Abstract
Ewing sarcoma (ES) is characterized by EWS::FLI1 or EWS::ERG fusion proteins. Knowing that ion channels are involved in tumorigenesis, this work aimed to study the involvement of the KCNN1 gene, which encodes the SK1 potassium channel, in ES development. Bioinformatics analyses from databases were used to study KCNN1 expression in patients and cell lines. Molecular approaches and in vitro assays were used to study the transcriptional regulation of KCNN1 and its involvement in the regulation of ES cell proliferation. KCNN1 is overexpressed in ES patient biopsies, and its expression is inversely correlated with patient survival. EWS::FLI1, like EWS::ERG, promotes KCNN1 and SK1 expression, binding to GGAA microsatellites near the promoter of KCNN1 isoforms. KCNN1 is involved in the regulation of ES cell proliferation, with its silencing being associated with a slowing of the cell cycle, and its expression modulates membrane potential and therefore calcium flux. These results highlight that KCNN1 is a direct target of EWS::FLI1 and EWS::ERG and demonstrate that KCNN1 is involved in the regulation of intracellular calcium activity and ES cell proliferation, making it a promising therapeutic target in ES.
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Introduction
Ewing sarcoma (ES), the second-most common primary malignant bone tumor in children, adolescents and young adults [1], is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family [2], leading to the EWS::FLI1 fusion protein in 85% of cases [3], and to the EWS::ERG protein in 10% of cases [4]. Current treatment consists of chemotherapy [5] and surgical resection of the tumor. This treatment increases patient survival to 70%, which unfortunately decreases to less than 30% if patients are resistant to chemotherapy or when metastases are present at diagnosis [6].
Studies have recently emerged regarding the involvement of ion channels in tumorigenesis [7,8,9,10]. Some potassium channels have thus been shown to be involved in gastrointestinal cancer [11], colorectal cancer [12] or myeloma [13]. These potassium channels, that form the largest family of ion channels, are subdivided into four classes, according to their numbers of transmembrane segments and pore domains [14]. Among them, calcium-activated potassium channels (KCa) are divided into three subclasses, namely small conductance (SKCa: SK1, SK2, and SK3), intermediate conductance, and big conductance, based on their unitary conductance [14]. SK3, whose α subunit is encoded by the KCNN3 gene, has been shown to be involved in the development of bone metastasis in breast cancer [15], and overexpressed in prostate cancer [16, 17] or melanoma [18, 19]. KCNN2 was shown overexpressed in melanoma [19] and in pancreas cancer [20].
Regarding the role of potassium channels in the development of ES, two studies have demonstrated the role of KCNA5, which encodes Kv1.5 [21, 22]. Specifically, these studies have demonstrated that DNA hypermethylation contributes to epigenetic repression of the KCNA5 locus, and that the consequent suppression of the Kv1.5 ion channel promotes cancer cell proliferation [21]. A single study regarding SKCa channels in ES has been published [23]. The authors demonstrated abnormal expression of KCNN1, which encodes SK1 in ES cells. They also showed that the EWS::FLI1 fusion protein regulates KCNN1 expression at the mRNA level, due to its ability to bind DNA near the KCNN1 promoter in an open chromatin region enriched with the GGAA sequence motif. Furthermore, it appears that KCNN1 expression renders ES cells susceptible to hypoosmotic solutions.
As a continuation of this initial single study, the aim of our study was to extend this work to a larger cohort of patients and cell lines, to highlight the role of EWS::FLI1 and EWS::ERG in the regulation of KCNN1 expression, to identify the role of KCNN1 in the regulation of a key function of tumor development and to determine the role of KCNN1 in the control of membrane potential and calcium influx through Store-Operated Calcium Entry (SOCE) and Constitutive Calcium Entries (CCE).
Results
KCNN1 is overexpressed in ES patients and cell lines
The expression of the three genes encoding SKCa channels was first compared in ES biopsies, using the publicly available R2 databases (https://r2.amc.nl, Table S2). RNA-Seq data from a cohort of 117 ES patients show that KCNN1 is significantly more highly expressed in ES patients than KCNN2 or KCNN3 (Fig. 1A). Comparison of KCNN1 expression across multiple tumor biopsies shows that KCNN1 is only highly expressed in biopsies from ES patients (Fig. 1B), unlike KCNN2 and KCNN3 which are expressed in various tumors (Supplementary Fig. 1A, B). Of note, KCNN1 expression is significantly correlated with patient survival at 5 years after diagnosis using two different patient cohorts [24, 25] (Fig. 1C and Supplementary Fig. 1C).
A Gene expression (Log2 normalized expression) of genes encoding SKCa channels (KCNN1, KCNN2 and KCNN3) in ES patients (117 patients) (Brown–Forsythe and Welch Anova test). B Gene expression (Log2 normalized expression) of KCNN1 in biopsies from patients with different tumors (Kruskal–Wallis test). C Overall survival probability (Kaplan Meier curve) of ES patients according to KCNN1 expression (high or low). The Bonf p corresponds to the p-value after the Bonferroni correction for multiple comparisons. Data are derived from the GSE17679 (see Supplementary Table S2). The method used to define high or low expression of KCNN1 is performed using the Kaplan Scan where an optimum survival cut-off is established based on statistical testing (see R2: Genomics Analysis and Visualization Platform)”. D Clinical tumor samples of 18 ES patients were fixed, embedded in paraffin, sectioned and immunostained with SK1 antibody. One representative photomicrograph is shown. E Expression of KCNN1 in normal tissues from GTEx database. TPM transcripts per million. F Gene expression (Log2 normalized expression) of KCNN1 in Mesenchymal Stem Cells (MSCs) coming from thirty healthy donors (30 donors) and in patients suffering from ES (117 patients) (Unpaired t-test). G Expression of KCNN1 in tumor-derived cell lines. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001. (In A, B, F, the boxes boundaries indicate the first and third quartiles - and the midline represents the median – of genes expression. The whiskers indicate the fifth and the ninety-fifth percentiles. In E, G, boxes represent the interquartile range, upper and lower whiskers the largest and smallest values, respectively. TPM transcript per million).
