Plasma membrane SK2 channel activity regulates migration and chemosensitivity of high‐grade serous ovarian cancer cells

No data are currently available on the functional role of small conductance Ca2+‐activated K+ channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non‐cell‐permeant SK2 inhibitor Lei‐Dab7, we identified functional SK2 channels at the plasma membrane, regulating store‐operated Ca2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei‐Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei‐Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro‐migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression‐free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.


Introduction
High-grade serous ovarian cancer (HGSOC) is the most common subtype of epithelial ovarian cancer.Although referred to as ovarian cancer, the origin of the serous subtype was long debated.The histology of these tumors seems to be not strictly restricted to ovarian epithelium and it is now widely acknowledged that the majority originate from the epithelium of the fallopian tube (for review Lisio et al. [1]).In early stages of the pathology (stages I and II), HGSOC is localized on the ovary.However, the silent progression of HGSOC frequently leads to late detection and diagnosis once the cancer has spread to the peritoneal cavity (Stages III and IV) [1].In the ascites and plasma of ovarian cancer patients, an increase in lysophosphatidic acid (LPA) level generally accompanies tumor development, and LPA was proposed as a diagnostic and prognostic marker for ovarian cancer [2][3][4].In vitro, LPA, a bioactive phospholipid, enhances ovarian cancer cell migration and invasion [5][6][7].HGSOC treatment combines a surgical procedure called 'debulking surgery' and taxane and platinum-based chemotherapy [8,9].Unfortunately, ovarian cancer cells frequently develop drug resistance and the 5-year survival rate is 20-30% [10][11][12].
Calcium-activated potassium channels (KCa) are subdivided into three families based on their conductance: small (SKCa), intermediate (IKCa), and big (BKca).The SKCa subfamily is comprised of three members, SK1 (KCNN1), SK2 (KCNN2), and SK3 (KCNN3).These channels are formed by the association of four αsubunits.These channel families have been demonstrated to be activated by increases in cytosolic Ca 2+ concentration rather than through a voltage-gated mechanism [13].We previously showed that SK3 channel activity can hyperpolarize the plasma membrane, increasing the driving force for Ca 2+ and potentiating the store-operated Ca 2+ entry (SOCE) and/or the constitutive Ca 2+ entry (CCE) pathways and contributing to the migration of cancer cells [14,15].Synthetic lipids (Ohmline) can dissociate plasma membrane-localized SK3 from the SOCE complex, leading to a decrease in cell migration in colon cancer cells [15].
In human cells, plasma membrane SKCa activity has been notably described in the regulation of neuron excitability [16].SK2 channels were also identified in intracellular compartments, such as mitochondria, the endoplasmic reticulum (ER), or vesicles, and notably controlling cell death [17][18][19][20].Although there is little information on SKCa channels in cancers, these channels participated in cell proliferation and cell migration [21][22][23].Our laboratory has described the role of plasma membrane SK3 in breast, melanoma, prostate, and colon cancer cell migration in vitro and in breast cancer metastasis in vivo [14 (p. 20), 15,21,24].One recent article indicated that plasma membrane SK2 was responsible for pancreatic cancer cell migration and invasion [22] and involved in the proliferation of glioblastoma cells and in hypoxia-induced proliferation of melanoma cells [25,26].To date, there is no data available on the role of SK2 channels in ovarian cancer cells and chemoresistance.
In this study, we explored the role of plasma membrane-localized SK2 channels in HGSOC cell migration and chemoresistance.Additionally, we assessed the effects of LPA on SK2 channel activity in HGSOC cells.

Survival assay
Cell viability was analyzed using the MTT assay over a 7-day period, as mentioned previously [28].Cell lines were seeded in 48-well plates (10 000 cells per well).Cells were then treated by a range of concentrations of Taxol ® (0 to 160 nM) in their respective cell culture medium at 24 and 96 h after seeding.Cell survival rates in Taxol ® conditions were normalized to control condition (without drug).An increase of EC 50 , the dose for which 50% of the drug's maximal effect is obtained, is associated to drug resistance.

