KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.


Introduction
Androgen deprivation therapy (ADT) is the standard care for the initial management of advanced and metastatic prostate cancer (PC); however, progression to castrationresistant PC (CRPC) occurs within a few years of the initiation of ADT [1]. The agents currently available for the treatment of CRPC include (1) chemotherapy agents, such as docetaxel (DTX), and (2) antiandrogens, including bicalutamide (BCT) and enzalutamide (EZT) [2,3]. However, PC patients with metastatic CRPC eventually become resistant to these agents [2,3].CRPC under ADT causes genetic changes in androgen receptors (AR), such as overexpression, mutations, and splice variants [4]. Moreover, CRPC is also driven from PC stem cells (PCSCs), and the sustained prevention of AR protein expression by their degradation is one potential mechanism by which PCSCs acquire antiandrogen resistance [5]. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors, such as olaparib, novel AR antagonists, such as darolutamide, and radio-ligand therapy with lutetium prostate-specific membrane antigen (Lu-PSMA) have emerged as potentially effective therapeutic options in patients with CRPC [6].
Multicellular tumor three-dimensional (3D) spheroid models are spherical self-assembled aggregates of cancer cells. They are a valuable tool for studying the tumor microenvi-

Acquired Resistance of LNCaP Spheroids to Antiandrogens and DOX Overcome by KCa1.1 Inhibition
The optimization of the initial cell seeding density is essential for the WST-1 liferation assay. Especially, it affects various parameters, such as metabolic acti drug penetration in a 3D spheroid. Under the condition described in Section cells/mL at day 0), LNCaP cells cultured in a 2D monolayer almost doubled ev from day 0 to day 3. Moreover, the metabolic activity of the WST-1 reagent in tured in a 3D spheroid was reduced by almost 30% in 2 days; however, it was kep additional 7 days. We further examined the WST-1 assay in 3D spheroids with th ent initial cell densities (2 × 10 4 , 5 × 10 4 , 10 5 , 2 × 10 5 , and 5 × 10 5 cells/mL); how drug sensitivities were almost same among them. As shown in Figure 3A-D, LNC roids acquired chemoresistance to DTX, PTX, DOX, and CIS following exposure (p < 0.01). In LNCaP spheroids, the resistance to DOX alone was reversed by the ment with 10 μM PAX for 24 h (p < 0.01) ( Figure 3E-H). No significant chang viability were found by the treatment with PAX alone for 48 h (1.032 ± 0.029 in cell viability (n = 5, p > 0.05)).