Immunohistochemical experiments were then performed to analyze the levels of SK1 in 18 clinical samples of patients with ES (Table S5). The intensity of staining varies from patient to patient. Specifically, in 7 of the 18 biopsies, areas with high SK1 levels were detected (Fig. 1D). In 5 biopsies no SK1 expression was demonstrated, and areas with moderate expression were shown in 6 biopsies (Supplementary Fig. 1D). Tumor heterogeneity in the same patient is illustrated by the presence of areas with no staining and areas with either high or moderate staining. Interestingly, when SK1 expression is high, a pericellular reinforcement of the label is observed, suggesting membrane localization (Supplementary Fig. 1D). Of note, the relatively small number of patients made it impossible to associate SK1 expression levels with patients’ clinical data. Regarding KCNN1 expression in healthy tissues, analysis across RNA-Seq samples, from the Genotype-Tissue Expression (GTEX, normal tissue, Fig. 1E) and in bone marrow-Mesenchymal Stem Cells (MSCs, R2 database), the putative cell type of origin of ES (Fig. 1F), shows higher expression of KCNN1 only in brain tissues. Other healthy tissues (Fig. 1E) or MSCs (Fig. 1F) barely express KCNN1.
At the cellular level, KCNN1 expression data assessed in numerous tumor-derived cell lines indicates restricted high expression in ES cells (Fig. 1G). These results were confirmed by comparing KCNN1, KCNN2, and KCNN3 expression in seven ES and six OS cell lines (GSE229906, Supplementary Fig. 1E, F).
Taken together, these results show that (i) KCNN1 is overexpressed in biopsies from ES patients and in ES cells, and that (ii) KCNN1 expression is associated with patient survival at 5 years after diagnosis.
KCNN1 and SK1 are transcriptionally regulated by the EWS::FLI1 fusion protein
The regulation of KCNN1 expression by EWS::FLI1 or EWS::ERG was first studied, using various ES cells, stably transduced with doxycycline-inducible shRNAs targeting EWS::FLI1 or EWS::ERG (Fig. 2A and Supplementary Fig. 2A, B). Transcriptomics data indicate that reduction of EWS::FLI1 expression significantly reduces KCNN1 expression in the 12 cell lines studied (Fig. 2A and Supplementary Fig. 2B). Notably, reduction of EWS::ERG expression significantly reduces KCNN1 expression in the two cell lines tested (Supplementary Fig. 2A). The regulation of KCNN1 expression was then studied using RT-qPCR and western-blot assays. Decreased EWS::FLI1 expression leads to decreased KCNN1 expression at the mRNA and protein levels (Fig. 2B). Of note, decreased EWS::FLI1 protein level is observed as early as 8 h after treatment of the cells with doxycycline (Supplementary Fig. 2C). To confirm these results in other ES cell lines, a pool of siRNAs targeting EWS::FLI1 was used in three cell lines. Decrease EWS::FLI1 expression leads to a decrease in KCNN1 expression of 75%, 40%, and 50% in A-673, RD-ES and SK-ES-1, respectively (Fig. 2C). The observed effects at the mRNA level are also translated to the protein level (Fig. 2D).
A KCNN1 expression (Log2 normalized expression) in A-673 cell line stably transduced with a doxycycline-inducible shRNA targeting EWS::FLI1 in control condition (−Doxycycline) or after doxycycline treatment (+Doxycycline). B. Normalized and relative EWS::FLI1 (left panel) and KCNN1 (middle panel) gene expressions in A-673/TR/shEF cell line after doxycycline treatment (1 µg/mL) (N = 5). Western-blots of SK1 and EWS::FLI1 (right panel) in A-673/TR/shEF cell line after doxycycline treatment (1 µg/mL) (N = 2). C Normalized and relative EWS::FLI1 and KCNN1 gene expressions in A-673 (N = 3), RD-ES (N = 4), and SK-ES-1 (N = 3) cell lines after 48 h of transient transfection with a pool of siRNAs targeting EWS::FLI1 (siRNA EWS::FLI1) or a siRNA control (siRNA CT) (upper panels). D Western blots of SK1 and EWS::FLI1 in A-673, RD-ES and SK-ES-1 cell lines after 72 h of transient transfection with a pool of siRNA targeting EWS::FLI1 (siRNA EWS::FLI1) or a siRNA control (siRNA CT) (N = 2) (lower panels). E Different KCNN1 transcripts according to their position on the genome. Brown rectangles correspond to transcribed and translated exons, while the salmon rectangles correspond to transcribed but not translated exons. White spaces are intronic sequences. F Normalized expression of KCNN1 transcripts (FPKM) in A-673 cell line (N = 3). G Normalized expression of KCNN1 transcripts (FPKM) in A-673/TR/SHEF cell line at day 0 and day 7 of doxycycline treatment (1 µg/mL). *: p < 0.05; **: p < 0.01 (Mann–Whitney tests). (In A, the boxes boundaries indicate the first and third quartiles - and the midline represents the median – of genes expression. The whiskers indicate the fifth and the ninety-fifth percentiles. In B, C, bars indicate means ± SD of relative and normalized EWS::FLI1 or KCNN1 mRNA expression determined by RT-qPCR. In E, F, FPKM Fragment Per Kilobase Per Million reads.).
Analysis of RNA-Seq data, based on an alignment to the transcriptome sequence, shows that A-673 cells express four of the eight KCNN1 transcripts described in the literature (NCBI RefSeq) (Fig. 2E), namely A (NM_002248.5), B (NM_001386976.1), C (NM_001386977.1) and E (NM_001386974.1) (Fig. 2F). The other six ES lines that were studied significantly expressed only the A, B and C transcripts (Supplementary Fig. 3). EWS::FLI1 silencing in A-673/TR/SHEF cells results in a decrease in expression level of the three major KCNN1 transcripts (Fig. 2G).