Migration assay
Cells were seeded into migration inserts (8 μM pore size, in 24-well plates, Falcon #353097, France), as described previously [14] and cell migration was induced by a 0-10% FBS gradient in the upper and lower compartment, respectively.Equal drug concentrations of Lei-Dab7 (10 nM) were added in both compartments.After 24 h, an automatic count was performed on nuclei stained with DAPI (1 μgÁmL À1 , Sigma-Aldrich) [29].For a total of 50 000 cells seeded, we counted around 1325 migrated cells for COV504 and 1100 cells for OVCAR3.

Store-operated Ca 2+ entry
To measure store-operated Ca 2+ entry (SOCE), Ca 2+ release from the endoplasmic reticulum was induced with Thapsigargin (4 μM, Invitrogen™) in PSS-free--Ca 2+ solution.At 500 ms, the injection of 5 mM CaCl 2 , Ca 2+ entry across the plasma membrane was measured as the maximum fluorescence ratio (F340/F380) minus the baseline value, which was normalized to the control condition.

Constitutive Ca 2+ entry measured by Mn 2+ quenching method
Constitutive Ca 2+ entry (CCE) was measured as described previously [30].Fura-2-labeled cells were incubated with PSS Ca 2+ -free solution, and 5 mM manganese (Mn 2+ ) (Sigma-Aldrich) was injected.The fluorescence emission was measured at 510 nm with excitation at 360 nm.The influx was analyzed by measuring the decreasing slope of fluorescence for 100 s after Mn 2+ injection.The absolute value of the slope was normalized to the control condition.

Patch clamp experiments
Electrophysiological recordings of SKCa channel currents on COV504 were performed with the whole cell patch clamp technique, as described previously [14,21,31].Data acquisition and analysis were performed using PCLAMP7 software (Axon Instruments, Union City, CA, USA).SK2 currents were generated by stepwise 10 mV depolarizing pulses (500 ms duration; 4 s intervals) from a constant holding potential of À90 mV up to +80 mV.To build IV relation, the average of steady state current at the end of the pulse (the last 50 ms) was chosen for each membrane potential.

Progression-free survival analysis
We used the Kaplan-Meier plotter data base (kmplot.com) [32] to investigate the progression-free survival between low and high KCNN2 expression levels in serous ovarian cancer.Gene expression data and relapse-free and progression-free survival information are downloaded from GEO, EGA and TCGA.The database is handled by a PostgreSQL server, which integrates gene expression and clinical data simultaneously.To analyze the prognostic value of a particular gene, the patient samples are split into two groups according to various quantile expressions of the proposed biomarker (auto select best cutoff).The two patient cohorts are compared by a Kaplan-Meier survival plot, and the hazard ratio with 95% confidence intervals and logrank P value are calculated.The best-performing threshold was used as a cutoff.We selected patients by type of treatment (Taxol ® or platin) and stages of the disease: early (stages I and II) and late (stages III and IV) [33].

Immunofluorescence
Approximately 5000 cells were seeded in presence or absence of LPA (10 μM) on Lab-Tek II chamber slides(ref 154534 Fisher Scientific, Illkirch, France) 72 h prior to the experiment.After washing with PBS, cells were fixed with PFA (4%) at room temperature for 15 min.Thereafter, cells were washed and permeabilized with Triton™ X-100 (0.1% in PBS; Sigma) and blocked with BSA 3% for 30 min.Primary antibody (IHC-plus KCNN2 antibody, Clinisciences, Nanterre, France) was diluted at 1 : 500 and incubated overnight at 4 °C.Secondary antibody was added at 1 : 1000 at room temperature for 1 h (Goat anti-rabbit Alexa488, AbCam, Cambridge, UK).Fluorescence was visualized using High Resolution Laser Scanning Leica SP8 gSTED Confocal Microscope (Leica) and LAS-X v2.0 software (Leica, Wetzlar, Germany).Images were analyzed using FIJI (IMAGEJ software, National Institutes of Health, US).

