Ca²⁺‐activated K⁺ channel K_Ca1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

Abstract

The large‐conductance Ca2+‐activated K+ channel KCa1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three‐dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa1.1 activator‐induced hyperpolarizing responses were significantly larger in human osteosarcoma MG‐63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa1.1 and its role in chemoresistance using a 3D spheroid model. KCa1.1 protein expression levels were significantly elevated in the lipid‐raft‐enriched compartments of MG‐63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa1.1 protein degradation in breast cancer. The siRNA‐mediated inhibition of FBXW7 in MG‐63 cells from 2D monolayers upregulated KCa1.1 protein expression. Furthermore, a treatment with a potent and selective KCa1.1 inhibitor overcame the chemoresistance of the MG‐63 and human chondrosarcoma SW‐1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP‐binding cassette transporters, the expression of the multidrug resistance‐associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa1.1. Therefore, the pharmacological inhibition of KCa1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa1.1‐positive sarcomas.

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

“…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].…”

“…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].…”

“…2.5. Involvement of the Ubiquitin E3 Ligase, FBXW7, in Enhancements in K Ca 1.1 Activity Induced by LNCaP Spheroid Formation FBXW7 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).…”

“…The cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 . Flat-bottomed dishes and plates (Corning, Corning, NY, USA) were used for the 2D cell culture [21]. The PrimeSurface ® system (Sumitomo Bakelite, Tokyo, Japan) was used for the 3D spheroid culture.…”

“…Post-translational modifications by E3 ubiquitin ligases (E3s) play a crucial role in protein stabilization, and the dysregulation of ubiquitination is associated with cancer progression, metastasis, and therapy resistance [20]. In sarcoma spheroid models, the downregulation of F-box/WD repeat-containing protein 7 (FBXW7) decreased the protein degradation of K Ca 1.1, which increased K Ca 1.1 activity [21]. Cereblon (CRBN) plays an important role in the assembly and plasma membrane expression of functional K Ca 1.1 [22] and also promotes AR protein degradation in androgen-sensitive human PC cell line, LNCaP [23].…”