Transcriptional Up-Regulation of FBXW7 by K<sub>Ca</sub>1.1 K<sup>+</sup> Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model

Introduction PrimeSurface Research Papers

Transcriptional Up-Regulation of FBXW7 by K_Ca1.1 K⁺ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model

Susumu Ohya, Hiroaki Kito, Junko Kajikuri , Yohei Yamaguchi and Miki Matsui

Int. J. Mol. Sci. 2024, 25, 6019. | https://doi.org/10.3390/ijms25116019

Copyright © Authors 2024
This article is licensed under a Creative Commons Attribution 4.0 International License (CC BY).

Background

The tumor suppressor FBXW7 is known to reduce cancer stem cell properties by promoting the proteolysis of pluripotent stem cell markers. Meanwhile, K_Ca1.1 (calcium-activated potassium channel) has been reported to be involved in cancer cell proliferation and cancer stem cell properties, with its inhibition demonstrating tumor suppressor effects. The authors have previously demonstrated that FBXW7 is an essential regulator of K_Ca1.1 protein degradation in several cancers. However, the molecular mechanisms and detailed intracellular signaling pathways by which K_Ca1.1 inhibition affects cancer stem cell properties via FBXW7 transcriptional regulation remain unclear.

Research Achievements

This study aimed to investigate the K_Ca1.1 inhibition-mediated regulation of FBXW7 expression and its associated signaling cascades in K_Ca1.1-expressing cancer cells. The authors found that inhibition of the Ca²⁺-activated K channel K_Ca1.1 in 3D model of human prostate cancer (LNCaP cells) promotes FBXW7 transcriptional activity through the Akt-Nrf2 signal pathway.
PrimeSurface™ 96U plate was used to form 3D spheroids for gene and protein expression analyses and other assays showing significantly increased FBXW7 expression and reduced Nrf2 nuclear translocation after K_Ca1.1 inhibitor PAX treatment. Furthermore, siRNA-mediated suppression of FBXW7 expression was observed using miR223 mimics. These results suggested that FBXW7 plays a crucial role in transcriptional regulation. These findings contribute to elucidating the mechanism by which K_Ca1.1 inhibition suppresses cancer stem cell properties and indicate its potential as a novel therapeutic target.

Figure 1. Effects of the treatment with PAX on FBXW7 expression and effects of Akt and Nrf2 activation on the K<sub>Ca</sub>1.1 inhibition-induced up-regulation of FBXW7 in the 3D spheroid model of LNCaP cell

Figure 1.
Effects of the treatment with PAX on FBXW7 expression and effects of Akt and Nrf2 activation on the K_Ca1.1 inhibition-induced up-regulation of FBXW7 in the 3D spheroid model of LNCaP cell

Figure 8.
Effects of the treatment with PAX on c-Myc expression in the LNCaP spheroid model.

Use of PrimeSurfaceTM in this study

Plate: PrimeSurface™ 96U plate
Cell: LNCaP cells; Cell density: 1x10⁴ cells/well; Medium: RPMI 1640 medium (10% FBS)
Methods and Results:
3D spheroid culture more closely mimics the in vivo environment than 2D culture, so the spheroid model was used. Gene and protein expression were performed using real-time PCR and Western blot analysis. Treatment with PAX, a K_Ca1.1 inhibitor, for 12 hours significantly increased FBXW7 expression in the spheroids and decreased the protein expression of c-Myc, a pluripotency marker. Furthermore, Ark and the Nrf2 signal activators SC79 and NK252 also used to confirm FBXW7 protein expression. These results suggested that inhibition of K_Ca1.1 via the Akt-Nrf2 signaling pathway promotes FBXW7 transcriptional activity, accelerates protein degradation, and suppresses cancer stem cell stemness.

(For details, please refer to the paper)　