Statistics
The results from the calcium measurement, mRNA expression levels and migration assays were normalized to the control condition and represented as mean AE SEM.The efficacy dose EC 50 from survival assays, western blot and patch clamp experiments were analyzed as non-parametric and paired data (Wilcoxon test).Statistical significance was established for P < 0.05*, P < 0.01**, P < 0.001***, using GRAPHPAD 8.01 software (Boston, MD, USA).

SK2 channels contribute in SOCE and migration in HGSOC cells
We used whole cell recordings and Lei-Dab7 (10 nM) to identify a SK2 current at the plasma membrane of COV504 cells (Fig. S1A).We unmasked the Lei-Dab7 sensitive current with an inward rectification, characteristic of SKCa channels (Fig. 1A).Acute application of Lei-Dab7 on COV504 cells significantly decreased the global current from 59.13 AE 10.48 to 31.82AE 7.41 pA per pF at +30 mV (Fig. 1A).Combined with immunofluorescence analysis of SK2 expression (Fig. S2C), all these results supported the idea that SK2 channels were functional and localized at the plasma membrane of COV504 cells.Acute Lei-Dab7 treatment was responsible for a significant decrease in SOCE ($ 20%) in both COV504 and OVCAR3 cells (Fig. 1B).No difference in the constitutive Ca 2+ entry after Lei-Dab7 treatment was detected (Fig. S1B).Lei-Dab7 significantly decreased the migration of COV504 cells by $ 20% (Fig. 1C).With siRNA targeting KCNN2 (siRNA selectivity, Fig. S1C), the effect was more striking, with a $60% decrease in cell migration (Fig. 1C).In OVCAR3 cells, Lei-Dab7 did not alter cell migration, while SK2 silencing significantly decreased the migration by $ 50% (Fig. 1D).Furthermore, the activation of SKCa channels by CyPPA significantly increased migration of COV504 but not OVCAR3 cells (Fig. S1D).
Taken together, our results showed that functional SK2 channels were localized at the plasma membrane (Fig. S2C) and involved in SOCE in both COV504 and OVCAR3 cells.In COV504 cells, functional plasma membrane SK2 channels were involved in cell migration.SK2 silencing experiments in both COV504 and OVCAR3 cells suggest the involvement of an intracellular pool of SK2 in cell migration (Fig. 1E).

LPA increases the activity of plasma membrane SK2 channels and cell migration in a SOCE-independent manner
Cells were exposed to 10 μM of lysophosphatidic acid (LPA) for 72 h.In COV504 cells, we noticed a significant increase in outward current (Fig. 2A, a) from 10.55 AE 3.16 to 31.79 AE 5.09 pA per pF at +30 mV (Fig. 2A,B), and acute injection of Lei-Dab7 abolished LPA-induced outward current from 31.79 AE 5.09 to 11.07 AE 1.91 pA per pF at +30 mV.LPA tended to hyperpolarize the membrane potential (P = 0.05) and Lei-Dab7 application did not alter it (Fig. 2A,C).These data show for the first time that SK2 channel activity is regulated by LPA.In COV504, LPA pre-treatment increased KCNN2 mRNA by 35 fold and SK2 protein level by 2.7 fold (Fig. 2B).In OVCAR3, LPA enhanced KCNN2 mRNA expression levels by 2 fold but did not affect SK2 protein levels (Fig. S2A).The expression level of KCNN1 and KCNN3 after LPA treatment is shown in Fig. S3A.Despite of KCNN2 mRNA and SK2 level changes, we surprisingly observed no increase in SOCE activity, and SOCE sensitivity to Lei-Dab7 was lost after LPA treatment in both cell lines (Fig. 2C and Fig. S2B).These results suggest that SK2 no longer participates to SOCE in LPA-treated cells.
As expected, and previously described in literature, LPA pre-treatment significantly increased cell migration by $ 40% in COV504 and $ 50% in OVCAR3 (Fig. 2D).SK2 inhibition by Lei-Dab7 abolished LPAinduced cell migration in both cell lines (Fig. 2D).Our data show for the first time that LPA strongly induces SK2 activity at the plasma membrane and LPAinduced cell migration is totally suppressed by inhibition of SK2 activity in both COV504 and OVCAR3, demonstrating that SK2 is a key regulator of LPA pro-migratory effect.As shown by immunofluorescence analysis, SK2 localization, in both cells lines, seems to be modified after LPA treatment (Fig. S2C).Furthermore, we showed that the LPA-treated cell migration was SOCE-independent, as the pharmacological inhibition of SOCE by synta66 did not modified LPA-treated cell migration (Fig. S3B).These results suggest that LPA altered the membrane localization of SK2 and its association with SOCE channel complexes (Fig. 2E).