Acquired Resistance of LNCaP Spheroids to Antiandrogens and DOX Overcome by K Ca 1.1 Inhibition
The optimization of the initial cell seeding density is essential for the WST-1 cell proliferation assay. Especially, it affects various parameters, such as metabolic activity and drug penetration in a 3D spheroid. Under the condition described in Section 4.2. (10 5 cells/mL at day 0), LNCaP cells cultured in a 2D monolayer almost doubled every 24 h from day 0 to day 3. Moreover, the metabolic activity of the WST-1 reagent in cells cultured in a 3D spheroid was reduced by almost 30% in 2 days; however, it was kept for the additional 7 days. We further examined the WST-1 assay in 3D spheroids with the different initial cell densities (2 × 10 4 , 5 × 10 4 , 10 5 , 2 × 10 5 , and 5 × 10 5 cells/mL); however, the drug sensitivities were almost same among them. As shown in Figure 3A-D, LNCaP spheroids acquired chemoresistance to DTX, PTX, DOX, and CIS following exposure for 48 h (p < 0.01). In LNCaP spheroids, the resistance to DOX alone was reversed by the pretreatment with 10 µM PAX for 24 h (p < 0.01) ( Figure 3E-H). No significant changes in cell viability were found by the treatment with PAX alone for 48 h (1.032 ± 0.029 in relative cell viability (n = 5, p > 0.05)).
LNCaP spheroids also exhibited resistance to the antiandrogens BCT and EZT (p < 0.01) ( Figure 4A,B). The PAX pretreatment significantly reversed the antiandrogen resistance acquired by LNCaP spheroids (p < 0.01) ( Figure 4C,D). There results suggest that the inhibition of K Ca 1.1 may represent a novel strategy for overcoming antiandrogen resistance in patients with CRPC.
Among the 10 ABC transporter candidates (MDR1, MDR3, MRP1-6, and ABCG1-2) possibly involved in chemoresistance, the MRP1, 3, 4, and 5 transcripts were expressed at high levels in LNCaP spheroids, with the levels of transcripts other than MRP4 significantly increasing with the spheroid formation ( Figure 5A-D). The expression levels of the other candidates were much lower in LNCaP spheroids (less than 0.002 in arbitrary units to ACTB). As shown in Figure 5E-H, the PAX treatment for 24 h significantly reversed the expression of MRP5 only. Western blot (WB) examinations also showed that increases in MRP5 protein expression levels in LNCaP spheroids were significantly reduced by the PAX treatment (p < 0.05) (Supplementary Figure S3A-D). No significant changes in MRP1, MRP3, and MRP4 protein expression levels were found by the PAX treatment (Supplementary Figure S4). ent initial cell densities (2 × 10 4 , 5 × 10 4 , 10 5 , 2 × 10 5 , and 5 × 10 5 cells/mL); however, the drug sensitivities were almost same among them. As shown in Figure 3A-D, LNCaP spheroids acquired chemoresistance to DTX, PTX, DOX, and CIS following exposure for 48 h (p < 0.01). In LNCaP spheroids, the resistance to DOX alone was reversed by the pretreatment with 10 μM PAX for 24 h (p < 0.01) ( Figure 3E-H). No significant changes in cell viability were found by the treatment with PAX alone for 48 h (1.032 ± 0.029 in relative cell viability (n = 5, p > 0.05)). LNCaP spheroids also exhibited resistance to the antiandrogens BCT and EZT (p < 0.01) ( Figure 4A,B). The PAX pretreatment significantly reversed the antiandrogen resistance acquired by LNCaP spheroids (p < 0.01) ( Figure 4C,D). There results suggest that the inhibition of KCa1.1 may represent a novel strategy for overcoming antiandrogen resistance in patients with CRPC. Among the 10 ABC transporter candidates (MDR1, MDR3, MRP1-6, and ABCG1-2) possibly involved in chemoresistance, the MRP1, 3, 4, and 5 transcripts were expressed at high levels in LNCaP spheroids, with the levels of transcripts other than MRP4 significantly increasing with the spheroid formation ( Figure 5A-D). The expression levels of the other candidates were much lower in LNCaP spheroids (less than 0.002 in arbitrary units to ACTB). As shown in Figure 5E-H, the PAX treatment for 24 h significantly reversed the   Furthermore, a co-treatment with the MRP4/5 inhibitor, MK571 and DOX overcame DOX resistance in LNCaP spheroids, whereas that with the selective MRP4 inhibitor, ceefourin 1 and DOX, did not ( Figure 6C,G). Neither inhibitor overcame resistance to the other chemotherapy drugs ( Figure 6). No significant changes in cell viability were observed with single treatments with MK571 or ceefourin 1 for 48 h (0.973 ± 0.031 and 0.965 ± 0.045, respectively, in relative cell viability (n = 5, p > 0.05)). These results strongly suggest that MRP5 plays a critical role in the acquisition and K Ca 1.1 inhibition-induced overcoming of resistance by LNCaP spheroids to DOX.

