Abstract

   Potassium channels are important regulators of cellular homeostasis and
   targeting these proteins pharmacologically is unveiling important
   mechanisms in cancer cell biology. Here we demonstrate that
   pharmacological stimulation of the Kv11.1 potassium channel activity
   results in mitochondrial reactive oxygen species (ROS) production and
   fragmentation in breast cancer cell lines and patient-derived organoids
   independent of breast cancer subtype. mRNA expression profiling
   revealed that Kv11.1 activity significantly altered expression of genes
   controlling the production of ROS and endoplasmic-reticulum (ER)
   stress. Characterization of the transcriptional signature of breast
   cancer cells treated with Kv11.1 potassium channel activators
   strikingly revealed an adaptive response to the potentially lethal
   augmentation of ROS by increasing Nrf2-dependent transcription of
   antioxidant genes. Nrf2 in this context was shown to promote survival
   in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell
   death. In conclusion, we found that the Kv11.1 channel activity
   promotes oxidative stress in breast cancer cells and that suppression
   of the Nrf2-mediated anti-oxidant survival mechanism strongly
   sensitized breast cancer cells to a lethal effect of pharmacological
   activation of Kv11.1.

   Keywords: NRF2, Potassium channels, Mitochondria, Cancer cell survival

1. Introduction

   Breast cancer is a heterogeneous disease both biologically and
   clinically with highly variable outcomes. Different types of breast
   cancer are commonly characterized by the expression levels of estrogen
   (ER+) and progesterone (PR+) receptors and/or the proto-oncogene
   receptor protein tyrosine kinase HER2/neu (HER2+) [[41]1]. Breast
   tumors that lack expression of ER and HER2/neu proteins, known as
   triple negative breast cancer (TNBC), are aggressive [[42]2,[43]3],
   highly metastatic, and have the worst outcome of all BC subtypes.
   Breast cancer progression is accompanied by genetic alteration of a
   multitude of genes which alone or in combination can significantly
   alter a variety of cellular events [[44]4]. Although several therapies
   have been developed against breast cancer [[45]5], it remains the
   second leading cause of cancer-related death in women worldwide,
   claiming more than 550,000 lives per year. Treatment options are often
   inadequate due to the difficulty in identifying proteins governing
   crucial biochemical signaling pathways and lack of approved targeted
   therapies, particularly for TNBC.

   Ion channels are the molecular regulators of ion exchange across
   membranes of all cells, and they have emerged as important players in
   cancer biology. Several members of the voltage-gated potassium (Kv)
   channel family, including Kv11.1, have been identified as potential
   targets for cancer therapy [[46]6,[47]7]. Anatomically, human Kv11.1 is
   expressed mostly in the brain and in the heart. These tissues are
   formed by highly differentiated cells that are primarily
   non-proliferative. Kv11.1 channels are also found in cancers with
   different histological characteristics and tissues of origin, including
   breast cancers [[48][7], [49][8], [50][9], [51][10], [52][11],
   [53][12]], where Kv11.1 activity controls several functional hallmarks
   of cancer cells such as proliferation, migration, metabolism and
   survival. However, the biochemical pathways linking K11.1 to these
   events remain largely unknown.

   Mitochondria are the major source of reactive oxygen species (ROS) as
   byproduct of the respiratory chain which relies heavily on ion
   homeostasis. Nevertheless, the interaction among ions and ROS can be
   bidirectional as ions can regulate ROS production and ROS can control
   activities of several ion channels [[54][13], [55][14], [56][15]] that
   can be expressed on both mitochondria and surface cell membranes.
   Increasing evidence indicate that the cross-talk between ions and ROS
   can play a major role in both physiological and pathological condition.
   For example, mitochondrial Ca^2+ homeostasis is fundamental to generate
   important metabolic processes however, high mitochondrial Ca^2+ level
   can initiate cell death pathways [[57]16,[58]17].

   We previously reported that the Kv11.1 potassium channel is expressed
   in breast cancers independently of their molecular and/or histological
   characterization [[59]18]. Furthermore, pharmacological stimulation of
   Kv11.1 results in arresting tumor growth by activation of a cellular
   senescence phenotype [[60]7,[61][18], [62][19], [63][20], [64][21]].
   Although senescent cells downregulate several oncogenes while
   significantly upregulating tumor suppressors they are metabolically
   active. Therefore, in this context, senescent phenotype could be
   considered as a survival mechanism. In this work, we characterized a
   cellular signaling mechanism linking Kv11.1 activation-dependent
   increase in mitochondrial ROS production to a NRF2-dependent
   antioxidant response that overcomes potentially lethal pharmacological
   treatment.

2. Results

2.1. Activation of Kv11.1 alters mitochondria structure

   We assessed the impact of Kv11.1 activation on mitochondrial structure.
   We performed experiments using the mitochondria-specific fluorescent
   label Mitotracker green ([65]Fig. 1A). Surprisingly, significant
   mitochondrial swelling was visible in the NS1643 treated cells already
   5 min after exposure to different concentrations of the drug. To
   further characterize this effect, we used Tetramethylrhodamine Methyl
   Ester (TMRM) fluorescent dye to monitor mitochondria metabolism upon
   NS1643 application ([66]Fig. 1B). We found that NS1643 produces a
   strong depolarization ([67]Fig. 1C) of mitochondria ΔΨ[m] which
   indicates loss of mitochondria function. Also, live cell imaging
   revealed that NS1643 treatment shortens the mitochondria ([68]Fig. 1D)
   confirming the potent effect of Kv11.1 activation on mitochondria
   morphological structure.

Fig. 1.

   [69]Fig. 1
   [70]Open in a new tab

   Activation of Kv11.1 affects mitochondria structure. A) Mitochondrial
   morphology highlighted in MDA-MB-231 cells using 200 nM of Mitotracker
   green for 20 min at 37 °C. Mitochondrial swelling was visible in the
   NS1643 treated cells 5 min after compound addition. Representative
   images and magnification of three independent experiments are shown. B)
   Live cell imaging of MCF7 cells treated with 2 μl DMSO or 50 μM NS1643
   before image acquiring. Yellow insert is magnified on the panel below.
   Pseudo-color masks represent mitochondria in different length group:
   Green (<1.5 μm), Red (1.5–10 μm) and Yellow (>10 μm). E) Fluorescence
   intensity of TMRM was measured to represent the change of mitochondrial
   membrane potential (DΨm). F) Mitochondria categorized into different
   length groups, the short (<1.5 μm), medium (1.5–10 μm) and elongated
   (>10 μm). The percentage of each group was calculated by dividing sum
   area of each group with total mitochondrial area. (For interpretation
   of the references to color in this figure legend, the reader is