chain reaction; RT-qPCR, reverse transcription quantitiative polymerase chain reaction; siCTL, si control; UHPLC-MS, ultra-high performance liquid chromatography tandem mass spectrometry.

Fig. 5

Alkyl- and alkenyl-EL promote KCNN3 expression while only alkyl-EL increases SK3 channel activity. A and B: Supplementation with both alkyl- and alkenyl-EL increases KCNN3 expression and is able to restore partially its expression reduced after PEDS1 knockdown. Cells were treated daily with 20 μM of EL in liposomes for 96 h. Cells to be transfected were pretreated with EL liposomes for 24 h before transfection with siCTL or siPEDS1. After 6 h, the transfected cells were treated daily during the remaining 72 h. KCNN3 mRNA level measured by RT-qPCR is increased after supplementation with EL (median ± interquartile range, Mann–Whitney test, ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, and ∗P < 0.5). C: Expression of miR-499a-5p–targeting KCNN3 mRNA is reduced after lyso-alkyl-EL and lyso-alkenyl-EL supplementation. MDA-MB-435s cells were treated daily with 20 μM of LPC(O-16:0) (left) and LPC(P-16:0) (right) in liposomes for 48 h, after which miR-499a-5p was quantified by RT-qPCR. D–H: Acute application of EL on SK3 currents. Whole-cell currents in HEK293T cells expressing recombinant human SK3 were generated by a ramp protocol from −100 to 100 mV in 500 ms from a constant holding of 0 mV with a pCa 6. D: Graphs representing the current recorded at 0 mV after acute application of alkyl-EL (3 μM). Data are relativised to the current recorded before alkyl-EL application. The black line indicates the median, each point represents SK3 current fold change after alkyl-EL application to one cell; bars, median. Wilcoxon signed-rank test. E: representative whole-cell currents recorded after application of vehicle (black trace, 7 min), after application to PC(O-16:0/22:6) (green trace, 3 μM, 7 min) and after addition of apamin (blue trace, 100 nM, less than 2 min) to inhibit SK3 currents. F: Graphs showing a representative time course at 0 mV of whole-cell currents (same cell than in E). G: Graphs representing the current recorded at 0 mV after acute application of alkenyl-EL (3 μM). Data are relativised to the current recorded before alkenyl-EL application. The black line indicates the median, each point represents SK3 current fold change after alkenyl-EL application to one cell; bars, median. Wilcoxon signed-rank test. H: Graphs showing a representative time course at 0 mV of whole-cell currents during application of vehicle, PC(P-16:0/C16:0), and apamin. AGPS, alkylglycerone phosphate synthase; EL, ether lipid; LPC, lysophosphatidylcholine; miRNA, micro RNA; PC, phosphatidylcholine; PEDS1, plasmanylethanolamine desaturase 1; qPCR, quantitiative polymerase chain reaction; RT-qPCR, reverse transcription quantitiative polymerase chain reaction; siCTL, si control.

Fig. 6

Schematic overview of SK3 regulation by EL. Both alkyl- and alkenyl-EL, requiring AGPS and PEDS1 for their endogenous synthesis, promote SK3 expression, through the downregulation of miRNAs targeting KCNN3 transcripts (miR-SK3). Alkyl-EL, and not alkenyl-EL, also promotes SK3 currents. This could lead to a fine regulation of SK3-dependent biological functions involved in calcium entry, cell migration, invasion, and adhesion, thus promoting aggressive features of cancer cells. AGPS, alkylglycerone phosphate synthase; EL, ether lipid; miRNA, micro RNA; PEDS1, plasmanylethanolamine desaturase 1.