Decreases in AR Protein Expression in Antiandrogen-Resistant-LNCaP Spheroids
We compared the gene and protein expression levels of AR between 2D monolayers and 3D spheroids of LNCaP cells using real-time PCR and WB examinations. The protein expression level of AR with a molecular weight of approximately 110 kDa decreased in lipid-raft fractions of LNCaP spheroids (p < 0.01) ( Figure 7B,C), without changes in that of AR transcripts (p > 0.05) ( Figure 7A). Consistent with the acquisition of resistance to antiandrogens being overcome by the PAX treatment ( Figure 4), a pretreatment with 10 µM PAX significantly increased AR protein levels in LNCaP spheroids (p < 0.01) ( Figure 7E,F), without changes in AR transcript levels (p > 0.05) ( Figure 7D). The MRP4/5 inhibitor, MK571, did not overcome resistance to antiandrogens by LNCaP spheroids (Supplementary Figure S3E,F). These results suggest that AR protein degradation plays a role in the acquisition of antiandrogen resistance in LNCaP spheroids, and that K Ca 1.1 inhibitors may prevent the activity and/or expression of E3s involved in AR degradation-mediated antiandrogen resistance by PCSCs.
PAX significantly increased AR protein levels in LNCaP spheroids (p < 0.01) ( Figure 7E,F), without changes in AR transcript levels (p > 0.05) ( Figure 7D). The MRP4/5 inhibitor, MK571, did not overcome resistance to antiandrogens by LNCaP spheroids (Supplementary Figure S3E,F). These results suggest that AR protein degradation plays a role in the acquisition of antiandrogen resistance in LNCaP spheroids, and that KCa1.1 inhibitors may prevent the activity and/or expression of E3s involved in AR degradation-mediated antiandrogen resistance by PCSCs.  Summarized results were obtained as the optical density of AR and ACTB band signals, respectively (n = 4 for each). After compensation for the optical density of the AR protein band signal with that of the ACTB signal, the AR signal in '2D' (C) or 'vehicle' (F) was expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. '2D'; # : p < 0.05 vs. vehicle control.

Decrease in the Expression of the AR-Targeted Ubiquitin E3 Ligase, MDM2, in LNCaP Spheroids
Several E3s that degrade the AR protein have been identified in PC cells: SKP2 (S-phase kinase-associated protein 2), RNF6 (RING (really interesting new gene) finger protein 6), UBE3A (ubiquitin protein ligase E3A), SIAH2 (seven in absentia (Drosophila) homolog 2), CRBN, MDM2, NRIP (nuclear receptor interaction protein), and USP14 (ubiquitin-specific protease 14) [22][23][24]29]. The transcriptional expression levels of six candidates, except for MDM2 and CRBN, was not downregulated by the PAX treatment for 24 h (Supplementary Figure S5). Moreover, the expression levels of the MDM2 and CRBN transcripts and proteins were inversely correlated with AR protein expression (2D vs. 3D, Figure 8A-E; vehicle-treated vs. PAX-treated, Figure 8F-J). tein 6), UBE3A (ubiquitin protein ligase E3A), SIAH2 (seven in absentia (Drosophila) homolog 2), CRBN, MDM2, NRIP (nuclear receptor interaction protein), and USP14 (ubiquitinspecific protease 14) [22][23][24]29]. The transcriptional expression levels of six candidates, except for MDM2 and CRBN, was not downregulated by the PAX treatment for 24 h (Supplementary Figure S5). Moreover, the expression levels of the MDM2 and CRBN transcripts and proteins were inversely correlated with AR protein expression (2D vs.  To elucidate the roles of CRBN and MDM2 in antiandrogen resistance through the promotion of AR protein degradation in LNCaP spheroids, we examined the effects of the pharmacological and siRNA-mediated inhibition of CRBN and MDM2 on acquired antiandrogen resistance. A pretreatment with the MDM2 inhibitor, nutlin-3a, for 24 h To elucidate the roles of CRBN and MDM2 in antiandrogen resistance through the promotion of AR protein degradation in LNCaP spheroids, we examined the effects of the pharmacological and siRNA-mediated inhibition of CRBN and MDM2 on acquired antiandrogen resistance. A pretreatment with the MDM2 inhibitor, nutlin-3a, for 24 h significantly reversed the resistance to BCT and EZT in LNCaP spheroids (p < 0.01) ( Figure 9A), whereas that with the CRBN inhibitor, iberdomide, did not (p > 0.05) ( Figure 9B). Similarly, the siRNA-mediated inhibition of MDM2 reversed the resistance to antiandrogens in LNCaP spheroids (p < 0.01) ( Figure 9C), whereas that of siRNA-mediated CRBN inhibition did not (p > 0.05) ( Figure 9D). Correspondingly, the siRNA-mediated inhibition of MDM2, but not CRBN, significantly increased AR protein levels in LNCaP spheroids (p < 0.05 in si-MDM2; p > 0.05 in si-CRBN) ( Figure 9E-H). These results strongly suggest that MDM2 is responsible for the antiandrogen resistance induced by spheroid formation in LNCaP cells through the promotion of AR protein degradation, and also that the transcriptional repression of MDM2 mediated by the inhibition of K Ca 1.1 overcame the antiandrogen resistance acquired by LNCaP spheroids through the increase in AR protein levels.
Similarly, the siRNA-mediated inhibition of MDM2 reversed the resistance to antiandrogens in LNCaP spheroids (p < 0.01) ( Figure 9C), whereas that of siRNA-mediated CRBN inhibition did not (p > 0.05) ( Figure 9D). Correspondingly, the siRNA-mediated inhibition of MDM2, but not CRBN, significantly increased AR protein levels in LNCaP spheroids (p < 0.05 in si-MDM2; p > 0.05 in si-CRBN) ( Figure 9E-H). These results strongly suggest that MDM2 is responsible for the antiandrogen resistance induced by spheroid formation in LNCaP cells through the promotion of AR protein degradation, and also that the transcriptional repression of MDM2 mediated by the inhibition of KCa1.1 overcame the antiandrogen resistance acquired by LNCaP spheroids through the increase in AR protein levels.    promoted the protein degradation of K Ca 1.1 in human breast cancer and sarcoma cell lines [18,21]. The protein expression levels of FBXW7 were markedly decreased by LNCaP spheroid formation (p < 0.01) ( Figure 10A,B), and the siRNA-mediated inhibition of FBXW7 significantly increased the protein expression levels of K Ca 1.1 in 2D-cultured LNCaP cells (p < 0.05) ( Figure 10C,D). These results suggest that FBXW7 plays a role in K Ca 1.1 protein degradation in LNCaP cells.