ChIP-Seq data from the A-673 cell line (Fig. 3A, left panel) show a significant peak for H3K4me3 (a histone modification marking active promoters [26]), as well as a significant peak for H3K27ac (a histone modification marking active promoters and enhancers [27]) and a FLI1 peak (with significant q. value) in close proximity to the KCNN1 promoter regions of the short transcripts. Our results appear to correspond to the positions observed by Fuest and colleagues, concerning the binding of EWS::FLI1 to an alternative enhancer in the second intron of the KCNN1 A transcript [23]. The same results can be observed for the 11 other ES cell lines characterized by the EWS::FLI1 fusion protein (Supplementary Fig. 4A). Of note, a similar regulatory mechanism by the EWS::ERG fusion protein can be observed for EW-3 and TC-106, two cell lines characterized by the presence of the EWS::ERG fusion protein (Fig. 3A, right panel and Supplementary Fig. 4A). These EWS::FLI1/EWS::ERG binding sites contain at least 12 GGAA microsatellites repetitions, indicated by the red vertical lines (Fig. 3A–D and Supplementary Fig. 4A).
A H3K4me3, H3K27ac and FLI1 ChIP-Seq data with quantitative value of peaks around KCNN1 promoters and sequences of EWS::FLI1 binding sites in A-673 (left panel) and TC106 (right panel) cell lines. B H3K27ac ChIP-Seq of A-673/TR/shEF cell line at day 0 and day 7 of doxycycline treatment with quantitative value of peaks around KCNN1 promoters. C FLI1 ChIP-Seq of A-673/TR/shEF cell line with quantitative value of FLI1 peaks around KCNN1 promoters, at day 7 of doxycycline treatment, and 3 days (day 7 + 3), 4 days (day 7 + 4), 7 days (day 7 + 7) and 10 days (day 7 + 10) after discontinuation of doxycycline treatment. D H3K27ac and FLI1 ChIP-Seq data on MSCs transfected with control plasmid, FLI1 overexpression plasmid or EWS::FLI1 overexpression plasmid around KCNN1 promoters.
To confirm the binding of EWS::FLI1 around KCNN1 promoter regions, publicly available H3K27ac (Fig. 3B) and FLI1 (Fig. 3C) ChIP-Seq data from the A-673/TR/shEF cell line were then used. Interestingly, the amplitude of the H3K27ac peak near the KCNN1 promoter decreases after seven days of treatment, that is, after silencing of EWS::FLI1 (Fig. 3B, lower panel), confirming the decreased expression of KCNN1 shown in Fig. 2A. FLI1 ChIP-Seq data show EWS::FLI1 binding at this chromosomal region (Fig. 3C), with a FLI1 peak associated with the H3K27ac activation marker (Fig. 3B) which gradually reappears after re-expression of EWS::FLI1 following the interruption of treatment with doxycycline (Fig. 3C).
It can be noted that EWS::FLI1 silencing leads to an increase in KCNN3 expression (Supplementary Fig. 4B) with EWS::FLI1 binding being observed in a chromosomal area in the vicinity of the KCNN3 promoter (Supplementary Fig. 4C). Following a shRNA-mediated silencing approach in ES cells, an approach of overexpression of EWS::FLI1 in the putative cell of origin of ES, MSCs, was used. Neither H3K27ac nor the FLI1 peak is observed around KCNN1 promoter areas in MSCs transfected with the control plasmid (Fig. 3D, upper panels) or FLI1 overexpression plasmid (Fig. 3D, middle panels). In contrast, when EWS::FLI1 was overexpressed, H3K27ac and EWS::FLI1 peaks appear around the promoter ___domain of KCNN1 transcript C (Fig. 3D, lower panels).
Altogether, these results demonstrate transcriptional regulation of KCNN1 by EWS::FLI1 or EWS::ERG, enabled by their binding to GGAA microsatellites.
KCNN1 is involved in the regulation of ES cell proliferation
The involvement of KCNN1 in the regulation of ES cell cycle was studied, using doxycycline-inducible shRNAs targeting KCNN1 in A-673 cells. The efficacy of shRNAs was first evaluated on KCNN1 expression after 72 h using RT-qPCR (Fig. 4A, shRNA 511, 908, and 576) and RNA-Seq (Supplementary Fig. 5A, GSE229906, shRNA908, left panel). The observed effects at the mRNA level are translated to the SK1 protein level (Supplementary Fig. 5A, shRNA 908, right panel). A similar decrease of KCNN1 expression at the mRNA level was obtained after 7 days of doxycycline treatment (Supplementary Fig. 5B).
A KCNN1 expression in A-673 cell line stably transduced with doxycycline-inducible shRNA control cells (shRNA CT) or 3 shRNA cells targeting KCNN1 (shRNA 511, shRNA 908 and shRNA 576) after 72 h of treatment with doxycycline (0.5 µg/mL). Bars indicate mean ± SD of relative and normalized KCNN1 mRNA expression obtained after RT-qPCR (N = 5 for shRNA CT and shRNA 908, N = 4 for shRNA 511 and N = 3 for shRNA 576). B Clonogenicity assays of A-673 cell line stably transduced with shRNA control (shRNA CT) or three shRNA targeting KCNN1 cells (shRNA 511, shRNA 908 and shRNA 576) after 72 h of doxycycline treatment (0.5 µg/mL), with the colonies numbers observed and the relative surface area of clones. Bars indicate mean ± SD of counted colonies or the clones surface area observed on the left panel relative to the untreated cells (N = 6 for shRNA CT, N = 3 for shRNA 511 and shRNA 908 and N = 7 for shRNA 908). C Cell cycle assays on shRNA 908 cells treated (shRNA 908 +Doxycycline) or not (shRNA 908 −Doxycycline) with doxycycline 0, 6, 8 or 10 h after thymidine double block. Dark blue: G0/G1 phase, Light blue: S phase, Purple: G2/M phase. D Percentage of cell distribution 6, 8, and 10 h after thymidine double block (N = 5 for 6 and 10 h after thymidine double block; N = 6 for 8 h after thymidine double block, Wilcoxon test). E Western blots of p21 and Aurora A and their quantification normalized to β-tubuline 10 h after the thymidine double block (N = 3). *p < 0.05; **p < 0.01 (A, B, D, E: Mann–Whitney test).
