Abstract
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.
Highlights
Androgen deprivation therapy (ADT) is the standard care for the initial management of advanced and metastatic prostate cancer (PC); progression to castrationresistant PC (CRPC) occurs within a few years of the initiation of ADT [1]
The MRP4/5 inhibitor, MK571, did not overcome resistance to antiandrogens by LNCaP spheroids (Supplementary Figure S3E,F). These results suggest that androgen receptors (AR) protein degradation plays a role in the acquisition of antiandrogen resistance in LNCaP spheroids, and that KCa 1.1 inhibitors may prevent the activity and/or expression of E3 ubiquitin ligases (E3s) involved in AR degradation-mediated antiandrogen resistance by PC stem cells (PCSCs)
The siRNA-mediated inhibition of murine double minute 2 (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
Summary
Androgen deprivation therapy (ADT) is the standard care for the initial management of advanced and metastatic prostate cancer (PC); progression to castrationresistant PC (CRPC) occurs within a few years of the initiation of ADT [1]. CRPC is 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]. Multicellular tumor three-dimensional (3D) spheroid models are spherical self-assembled aggregates of cancer cells. They are a valuable tool for studying the tumor microenvironment (TME) in solid tumors and are important for investigating the characteristics of CSCs, such as tumor initiation, metastasis, and resistance to chemotherapies (chemoresistance) [7,8]. Previous studies demonstrated that the expression of CSC markers, such as CD44 and NANOG, was upregulated in aggregated PC spheroid models and contributed to their acquisition of chemoresistance and antiandrogens [9,10]. Ultra-low attachment plates and dishes pre-coated with an ultra-hydrophilic polymer promoted the spontaneous formation of cancer spheroids
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