Plasma membrane SK2 channel activity increases sensitivity to chemotherapy
We investigated the role of SK2 in Taxol ® resistance by using cell survival assays for 7 days.Inhibition of SK2 channel activity by Lei-Dab7 significantly increased EC 50 by $ 60% in OVCAR3 cells (Fig. 3A).In COV504 cells (Fig. 3B), EC 50 tended to increase by $ 43% (P = 0.094).
Then, we generated Taxol ® -resistant sublines, COV504 TX and OVCAR3 TX, derived from COV504 and OVCAR3 cells.EC 50 was increased by 6 fold and 1.5 fold in COV504 TX and OVCAR3 TX, respectively (Fig. S4A).In COV504 TX, KCNN2 mRNA expression level increased by 7 fold and SK2 protein level increased by 1.5 fold (Fig. S4B).In OVCAR3 TX, KCNN2 mRNA expression level increased by 4 fold but SK2 protein level was unchanged (Fig. S4C) Similarly to sensitive cell lines (Fig. 1B), we confirmed that Lei-Dab7 significantly reduced SOCE in the resistant sublines respectively by $ 15% and $ 30% in COV504 TX and OVCAR3 TX cells (Fig. S4D).These results showed SK2 channels remained associated to SOCE complex in resistant cell lines.
Inhibition of SK2 activity by Lei-Dab7 increased Taxol ® resistance in both COV504 TX and OVCAR3 TX (Fig. 3).In OVCAR3 TX cells, the resistance developed by Lei-Dab7 administration was so large that the cell viability decrease did not appear with the range concentration of Taxol ® we used (EC 50 greater than 40 nM) (Fig. 3C).In COV504 TX, the EC 50 value increased by 2.8 fold after Lei-Dab7 treatment (Fig. 3D).In both resistant and sensitive cell lines, Lei-Dab7 at 10 nM did not affect the cell viability measured in the same conditions after a 7-day culture (Fig. S4E).These data provided the first evidence that plasma membrane SK2 activity increased Taxol ® sensitivity in HGSOC cells.
plotter database.We noticed that in the early stages (I and II), patients treated with Taxol ® or platinumbased drugs received better prognoses when high KCNN2 levels were present in the tumors (Fig. 4A).However, KCNN2 mRNA level was not related to the progression-free survival of patients with late stages (III and IV) who underwent either of the chemotherapies (Fig. 4B).
Thus, high KCNN2 mRNA level could be associated with a better outcome from chemotherapy treatment in the early stages of serous ovarian cancer, but the benefits were lost in more advanced forms of the disease.

Discussion
Our results show that plasma membrane-localized SK2 participates to SOCE in HGSOC cells and plays a key role in LPA-induced cell migration in a SOCE independent manner.Furthermore, we discovered that plasma membrane SK2 inhibition increased chemoresistance to Taxol ® and that KCNN2 mRNA expression level may constitute a prognostic marker for early stages of serous ovarian cancer.