Induced by LNCaP Spheroid Formation
FBXW7 promoted the protein degradation of KCa1.1 in human breast cancer and sarcoma cell lines [18,21]. The protein expression levels of FBXW7 were markedly decreased by LNCaP spheroid formation (p < 0.01) ( Figure 10A,B), and the siRNA-mediated inhibition of FBXW7 significantly increased the protein expression levels of KCa1.1 in 2D-cultured LNCaP cells (p < 0.05) ( Figure 10C,D). These results suggest that FBXW7 plays a role in KCa1.1 protein degradation in LNCaP cells.

Discussion
Recent studies demonstrated that K + channels are important contributors to drug resistance being overcome in solid cancers [14,30]. KCa1.1 gene amplification plays an important role in PC progression [15]. The main results of the present study are as follows: (1) the inhibition of KCa1.1 overcame acquired resistance to antiandrogens in LNCaP spheroids ( Figure 4); (2) AR protein degradation by the upregulation of MDM2 was associated with the acquisition of antiandrogen resistance in LNCaP spheroids, and the pharmacological inhibition of KCa1.1 reversed MDM2-mediated AR degradation, which overcame antiandrogen resistance (Figures 7-9); and (3) the inhibition of KCa1.1 overcame acquired resistance to DOX in LNCaP spheroids (Figure 3), and MRP5 was associated with both the acquisition of DOX resistance and it being overcome by the inhibition of KCa1.1 (Figures 5  and 6). We also found that KCa1.1 proteins were overexpressed in LNCaP spheroids, together with a decrease in FBXW7-mediated KCa1.1 protein degradation (Figures 1 and 10). Collectively, the present results demonstrated the impact of KCa1.1 on the resistance to antiandrogens and DOX in LNCaP spheroid models.
The E3, MDM2, promotes cancer stemness with an AR-negative signature in PCSCs by selectively degrading AR proteins [24]. In contrast, the loss of MDM2 promotes CSC differentiation by reversing the processes of PC stemness [24]. In the present study, several PCSC markers were overexpressed by LNCaP spheroid formation (Figure 2). NANOG has been implicated in the acquisition of cancer therapy resistance [31], and its

