Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells

Abstract

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.

Ca2+‐activated K+ channel KCa1.1 as a therapeutic target to overcome chemoresistance in three‐dimensional sarcoma spheroid models

“…To identify candidates involved in the protein degradation of K Ca 1.1 in MG‐63 cells (see Section 1), we compared the expression levels of FBXW7, MDM2, and cereblon (CRBN) between the 2D monolayers and 3D spheroids of both sarcomas 27,30 . As shown in Figure 3A‐C, the expression levels of FBXW7 transcripts and proteins were significantly decreased by spheroid formation in MG‐63 cells.…”

“…PCR primers of human origin are listed in Table . The relative expression levels were calculated using the 2 −ΔΔCt method and normalized to β‐actin (ACTB) 27 …”

ARe we there yet? Understanding androgen receptor signaling in breast cancer

“…Further, inhibition of AR resulted in decreased levels of phospho-Elk1, phospho-RSK, and c-FOS in xenograft tumors and in patient tumors, corresponding to a decrease in ERK target proteins 46 . In TNBC, AR inhibition has also been shown to modulate the activity of the Ca 2+ -activated K + channel, K Ca 1.1, which is associated with breast cancer invasion and metastasis 47,48 . Multiple groups have also studied the role of cytoplasmic AR phosphorylation 49,50 ; however, additional work is required to understand how AR modifications influence cellular function and localization.…”

The Divergent Function of Androgen Receptor in Breast Cancer; Analysis of Steroid Mediators and Tumor Intracrinology

“…A recent study in triple negative breast cancer has identified the Ca 2+ activated K + channel—K ca 1.1 as an androgen target gene. K ca 1.1 is associated with breast cancer invasion and metastasis and treatment with anti-androgens prevents its activity ( 116 ). Studies in prostate cancer have reported on many mechanisms of rapid non genomic 2nd messenger signaling following exposure to androgens ( 117 , 118 ).…”