Seventy-two hours after doxycycline treatment of the transduced cells, a decrease in the ability of KCNN1-targeted-shRNA cells to form colonies is observed (Fig. 4B). Indeed, a significant decrease in the number of clones is observed with a decrease of at least 50% for shRNA 908 and shRNA 576 cells (Fig. 4B, left and middle panel). Clones size is also significantly smaller for all three shRNA samples, compared to control cells (shRNA CT, Fig. 4B, left and right panels). These results were confirmed with viability assays performed on MHH-ES-1 cells transfected with a pool of siRNAs targeting KCNN1 (Supplementary Fig. 5C). To better understand the involvement of KCNN1 in the regulation of ES cell proliferation, cell cycle assays were then performed on synchronized cells (Fig. 4C,D). On average, 95% of cells are synchronized in S phase at the end of the thymidine double block (Fig. 4C). A slowdown of the cell cycle is observed 10 h after the end of the double block, with the majority of shRNA 908 cells treated with doxycycline still being in the S phase (49.13% vs 33.55%).
Measurement of expression of p21 and Aurora A, known to accumulate in G2/M phase, confirms the slowdown of the cell cycle after KCNN1 silencing (Fig. 4E). Specifically, 10 h after the end of the thymidine double block, a higher expression of p21 and Aurora A is measured, illustrating a faster return of cells to G2/M phase in control condition than after KCNN1 silencing (Fig. 4E and Supplementary Fig. 5D). It can be noted that KCNN1 silencing by a pool of siRNA against KCNN1 also leads to an increase in cell sensitivity to doxorubicin (Supplementary Fig. 6A). Indeed, after KCNN1 silencing, a significant decrease in cell viability was observed in cells treated with 10 nM doxorubicin, whereas no effect was observed in control conditions. In addition, when cells were treated with 30 nM doxorubicin, KCNN1 silencing significantly increased cell response to doxorubicin. In contrast no modification in the ability of A-673 cells to migrate was observed (Supplementary Fig. 6B).
Together, these results demonstrate the involvement of KCNN1 expression in the regulation of key functions in tumor development, in particular in the control of the ES cell cycle and the ability of cells to form colonies.
KCNN1 expression modulates calcium flux
Because it is known that SKCa controls the membrane potential of various cell types including cancer cells, KCNN1 involvement in membrane potential regulation was investigated. Functionally, KCNN1 silencing leads to plasma membrane depolarization in a 2D or 3D cell model (Fig. 5A,B). Indeed, the DiBAC4(3) fluorescence measurement is significantly higher in shRNA 511, shRNA 908 and shRNA 576 cells than in shRNA CT cells (Fig. 5A). In addition, measurements of the membrane potential of shRNA 908 spheroids show a significant depolarization of 20 mV compared to the shRNA CT spheroids (Fig. 5B). As expected, DiBAC4(3) fluorescence measurements showed the ability of compound GW542573X, an activator of the SK1 channel to hyperpolarize the cell membrane (Fig. 5C).
A Measures of the plasma membrane depolarization in A-673 cell line stably transduced with shRNA control cells (shRNA CT) or three shRNA cells targeting KCNN1 after 7 days of doxycycline treatment (0.5 µg/mL). (N = 48 for shRNA CT and shRNA 908, N = 46 for shRNA 511, N = 45 for shRNA 576). B Membrane potential measures of shRNA CT or shRNA 908 spheroids treated 7 days with doxycycline (0.5 µg/mL) (N = 7). C Plasma membrane depolarization measures in A-673 cells treated with the SK1 channel activator GW542573X (N = 50). D Effect of KCNN1 knockdown on SOCE in A-673 cell line stably transduced with shRNA control (shRNA CT) or three shRNA targeting KCNN1 and the relative fluorescence measurement of SOCE induced by TG (N = 26). E Effect of KCNN1 knockdown on CCE using Mn2+ quenching assay and the normalized Mn2+ quenching slope (N = 21 for shRNA CT, N = 20 for shRNA 511 and N = 22 for shRNA 908 and shRNA 576). F Effect of the SK1 channel activator GW542573X on SOCE in A-673 cells and the relative fluorescence measurement of SOCE induced by TG in GW542573X conditions (N = 14 for Control and N = 21 for G542573X condition). G Effect of GW542573X on CCE using Mn2+ quenching assay and the normalized Mn2+ quenching slope (N = 28 for Control and N = 20 for GW542573X condition). *p < 0.05; **p < 0.01; ***p < 0.001 (boxes indicate the first and third quartiles—and the median inside—of the different measures. The whiskers indicate the fifth and the ninety-fifth percentiles. A, D, E Anova test; B, F, G Mann–Whitney test and C: Unpaired t-test).
Next, to identify the molecular mechanisms by which the KCNN1 expression affects ES cell proliferation, the effect of its silencing on calcium signaling was investigated. SOCE and Constitutive Calcium Entry (CCE) were measured using Fura-2AM and the Mn2+ quenching techniques, respectively. A significant decrease in calcium entry measured after SOCE activation by thapsigargin (TG) and following addition of 2 mM calcium to the extracellular solution, in KCNN1 knocked-down cells in comparison to the shRNA CT cells (Fig. 5D). Silencing KCNN1 expression after treatment of the cells with doxycycline also reduces the Mn2+ quenching rate (Fig. 5E) in all three shRNA cells, compared to the shRNA CT cells, indicating decreased CCE. On the other hand, in the presence of the SK1 channel activator GW542573X, an increase of both SOCE and CCE is observed in the A-673 cell line (Fig. 5F,G). A decrease in calcium entries is also observed in KCNN1-knocked-down SK-ES-1 cells compared with the shRNA CT cells (Supplementary Fig. 7A, B).