Discussion
Recent studies demonstrated that K + channels are important contributors to drug resistance being overcome in solid cancers [14,30]. K Ca 1.1 gene amplification plays an important role in PC progression [15]. The main results of the present study are as follows: (1) the inhibition of K Ca 1.1 overcame acquired resistance to antiandrogens in LNCaP spheroids ( Figure 4); (2) AR protein degradation by the upregulation of MDM2 was associated with the acquisition of antiandrogen resistance in LNCaP spheroids, and the pharmacological inhibition of K Ca 1.1 reversed MDM2-mediated AR degradation, which overcame antiandrogen resistance (Figures 7-9); and (3) the inhibition of K Ca 1.1 overcame acquired resistance to DOX in LNCaP spheroids (Figure 3), and MRP5 was associated with both the acquisition of DOX resistance and it being overcome by the inhibition of K Ca 1.1 (Figures 5 and 6). We also found that K Ca 1.1 proteins were overexpressed in LNCaP spheroids, together with a decrease in FBXW7-mediated K Ca 1.1 protein degradation (Figures 1 and 10). Collectively, the present results demonstrated the impact of K Ca 1.1 on the resistance to antiandrogens and DOX in LNCaP spheroid models.
The E3, MDM2, promotes cancer stemness with an AR-negative signature in PCSCs by selectively degrading AR proteins [24]. In contrast, the loss of MDM2 promotes CSC differentiation by reversing the processes of PC stemness [24]. In the present study, several PCSC markers were overexpressed by LNCaP spheroid formation (Figure 2). NANOG has been implicated in the acquisition of cancer therapy resistance [31], and its overexpression in PC cells has been shown to reduce AR levels [32]. MDM2 and CRBN were included among the 6490 target genes of the NANOG transcription factor obtained from the chromatin immunoprecipitation (ChIP) enrichment analysis (ChEP) transcriptional factor targets dataset [33]. These findings suggest that NANOG is a transcription factor of MDM2 in PCSCs; however, further studies are needed to obtain direct evidence to show that MDM2 is a NANOG downstream gene in PCSCs. Additionally, several studies have shown that signal transducer and activator of transcription (STAT) signaling pathways play critical roles in the AR protein degradation and stabilization in PC cells [34,35]. Further studies will be needed to elucidate the possible involvement of K Ca 1.1 in the regulation of STAT pathway-mediated AR protein degradation.
The present study indicated that acquired resistance to DOX in LNCaP spheroids was due to upregulated MRP5 expression ( Figures 3C and 5D). Consistent with these results, DOX resistance was reversed by a treatment with the MRP4/5 inhibitor without affecting the selective MRP4 inhibitor ( Figure 6C,G). These results are in accordance with recent findings showing that MRP5 was associated with drug resistance to DOX, but not PTX or CIS [26,36]. Different from our recent study on sarcoma spheroids [21], the inhibition of K Ca 1.1 did not affect the expression levels of MRP1 ( Figure 5E) and Nrf2 (Supplementary Figure S6A) in LNCaP spheroids. In fibrosarcoma spheroids, the NANOG inhibition reverses DOX resistance [31]. As shown in Supplementary Figure S6B-D, the inhibition of K Ca 1.1 downregulated the expression of the CSC markers in LNCaP spheroids. It currently remains unclear whether MRP5 is a NANOG downstream gene in PCSCs; however, it was not included among the 6490 target genes of the NANOG transcription factor obtained from the ChEP transcriptional factor targets dataset. Furthermore, Ji et al. (2021) recently showed that the high expression of MRP5 reduced the sensitivity of LNCaP spheroids to EZT [37]. However, the present study showed no significant changes in antiandrogen resistance in LNCaP spheroids treated with MK571 (Supplementary Figure S3E,F).
Consistent with human sarcoma spheroid models [21], K Ca 1.1 protein degradation was suppressed by the downregulation of FBXW7 in LNCaP spheroids ( Figure 10) [21]. Recent studies have focused on the role of mitochondrial ion channels in chemoresistance in cancers [38,39]. Mitochondrial K Ca 1.1 may be responsible for the acquisition of resistance to antiandrogens and chemotherapies by PCSCs. A more detailed understanding of K Ca 1.1regulated resistance to antiandrogens and chemotherapies is needed for the development of novel treatment strategies for CRPCs associated with PCSCs.
The pro-inflammatory cytokines, IL-1β and IL-8, have been shown to repress AR mRNA expression in PC cells [40,41], and play a role in antiandrogen resistance in PC [42]. However, their levels in LNCaP spheroids were undetectable by an ELISA assay (less than 1 pg/mg protein). As shown in Figure 7D, the inhibition of K Ca 1.1 in LNCaP spheroids did not affect the expression level of AR; however, IL-1β and/or IL-8-producing, tumorinfiltrating non-cancerous cells, such as tumor-associated macrophages cells in the TME, may be responsible for the AR transcriptional repression-mediated antiandrogen resistance in PCSCs.
In the TME of PC, tumor-infiltrating, non-cancerous cells, such as regulatory T cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs), play a critical role in immunosuppression [43]. A recent study showed that AR-expressing CAFs can affect PC progression and metastasis [44]. In addition, TAM-derived IL-6 and IL-8 are important for the regulation of AR expression and antiandrogen resistance [44]. Studies to shed light on the cellular heterogeneity of TME using accurate TME-mimicking cancer organoid models and patient-derived organoids will be needed to elucidate more mechanisms underlying AR protein degradation and antiandrogen resistance in PCSCs.

