Acidosis induces RIPK1-dependent death of glioblastoma stem cells via acid-sensing ion channel 1a

Abstract

Eliciting regulated cell death, like necroptosis, is a potential cancer treatment. However, pathways eliciting necroptosis are poorly understood. It has been reported that prolonged activation of acid-sensing ion channel 1a (ASIC1a) induces necroptosis in mouse neurons. Glioblastoma stem cells (GSCs) also express functional ASIC1a, but whether prolonged activation of ASIC1a induces necroptosis in GSCs is unknown. Here we used a tumorsphere formation assay to show that slight acidosis (pH 6.6) induces necrotic cell death in a manner that was sensitive to the necroptosis inhibitor Nec-1 and to the ASIC1a antagonist PcTx1. In addition, genetic knockout of ASIC1a rendered GSCs resistant to acid-induced reduction in tumorsphere formation, while the ASIC1 agonist MitTx1 reduced tumorsphere formation also at neutral pH. Finally, a 20 amino acid fragment of the ASIC1 C-terminus, thought to interact with the necroptosis kinase RIPK1, was sufficient to reduce the formation of tumorspheres. Meanwhile, the genetic knockout of MLKL, the executive protein in the necroptosis cascade, did not prevent a reduction in tumor sphere formation, suggesting that ASIC1a induced an alternative cell death pathway. These findings demonstrate that ASIC1a is a death receptor on GSCs that induces cell death during prolonged acidosis. We propose that this pathway shapes the evolution of a tumor in its acidic microenvironment and that pharmacological activation of ASIC1a might be a potential new strategy in tumor therapy.

Introduction

Necroptosis is a form of regulated cell death that relies on a cascade involving phosphorylation and activation of serine/threonine receptor-interacting protein kinase 1 (RIPK1) and RIPK3 [1,2,3]. This leads to oligomerization and translocation of the mixed lineage kinase domain-like pseudokinase (MLKL) to the plasma membrane, which finally elicits membrane rupture [4, 5]. The canonical inhibitor of RIPK1 signaling is necrostatin 1 (Nec-1) [6]. Necroptosis can be induced by tumor-necrosis-factor receptor 1 (TNFR1) signaling [7], but TNFR-independent pathways for necroptosis induction via RIPK1 are less well understood [6, 8, 9].

Recently, it has been shown that necroptosis of neurons can be induced by the activation of acid-sensing ion channel 1a (ASIC1a) [10, 11] independently of TNFR1. ASIC1a is a neuronal Na⁺ channel that is expressed throughout the nervous system [12, 13] and is activated by extracellular protons with high sensitivity (half-maximal activation at pH 6.6) [14]. Physiologically, it contributes to sensing transient decreases in pH as they occur during synaptic transmission. Even though ASIC1a desensitizes within a few seconds, it can also signal sustained acidosis, for example during ischemic stroke. In animal models of ischemic stroke, ASIC1a is responsible for acid-induced cell death: the specific toxin inhibitor psalmotoxin 1 (PcTx1) [15] or knockout of the ASIC1a gene significantly reduces neuronal death [49,50]. On the other hand, and in agreement with an anti-tumorigenic role of ASIC1a, the expression of ASIC1a is associated with increased survival time in lower-grade glioma patients [26]. Because ASIC1a activation leads to increased cell death only early during sphere formation, in vivo studies are necessary to reveal whether this pathway is indeed anti-tumorigenic.

GBM is heterogeneous [51]. We, therefore, included two GSC lines from primary GBM with different transcriptional profiles: R54, with a pro-neural-like profile, and R8, with a mesenchymal-like profile [52]. It was remarkable that mild acidosis similarly reduced SFR for both GSC lines in a PcTx1 and Nec-1-dependent manner, suggesting that cell death induction by activation of ASIC1a is common to different GSCs. Sustained acidosis is not only a hallmark of the TME but accompanies many inflammatory and neurodegenerative diseases of the CNS, such as multiple sclerosis (MS), Alzheimer’s (AD), and Parkinson’s disease (PD) as well as traumatic brain injury. ASIC1a activity, causing cell death and inflammation, is increasingly associated with these diseases [53,54,55] and ASIC1a inhibition ameliorates cell damage [Transmission electron microscopy

GSCs were cultured at pH 7.4 or pH 6.6 for 1 or 7 days. Cells were collected by centrifugation, washed in DPBS, and immediately fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 h, followed by washing in the buffer for a further 24 h. Cell pellets were collected by centrifugation (1000 rpm, 5 min) and embedded in 2% low gelling temperature agarose (#A9414, Sigma). Small blocks of embedded cells were sliced and post-fixed in 2.5% glutaraldehyde for 24 h followed by washing in 0.1 M phosphate buffer for 24 h. Agarose blocks were then incubated in 1% OsO₄ (in 0.2 M phosphate buffer) for 3 h, washed twice in distilled water, and dehydrated using ascending alcohol concentrations (i.e., 25%, 35%, 50%, 70%, 85%, 95%, and 100%; each step for 5 min). Dehydrated blocks were incubated in propylene oxide followed by subsequent 20 min of incubation in a 1:1 mixture of epon (47.5% glycidether, 26.5% dodenylsuccinic acid anhydride, 24.5% methylnadic anhydride, and 1.5% Tris (dimethyl aminomethyl) phenol) and propylene oxide. The samples were then incubated in epoxy resin for 1 h at room temperature followed by polymerization (28 °C for 8 h, 80 °C for 2.5 h, and finally at RT for 4 h). Ultra-thin sections (70 nm) were mounted on grids, contrast-enhanced with uranyl acetate and lead citrate, and examined with an EM900 electron microscope (Zeiss, Germany). Images were captured using a Slow-scan CCD-Camera (TRS, Germany).