Overall, these results indicate that KCNN1 expression and SK1 activity are involved in the control of membrane potential and calcium influx in ES cells.
An increase in extracellular potassium concentration alters membrane potential, calcium influx and the ability of ES cells to form colonies
To demonstrate a possible link between plasma membrane potential and ES cell proliferation, A-673 cells were depolarized with 40 mM KCl, a condition previously shown to make the plasma membrane more positive and reduce the driving force for calcium entry (Fig. 6A). As expected, this reduction in the driving force for calcium entry led to a decrease in calcium influx (Fig. 6B, C) and a significant decrease in the number of clones (Fig. 6D, left and middle panel). Clone sizes were also significantly smaller (Fig. 6D left and right panel).
A Measurement of plasma membrane potential in A-673 cells cultured in potassium-enriched (40 mM KCl) or non-potassium-enriched (40 mM NaCl) medium (N = 25). B Effect of 40 mM KCl (high K+) on SOCE in A-673 cell line and the relative fluorescence measurement of SOCE induced by TG (N = 16). C Effect of 40 mM KCl (high K+) on CCE using Mn2+ quenching assay and the normalized Mn2+ quenching slope (N = 15). D Clonogenicity assays of A-673 cell line in presence of high concentration of extracellular potassium (40 mM, High K+) or low concentration of extracellular potassium (5 mM, low K+), with the colonies numbers observed and the relative surface area of clones. Bars indicate mean ± SD of counted colonies or the clones surface area observed on the left panel relative to the untreated cells (N = 3). Bars indicate means ± SD of colonies numbers and the surface occupation. *: p < 0.05; **: p < 0.01; ****: p < 0.001. In B and C, boxes indicate the first and third quartiles - and the median inside - of the different measures. The whiskers indicate the fifth and the ninety-fifth percentiles. A, B, C, and D: Mann–Whitney test.
Thus, these results indicate that the proliferation of ES cancer cells is regulated by plasma membrane potential.
Discussion
Intercellular communication, ensured in part by ion channels is essential for maintaining tissue homeostasis. Among these ion channels, the potassium channel family is the most important with, in particular, SKCa channels [28, 29]. As mentioned in the introduction, a single report has described abnormal expression of KCNN1 in ES [23]. Using RNA-Seq data from patients’ biopsies of various cancers and from multiple tumor cell lines, our work has demonstrated an overexpression of KCNN1 specifically in ES, supplementing the single previous study [23]. In addition, SK1 expression at the protein level was detected in a majority of the patient biopsies studied, in particular with membrane expression in areas where high SK1 expression was observed. Furthermore, our work has demonstrated very low expression of KCNN1 in healthy tissues, with the exception of the brain, complementing and reinforcing the study of Fuest and colleagues, who demonstrated specificity of KCNN1 expression in ES tumor compared to healthy bone tissue [23]. It can therefore be hypothesized that targeting KCNN1 with a strategy using a candidate that does not cross the blood-brain barrier would limit the development of side effects.
Several studies have shown that EWS::FLI1 is able to regulate the expression of various genes. Both FLI1 and the fusion protein EWS::FLI1 bind the canonical ETS binding motif, GGAA [30, 31]. It has also been proven that EWS::FLI1 is able to bind GGAA microsatellites [32, 33], which can be repeated up to 20 times. This fusion protein is known to be able to recruit chromatin remodeling complexes and the transcription machinery [34], which can lead to the emergence of variant transcripts [35]. Here using 12 ES cell lines, we have highlighted the previous observation showing that KCNN1 expression is regulated by EWS::FLI1. Indeed, in all these cell lines, EWS::FLI1 bound GGAA microsatellites close to the promoter of various KCNN1 transcripts, as seen in previous data [23]. In this context, it has been shown in previous studies that EWS::FLI1 may be able to change the 3D configuration of chromatin by binding GGAA microsatellites either by disturbing the Topological Associated Domains, which are epigenetics structures involved in the regulation of genes expression, or by modulating chromatin loops, and thus, altering genes transcription [36]. Furthermore, we have demonstrated that among the eight KCNN1 transcripts described in the literature, three are regulated by EWS::FLI1, namely A (NM_002248.5), (NM_001386976.1) and C (NM_001386977.1). On the other hand, the results from overexpression of EWS::FLI1 in MSCs proves that the transcription of the KCNN1 C transcript requires the expression of the fusion protein, as FLI1 was not sufficient to induce KCNN1 transcription. We have also demonstrated for the first time that KCNN1 expression is regulated by not only EWS::FLI1 but also by EWS::ERG, confirming the ability of this rarer fusion protein to induce genes expressed only in ES tissues, similar to EWS::FLI1 [37].
