The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.

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Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

The intermediate‐conductance Ca2+‐activated K+ channel KC a3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target and biomarker for cancer. Histone deacetylase inhibitors (HDACis) have considerable potential for cancer therapy, however, the effects of HDACis on ion channel expression have not yet been investigated in detail. The results of this study showed a significant decrease in KC a3.1 transcription by HDAC inhibition in the human breast cancer cell line YMB‐1, which functionally expresses KCa3.1. A treatment with the clinically available, class I, II, and IV HDAC inhibitor, vorinostat significantly downregulated KC a3.1 transcription in a concentration‐dependent manner, and the plasmalemmal expression of the KC a3.1 protein and its functional activity were correspondingly decreased. Pharmacological and siRNA‐based HDAC inhibition both revealed the involvement of HDAC2 and HDAC3 in KC a3.1 transcription through the same mechanism. The downregulation of KC a3.1 in YMB‐1 was not due to the upregulation of the repressor element‐1 silencing transcription factor, REST and the insulin‐like growth factor‐binding protein 5, IGFBP5. The significant decrease in KC a3.1 transcription by HDAC inhibition was also observed in the KC a3.1‐expressing human prostate cancer cell line, PC‐3. These results suggest that vorinostat and the selective HDACis for HDAC2 and/or HDAC3 are effective drug candidates for KC a3.1‐overexpressing cancers.

Recent studies have indicated that the intracellular concentration of chloride ions (Cl−) regulates gene expression in several types of cells and that Cl− modulators positively or negatively regulate the PI3K/AKT/mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT)3 signaling pathways. We previously reported that the Ca2+‐activated Cl− channel anoctamine (ANO)1 regulated human epidermal growth factor receptor 2 (HER2) transcription in breast cancer YMB‐1 cells. However, the mechanisms underlying ANO1‐regulated HER2 gene expression have not yet been elucidated. In the present study, we showed the involvement of intracellular organelle ClC‐3 Cl−/H+ transporter in HER2 transcription in breast cancer MDA‐MB‐453 cells. The siRNA‐mediated inhibition of ClC‐3, but not ANO1, markedly repressed HER2 transcription in MDA‐MB‐453 cells. Subsequently, treatments with the AKT inhibitor AZD 5363 and mTOR inhibitor everolimus significantly enhanced HER2 transcription in MDA‐MB‐453 cells, whereas that with the STAT3 inhibitor 5,15‐diphenylporphyrin (5,15‐DPP) inhibited it. AKT and mTOR inhibitors also significantly enhanced HER2 transcription in YMB‐1 cells. The siRNA‐mediated inhibition of ClC‐3 and ANO1 resulted in increased AKT phosphorylation and decreased STAT3 phosphorylation in MDA‐MB‐453 and YMB‐1 cells, respectively. The intracellular Cl− channel protein CLIC1 was expressed in both cells; however, its siRNA‐mediated inhibition did not elicit the transcriptional repression of HER2. Collectively, our results demonstrate that intracellular Cl− regulation by ANO1/ClC‐3 participates in HER2 transcription, mediating the PI3K/AKT/mTOR and/or STAT3 signaling pathway(s) in HER2‐positive breast cancer cells, and support the potential of ANO1/ClC‐3 blockers as therapeutic options for patients with resistance to anti‐HER2 therapies.