Plasma membrane and intracellular SK2 regulate cell migration
A differential effect between SK2 knockdown and the SK2 peptide inhibitor Lei-Dab7 (Fig. 1) was observed on cell migration.These data would suggest the existence of two pools of SK2 channels: intracellular pool and at the plasma membrane-localized pool.It is known that SKCa channels localized at the plasma membrane control neural excitability, cell migration in vitro and in vivo cancer metastasis [14,16,34].However, functional SKCa channels, in particular the SK2 and SK3 subtypes, were identified at the inner mitochondrial membrane of neuronal and cardiac cells and protected cells against death [35][36][37].Functional SK2 in endoplasmic reticulum (ER) unmasked by CyPPA (a cell permeant and potent activator of SKCa channels) was also observed to be protective against ER stress-induced cell death [19].Our SOCE assays suggest that SK2 channels can contribute to SOCE but this contribution is lost under LPA treatment.In neurons and cardiomyocytes, plasma membrane SK2 localization is dependent upon a large variety of partners, such as palmitoylated protein 2, alpha-actinbinding protein 2, or filamin A, which enable its trafficking to the plasma membrane through endosomes [18,20].Moreover, it has been reported that SK3 activity depends on trafficking by caveolae, regulating its localization and possibly the number of channels at the plasma membrane [38,39].LPA may relocate SK2 channels Altering the activity of cytoskeleton proteins [6] and thereby disrupt the regulation of vesicular transport.
Previously, we described the involvement of plasma membrane SK3 channels in SOCE and constitutive Ca 2+ entry and found that SK3 was localized within nanodomains of the plasma membrane [15,21,34].The association of SK2 channels with SOCE was suggested to be restricted to specific nanodomains of the plasma membrane [40] but this remained to be demonstrated in our cells.Nevertheless, the absence of a shift in the membrane potential after Lei-Dab7 treatment may suggest that SK2 potentiates SOCE by promoting a local hyperpolarization to enhance the driving force for Ca 2+ .However, current clamp experiments should be carried out to measure the resting membrane potential more accurately.
Under LPA treatment SK2 channels no longer participate to SOCE.Here, we cannot exclude that LPA may promote the phosphorylation of SK2 channels or their partners to provoke their plasma membrane delocalization.This phenomenon (phosphorylation/dissociation) was previously described for the ORAI/SK3 channel complex [40].It is also known that lipid environments can alter the curvature of the plasma membrane, delocalize channels, and promote their activation [34].Notably, LPA can activate TREK channels (2-pore domain K + channel) using a similar mechanism [41,42].From this perspective, a direct LPA insertion into the plasma membrane could lead to SK2 delocalization.Nevertheless, LPA interaction with its receptors up-regulated SK2 transcriptionally, as shown in COV504 cells.Additionally, LPA may also recruit intracellular SK2, originally trapped in endosomes and mobilized by cytoskeletal protein activation [6,20].Finally, LPA can promote ovarian cancer cell migration by activating an epidermal growth factor receptor (EGFR)-dependent pathway.Indeed, it was recently reported that SK2 activity promoted cell migration and invasion in pancreatic cancer cells by enhancing an integrin-EGFR-AKT (Protein kinase B) pathway [22,43].