Regarding the role of KCNN1 in a key function of tumor development, our results have shown that the silencing of KCNN1 expression decreases the ability of ES cells to form colonies and slows down the cell cycle. The mechanisms by which potassium channels regulate cell proliferation are the subject of three consensus theories [38]: (1) oscillation of the membrane potential that participates in the control of cell volume and thus allows cells to pass the “cell volume checkpoint” for successful cell cycle progression, (2) potassium efflux through potassium channels can lead to membrane hyperpolarization, which increases the driving force for calcium entry through a calcium-permeable channel at the plasma membrane. The increase in intracellular calcium concentration in turn promotes proliferation through calcium signaling. The third theory would be explained by non-canonical functions of potassium channels, that is independently of a functional channel [38]. Although Fuest and colleagues did not recorded SK1 currents in ES cells [23], our work have shown that KCNN1 silencing leads to membrane depolarization in a 2D and 3D cell models, and to a decrease in both SOCE and CCE, suggesting a mechanism of action according to the second hypothesis without excluding the third one. Indeed, the effect of an SK1 channel activator and opener [39, 40] on both membrane potential and calcium entries strongly suggests that KCNN1, which is translated to SK1 protein, may express an SK1 channel, that the control plasma membrane potential of ES cells and thus calcium entries through calcium channels. In this context, we can note that a study has identified oncocomplexes of SK3 with calcium channels in various cancer cells, these oncocomplexes controling calcium entry and thus cancer migration and metastasis development [9]. Furthermore, experiments carried out with high concentrations of extracellular potassium show that membrane depolarization induced by a decrease in potassium efflux is accompanied by a reduction in calcium influx and the ability of ES cells to form colonies. These results highlight a potential oncocomplex involving SK1 and calcium channels that may promote the proliferation of Ewing sarcoma. Further studies are needed to identify the calcium channel components (such as the Orai and TRP families) associated with SK1 in ES cell.
In addition to a crucial role for KCNN1/SK1 in controlling cell proliferation, KCNN1 expression also regulates the ability of ES cells to respond to chemotherapy. It can be hypothesized that the association between patient survival and KCNN1 expression, demonstrated in two independent cohorts of patients, is closely linked to the ability of KCNN1 to modulate the cellular response to chemotherapy, this process being strongly associated with patient survival [6].
Regarding the expression of other SKCa channel genes in ES cells, our work has shown that KCNN3 expression increases after EWS::FLI1 silencing. These results suggest that cells that strongly express EWS::FLI1 would strongly express KCNN1, which in turn would stimulate cell proliferation. Conversely, cells with low expression of EWS::FLI1 would more strongly express KCNN3, which would participate in the stimulation of cell migration as observed in bone metastases [15]. This hypothesis is consistent with the work of Franzetti et al., who have shown that ES cells highly expressing EWS::FLI1 are characterized by highly active cell proliferation, and ES cells with low EWS::FLI1 expression have a high propensity to migrate, invade and metastasize [41]. In addition, it has been demonstrated that the number of GGAA repeats in microsatellites influences the regulation of EWS::FLI1-targeted genes [42]. Indeed, they demonstrated that four GGAA repetitions – or more (within a limit of 20) – of GGAA activates EWS::FLI1-regulated genes, while genes repressed by EWS::FLI1 are associated with a canonical ETS binding site (a single GGAA motif) [42]. Only two GGAA repeats have been identified in the EWS::FLI1 binding site in the KCNN3 promoter, suggesting a repression of this gene by the fusion protein.
To summarize, our work has dissected the regulation of transcription of the various KCNN1 isoforms by not only EWS::FLI1 but also EWS::ERG. Our work has identified a major role for KCNN1 in the regulation of a key function in tumor development, cell proliferation, and has suggested the role of SK1 in modulating calcium fluxes through its hyperpolarizing effect on the plasma membrane. In conclusion, our results have demonstrated the overexpression of KCNN1 in ES patients, identifying KCNN1 as a novel potential therapeutic target in this tumor.
Materials and methods
Cell culture and reagents
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Basel, Switzerland) or in Rosewell Park Memorial Institute Medium (Lonza), supplemented with 10% Fetal Bovin Serum (FBS, Lonza). The A-673/TR/shEF cell line, is a stably transduced A-673 cell line with a doxycycline-inducible shRNA targeting EWS::FLI1 [43]. A list of ES cell lines and their fusion type is provided in Table S1. For experiments in high potassium (high K+) condition, the medium was supplemented with 40 mM of KCl (Sigma, St. Quentin Fallavier, France). GW542573X and doxycycline were purchased from Tocris (Bristol, United Kingdom) and Sigma-Aldrich, respectively. All cell lines were subjected to a complete validation process and were authenticated by Short Tandem Repeat (STR) profiling analysis (Eurofins, Germany). Experiments were performed using mycoplasma-free cell lines.
Bioinformatics analyses
The “R2: Genomics Analysis and Visualization Platform” website provides free access datasets from patient biopsies. Multiple datasets were used to compare KCNN1 expression and to study the survival rate of ES patients (Table S2). The GTEx (https://gtexportal.org/home/) and the Cancer Cell Line Encyclopedia (CCLE, https://sites.broadinstitute.org/ccle/) were used to compare KCNN1 expression levels in healthy tissues (GTEx version 8) and tumorous cell lines (CCLE - DepMap 22Q4), respectively.
RNA-seq analyses
RNA-seq was performed for ES and osteosarcoma cells and gene-level quantification was realized as previously described [44].
For transcript-level quantification, RNA-seq experiments for the A-673, EW-3, EW-24, MHH-ES-1, RD-ES, SK-ES-1, and TC-71 cell lines were performed by ActivMotif Inc. Libraries were prepared in triplicate using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Sequencing was then performed on an Illumina NextSeq 500 in a 2 × 42 bp (PE42) format. RNA-seq data for A-673/TR/SHEF without induction (day 0) and with induction of the shRNA targeting EWS::FLI1 (day 7) came from Gene Expression Omnibus (GEO) GSE164373 [45], also in triplicates for each condition. These data were filtered and aligned to the same reference genome, and quantified based on the annotation file from the RefSeq associated with that genome. Packages used for the analyses have been detailed in Table S3.