Materials and Reagents
The following chemicals and reagents were used: EZT, iberdomide, MK571 Medical & Biological Laboratories, Nagoya, Japan) antibodies, and then incubated with antirabbit and mouse horseradish peroxidase-conjugated IgG (Merck Millipore, Darmstadt, Germany). Strong band signal for flotillin-1 (45 kDa, anti-flotillin polyclonal (rabbit) antibody, GeneTex, Alton Pkwy Irvine, CA, USA) protein, a lipid-raft marker, was detected in ultra-RIPA soluble fractions, but not RIPA soluble ones. Image detections and the quantitative analyses of the optical densities of protein band signals were performed as previously reported [21].

Immunocytochemistry
Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA). K Ca 1.1 expression was expressed as means fluorescence intensity after the subtraction of that in cells stained by the secondary antibody [21].

Cell Viability Assay
Cell viability was assessed using the WST-1 assay [21]. Briefly, using a density of 10 5 cells/mL, cells were cultured in duplicate in 96-well plates for 7 days (for the 3D culture) and 1 day (for the 2D culture). The cells were then treated with drugs used in chemotherapy (DTX, PTX, DOX, and CIS) and antiandrogens (BCT and EZT) for 48 h. Two hours after the addition of WST-1 reagent to each well, the absorbance was measured using the microplate reader SpectraMax 384 (Molecular Devices Japan, Tokyo, Japan) at a test wavelength of 450 nm and reference wavelength of 650 nm.

Statistical Analysis
Statistical evaluation was performed with Statistical software XLSTAT (version 2013.1). Unpaired/paired Student's t-tests with Welch's correction or Tukey's tests were used to assess the significance of differences between two groups and among multiple groups. Results with a p value of less than 0.05 were considered to be significant. Data were presented as means ± SEM.

Conclusions
In conclusion, the present results suggest that K Ca 1.1 may be a key modulator of antiandrogen resistance in androgen-sensitive, K Ca 1.1 gene-amplified PC, and that K Ca 1.1 inhibitors may overcome acquired antiandrogen resistance in PCSCs. Furthermore, K Ca 1.1 played a vital role in the overcome of DOX resistance by downregulating MRP5 in LNCaP spheroids, suggesting the potential of K Ca 1.1 inhibitors as an effective therapeutic inter-vention in combination with antiandrogens. The targeting of K Ca 1.1 is promising for suppressing the progression of CRPC associated with PCSCs through the regulation of MDM2-mediated AR degradation.