SK2 contribution to chemosensitivity
Plasma membrane SK2 inhibition increased resistance to Taxol ® in HGSOC cell lines.Several studies reported inhibition or downregulation of K + channel led to chemoresistance in cancers [44].In the literature, a prominent hypothesis is that the increase of K + efflux and/or reduction of intracellular K + concentration can lead to the activation of pro-apoptotic factors, such as interleukin-1β (IL-1β), reactive oxygen species, or Caspase 1, thus promoting sensitivity to chemotherapies.In addition, promoting membrane hyperpolarization, the K + efflux can potentiate a cytosolic Ca 2+ increase, and contribute to the induction of mitochondrial Ca 2+ overload and cell death [45].For KCa specifically, their expression and activity potentiate cisplatin drug uptake into the cell leading to chemosensitivity.Unfortunately, the related mechanism of action through which KCa channels mediate this effect is still unclear [46].
In a concordant manner, the KM-plotter analysis showed that higher KCNN2 expression levels are associated with a better prognosis for patients treated with Taxol ® or platinum-based drugs.However, while this phenomenon was observed for early stages, it was not observed for late stages of serous ovarian cancer.These results agreed with analysis performed on sarcoma tumors [47].In addition, higher KCNN3 mRNA expression levels were associated with a better prognosis in patients with ovarian cancer [48].Thus, mRNA levels of KCNN2 and KCNN3 channels may constitute a promising prognostic marker or interesting targets to regulate chemosensitivity of several cancers.
Overall, both facets of SK2 could be exploited in the course of patient care.SK2 activators could be used as adjuvants to chemotherapy to enhance its efficacy, while SK2 inhibitors could be administered as monotherapy to limit the spread of cancer cells.

Conclusion
Our results showed for the first time that SK2 is involved in ovarian cancer cell migration and that plasma membrane SK2 plays a key function in LPAinduced cell migration, suggesting SK2 as potential target that should be inhibited in context of the tertiary prevention after the end of chemotherapy cycles.Additionally, SK2 activity increases chemotherapy efficacy and KCNN2 expression levels may constitute a prognostic marker for early stages of serous ovarian cancer.However, the mRNA levels of KCNN2 were not sufficient to predict SK2 activity, cellular localization and function.Thus, while plasma membrane SK2 should be blocked to control cancer cell migration, its activity should be potentiate during chemotherapy to sensitize ovarian cancer cells.The activation or inhibition of SK2 should be adapted according to the stages of the patient's care pathway.Currently available tools to modulate SKCa channels are rare and lack selectivity and their development could provide a useful strategy to improve ovarian cancer outcomes.

Fig. 3 .
Fig. 3. Plasma membrane SK2 activity increases Taxol ® sensitivity in sensitive and resistant cell lines.In the presence or absence of Lei-Dab7 (10 nM), cells were treated with a range of Taxol ® concentration to evaluate EC 50 values in a 7-day survival assay in OVCAR3 (N = 8) (A), and COV504 (N = 6) (B), OVCAR3 TX (N = 5) (C), and COV504 TX (N = 6) (D) cells.EC 50 is the dose for which we obtained 50% of the drug's maximal effect.EC 50 increase is associated with drug resistance.On the right, histograms represent EC 50 values in the presence of Lei-Dab7.Each point is the result of one experiment.For the statistical analysis of EC 50 in OVCAR3 TX + Lei-Dab7 condition, the maximal value of the Taxol ® range was used (40 nM) (representing by a dot line on C right panel).The survival assay results are expressed as mean AE SEM (Wilcoxon, P < 0.05*) (OVCAR3 TX and COV504 TX = Taxol ® resistant cells).

Fig. 4 .
Fig. 4. High KCNN2 mRNA expression was associated with better survival rates among patients with early stages of serous ovarian cancer.(A) Progression-free survival of patients with stages I and II of serous ovarian cancer depending on KCNN2 expression level.Kaplan-Meier curves represented patients treated with Taxol ® (left panel, P value 0.0289, FDR over 50%) or platinum-based drugs (right panel, P value 0.0266; FDR over 50%) (Taxol ® , N = 51; platinum-based drugs, N = 69).(B).Progression-free survival of patients with stages III and IV of serous ovarian cancer treated with Taxol ® (left panel, P value 0.2269, FDR 100%) or platinum-based drugs (right panel, P value 0.3228, FDR 100%) (Taxol ® , N = 578; platinum-based drugs, N = 907) depending on KCNN2 expression levels (kmplot.com).