ChIP-seq analyses
ChIP-Seq data were derived from the GSE176400 [46], GSE129155 [47], and GSE94278 [34] entries in the GEO database. GSE176400 contains ChIP-seq data from 14 ES lines with inducible fusion-type specific EWS::ETS knockdown. GSE129155 contains ChIP-seq data for inducible EWS::FLI1 knockdown in the cell line A673/TR/shEF. GSE94278 contains Bigwig files of normalized ChIP-Seq values of MSCs transfected with a control plasmid, a FLI1 plasmid, and an EWS::FLI1 plasmid. These data were filtered and aligned to the GRCh38.p14 reference genome analysis set from NCBI. Packages used for the analyses have been detailed in Table S4.
Patients data and paired slide immunohistochemistry
A cohort of 18 ES diagnostic tumors were collected retrospectively in University Hospitals of Strasbourg between 2005 and 2022. Those patients were treated in or according to the past Euro-E.W.I.N.G.99 and EURO EWING 2012 protocols [48, 49]. The research protocol was validated by the local institutional ethic committee at University Hospitals of Strasbourg for human tissue experiments and a CNIL declaration 1970 390 v0 was obtained. All patients and their parents gave their written informed consent. The diagnostic anonymized paraffin-embedded (PE) tumor samples were stored in the pediatric tumor bank at the Biological Resource Centre of Strasbourg. 3-µm sections of those ES PE tumor tissues were cut and stained for SK1 with anti-human SK1 antibody (Genetex, Irvin, CA, USA). After baking slides for 1 h at 60 °C, samples were processed routinely via deparaffinization, rehydration, antigen retrieval in Tris-EDTA buffer pH9 for 20 min at 97 °C. The endogenous peroxidase activity was then quenched, and non-specific binding was subsequently blocked with 2% (v/v) normal donkey serum (Jackson ImmunoResearch) and 1% (p/v) B.S.A. in 1× TBS Tween pH 7.4 for 25 min at room temperature. Samples were then incubated with appropriate antibody diluted at 1:50 in blocking solution for 1 h at room temperature. Negative control was achieved by omitting a primary antibody. Following the primary antibody incubation, sections were washed in 1× TBS Tween pH 7.4 and samples were incubated for 45 min at room temperature using a polyclonal biotinylated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch), and finally with an HRP conjugated streptavidin complex (Agilent) for another 45 min at room temperature. Revelation was conducted with DAB liquid chromogen (microm-microtech France) and counterstained with Gill-2, dehydrated through graded ethanol baths, cleared in OTTIX-plus and cover-slipped with Pertex mounting media.
Sections were analyzed using standard light microscopy. Each sample was examined twice by two experimentators blinded to the experiment. The expression and distribution of SK1 were assessed within the sections. The intensity of staining was assessed semi-quantitatively in reference to the most intensely stained slide as follows: no expression, high expression, and intermediate expression qualified as moderate.
RT-qPCR (Reverse Transcription – quantitative Polymerase Chain Reaction)
RNA was extracted using the NucleoSpin RNA Plus Kit (Macherey-Nagel, Duren, Germany). Reverse Transcription (RT) was performed from 1 µg of RNA with the “Maxima H Minus First Strand cDNA Synthesis Kit” (Macherey-Nagel). Quantitative Polymerase Chain Reaction was performed using SYBR Select Master Mix (ThermoFischer) and the primers listed in Table 1.
Western blot
Protein extraction and Western blots were performed as previously described [44]. The antibodies used targeted Aurora A (Cell Signaling Technology (CST), Danvers, MA, USA), p21 (CST), SK1 (Genetex, Irvin, CA, USA), FLI1 (Abcam, Cambridge, UK), β-actin (CST) and β-tubulin (CST). Antibody binding was visualized with a fluorescence system or an enhanced chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, ThermoSientific, Illkirch, France).
Transienty siRNA transfection
ES cells were transfected with a pool of five siRNAs targeting EWS::FLI1 (Dharmacon, Horizon, Perkin-Elmer, Villebon-sur-Yvette, France) or KCNN1 (SantaCruz, Dallas, TX, USA) or with a pool of five control siRNAs using Lipofectamine RNAiMax (ThermoFischer). RNAs and proteins were respectively harvested after 48 h and 72 h post transfection, respectively.
shRNA stable transduction
The A-673 and SK-ES-1 cell lines were transduced with three doxycycline-inducible shRNAs targeting KCNN1 (Dharmacon) or a doxycycline-inducible non-targeting control shRNA (Dharmacon). The expression of KCNN1 was measured by RT-qPCR after 72 h or 7 days of doxycycline treatment (Sigma-Aldrich). Before each experiment, cells were pre-treated with doxycycline for 72 h or 7 days and the treatment was maintained afterwards.
Membrane depolarization measurements
Extemporaneously, 0.18% glucose was added to Physiological Salin Solution (PSS, 137 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.42 mM Na2HPO4 (anhydrous), 0.44 mM Na2HPO4 (hydrated), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4.17 mM NaHCO3, pH 7.4). DiBAC4(3) stock solution was prepared following Thermofischer recommendations. Adherent cells were loaded for 15 min at 37 °C with the 1 µM DiBAC4(3) in PSS. Fluorescence was measured at 520 nm using a FlexStation Microplate Reader (Molecular Devices, San Jose, CA, USA), in response to an excitation wavelength of 485 nm. Then, a second fluorescence measurement was taken, after a 10 min incubation of the cells with 100 µL of 1 µM DiBAC4(3).
For membrane depolarization measurements in 3D, spheroids were formed through use of the scaffold-free method cell suspensions. Cell suspensions (100 μl of cell suspension at 4.104 cells/ml) were added to Ultra-Low Attachment microplates (Corning, Glendale, AZ, USA) and the plates were incubated in standard, stationary cell culture conditions for 72 h. Spheroids membrane potentials were measured as described previously [50].
Clonogenicity assays
Five hundred A-673 cells were incubated for 8–10 days after pre-treatment under standard conditions. Cells were fixed with 1% glutaraldehyde and stained by crystal violet. The number of colonies was then counted with a Cell Counter from Fiji Software and their surface was measured using a macro created by the Cellular and Tissular Imaging Core Facility of Nantes University (MicroPICell, Nantes, France).
Viability assays
After siRNA transfection, viability assays were performed on re-seeded cells, using an Omni device from CytoSMART (Axion Biosystems, Atlanta, GA, USA). Cell viability was monitored for 72 h in time-lapse, with a scan every 4 h. Data were collected on the CytoSMART Cloud, and the analyses were performed using the same software.
TranswellTM motility
Fourty-eight hours after siRNA transfection, 60,000 cells/well were seeded onto the upper surface of transwell inserts (Falcon, Franklin Lakes, NJ) and incubated at 37 °C for 8 h. At the end of the incubation period, cells on the upper surface of the inserts were wiped off, and the cells on the underside of the membrane were fixed, stained with crystal violet and counted by bright-field microscopy in five random fields.
Thymidine synchronization
After their adhesion, cells were treated with 2 mM thymidine (Sigma-Aldrich) for 18 h and left in DMEM containing 10% FBS for 8 h. The cells were then treated for a second time with 2 mM thymidine for 16 h before being left in DMEM containing 10% FBS. Cell distribution was observed by flow cytometry on the Platform Cytocell at Nantes University (FACSymphony A5, BD, Franklin Lakes, USA), after 0, 6, 8, and 10 h or not synchronized. Proteins were also extracted for western-blot analyses.
Cell cycle analyses
After thymidine synchronization, cells were harvested and fixed with 70% ethanol before being suspended in a phospho-citrate buffer (Na2HPO4 0.2 M, citric acid C6H8O7 0.1 M, pH 7.5). Cells were then resuspended in PBS, 0.12% Triton, 0.12 mM EDTA and 100 µg/mL RNAse A (Promega A797c, Promega) and incubated for 30 min at 37 °C. Propidium Iodide (50 µg/mL) was added for 20 min in the dark at 4 °C. Cell distribution was observed by flow cytometry on the Cytocell Platform at Nantes University (FACSymphony A5). Data were analyzed with Multicycle software.
Store operated Ca2+ entry measurement by Fura-2 AM
As previously described [51], SOCE was measured after Fura-2-AM loading of cells for 45 min at 37 °C. Maximum of fluorescence (peak of Ca2+ influx F340/F380) was measured and compared to normal condition.
Constitutive Ca2+ entry measurement by Mn2+ quenching
As previously described [51], CCE was measured by manganese (Mn2+) (Manganese chloride, Sigma) quenching after loading cells with Fura-2-AM for 45 min at 37 °C.
Statistical analyses
Statistical analyses were performed using GraphPad Prism Software. Comparison of SKCa (KCNN1, KCNN2, and KCNN3) gene expression in patients suffering from ES was performed by a Brown–Forsythe and Welch ANOVA and a Games–Howell multiple comparisons tests. The comparison of KCNN1 gene expression between ES patients and MSCs coming from healthy donors was performed by an unpaired t-test and a Welch correction. The comparison of KCNN1 gene expression among multiple tumors was performed by a Kruskal–Wallis test and a Dunn’s multiple comparisons test. To compare two groups from in vitro experiments, Mann–Whitney tests were performed and ANOVA tests were administered when N > 30 and a normality test had been passed. Wilcoxon tests were used during cell cycle assays. α = 0.05 and p < 0.05 is considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Data availability
The datasets generated and/or analysed during the current study are available in the Gene Expression Omnibus repository (GEO) under accession number: GSE229906, GSE129155, GSE94278, GSE164373, GSE176400. The computer code produced in this study is available on GitHub: (https://github.com/EpistressLab/KCNN1_ewing_sarcoma).
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Acknowledgements
We thank the Cytometry Facility « Cytocell » from Nantes for their expert technical assistance (Cytocell) and le Centre de Ressources Biologiques des Hôpitaux Universitaires de Strasbourg. This work was supported by INCA (# 2018-151), Cancéropôle Grand Ouest (DIACAN, REOCHILD), Ligue contre le cancer (CD 41, 44, 49, 56, and 85), M la vie avec Lisa, Imagine for Margo, Societé Française de lutte contre les cancers et les leucémies de l’enfant et de l’adolescent, le Rallye du Cœur, l’association Mimi pour la vie and LifePink.
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MD, MG, MP-C, AC; OD; FR, CV, FV conceived the project. MD, MG, AP were responsible for the data curation. MD, MG, AP, RT, CV, FV analyzed the data. MD, MG, AP, RB, JA, MM were in charge of the investigation during the project. MD was also responsible for the methodology of the project. MD, MP-C, AC and FV validated the data and MD and FV wrote the original draft. MG, RT, MM, LR, BB-LR, MB, SG, FL, BO, OD, FR, CV, CH and NEW wrote the review and edited the manuscript. NEW and CH have provided ES diagnostic biopsies and anonymized paired clinical data from patients. FV was in charge of funding acquisition, project administration, resources and supervision during the project. All authors read and approved the final version of the manuscript and are accountable for all aspects of the work.
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All methods used were performed in accordance with the relevant guidelines and regulations. 18 ES diagnostic tumors collected retrospectively in University Hospitals of Strasbourg between 2005 and 2022 were used. Those patients were treated in or according to the past Euro-E.W.I.N.G.99 and EURO EWING 2012 protocols. The research protocol was validated by the local institutional ethics committee at University Hospitals of Strasbourg for human tissue experiments and a CNIL declaration 1970 390 v0 was obtained. All patients and their parents gave their written informed consent.
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Dupuy, M., Gueguinou, M., Postec, A. et al. Chimeric protein EWS::FLI1 drives cell proliferation in Ewing Sarcoma via aberrant expression of KCNN1/SK1 and dysregulation of calcium signaling. Oncogene 44, 79–91 (2025). https://doi.org/10.1038/s41388-024-03199-7
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DOI: https://doi.org/10.1038/s41388-024-03199-7
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