(F-223) Examining the Apoptotic Mechanism of Piezo1 Activation and TRAIL Sensitization in Glioblastoma Cells

Introduction:: Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer in adults with a median survival of 14 months post-treatment [1]. The current standard of care for patients with GBM is temozolomide (TMZ), the first widely available oral chemotherapy drug [2]. However, TMZ has shown limited clinical success due to its non-specific selectivity of GBM cells and easily acquired drug resistance. Contrary to TMZ, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic that exhibits high specificity to kill malignant cancer cells while completely evading healthy cells [3]. Despite TRAIL’s insufficient anti-cancer efficacy in clinical trials, the King Lab recently discovered that TRAIL treatment combined with the activation of Piezo1, a mechanosensitive Ca2+ ion channel, significantly sensitizes prostate cancer cells to apoptosis [4]. Piezo1 activation induces Ca2+ influx into the cell, and activation can be triggered by mechanical stimulation, such as fluid shear stress (FSS), or treatment with its chemical agonist, Yoda1. We investigated the mechanism of Piezo1 sensitization to TRAIL-mediated apoptosis in GBM cells by inhibiting key proteases within the proposed cell signaling pathway, including bax, calpain, and Piezo1 (Fig. 1). Since GBM is particularly resistant to a TRAIL treatment in isolation, a combinatory treatment regimen involving Piezo1 activation and TRAIL is examined in this study to elucidate a new clinical therapeutic approach to extrinsically motivate GBM cell death.

Preferred Sub-Track: Cancer Drug Delivery


Materials and Methods:: To investigate the effects of Piezo1 activation on TRAIL-mediated apoptosis, two glioblastoma cell lines were studied – U-87 MG cells (TMZ sensitive) and LN-18 cells (TMZ resistant). Since Piezo1 is a mechanosensitive ion channel, Yoda1, Piezo1’s chemical agonist, was utilized to track intracellular Ca2+ mobilization in flow cytometry by ratiometric fluorescence of Fura Red (2mM) and Fluo-4 (1mM). U-87 MG and LN-18 cells were treated with 5 µM DMSO, 5 µM Yoda1, and 50 µM GsMTx-4, a stretch-activated ion channel inhibitor. Cell viability with calpain inhibition was measured after U-87 MG and LN-18 cells were treated with 25 ng/mL TRAIL, 10 µM DMSO, 10 µM Yoda1, and 5 µM Calpeptin, a calpain protease inhibitor. After treatment, an Annexin V assay was performed to evaluate cell death via flow cytometry. To further understand the effects of mitochondrial permeabilization and bax inhibition, both cell lines were treated with 25 ng/mL TRAIL, 10 µM DMSO, 10 µM Yoda1, and 0.25 µM Bax Channel Blocker (BCB), a bax protease inhibitor. After treatment, cells incubated for 15 minutes with 40 µM JC-1 dye. Mitochondrial outer membrane permeabilization (MOMP) was assessed via flow cytometry. Lastly, to validate Piezo1’s role in GBM cell sensitization to TRAIL, a mechanical shear stress of 5.0 dyn/cm2 was applied to both cell lines for one hour following TRAIL (25 ng/mL) treatment. Cells were stained with Annexin-V and Propidium Iodide fluorescent dyes and measured in flow cytometry.

Results, Conclusions, and Discussions:: In this study, we found that Piezo1 activation via its chemical agonist Yoda1 and mechanical stimulus FSS sensitizes U-87 MG and LN-18 GBM cells to TRAIL-mediated apoptosis (Fig. 1). Inhibition of Piezo1 via GsMTx-4 significantly decreases Ca2+ influx following Yoda1 treatment, where U87 cells required a higher concentration of GsMTx-4 to successfully inhibit Piezo1 (Fig. 2) [5]. Further, by inhibiting calpain, the immediate successor to Piezo1 activation and Ca2+ influx, and bax, the immediate precursor to mitochondrial permeabilization, there was a significant decrease in cell death and mitochondrial depolarization following combined TRAIL and Yoda1 treatment (Fig. 3A-B) [5]. Moreover, the FSS and TRAIL combination treatment resulted in a significant decrease in cell viability in U87 and LN18 cells when compared to the TRAIL-treated cells in the absence of FSS (Fig. 4A-B). Ongoing research includes the investigation of extrinsic activation methods that utilize chimeric antigen receptor (CAR) T-cells to maximize GBM cell death. This study determines the signal transduction mechanisms in which Piezo1 activation sensitizes GBM cells to TRAIL-mediated apoptosis via Piezo1, calpain, and bax inhibition. The synergistic “crosstalk” between TRAIL and Yoda1 significantly enhances GBM cell death. Although the brain possesses low levels of FSS, the mechanism in which Piezo1 activation incurs apoptosis has numerous therapeutic implications as anti-cancer targets. As a result, the success of this study leads to a highly specific and minimally invasive approach that overcomes TMZ resistance in GBM tumors by sensitizing cells to TRAIL via chemical activation or mechanical stimulation of Piezo1.

Acknowledgements (Optional): : This work was funded by the National Institute of Health, Grant Numbers R01CA256054 and R01CA203991 to M.R.K. This material is based on work supported under National Science Foundation Graduate Research Fellowship to J.A.D. and N.S.S. (1937963). Funding from the SyBBURE Searle Undergraduate Research Program was provided to S.H.D. and N.S.S.

References (Optional): : [1] Delgado-López, P. D., & Corrales-García, E. M. (2016). Survival in glioblastoma: a review on the impact of treatment modalities. Clinical & Translational Oncology, 18(11), 1062–1071. https://doi.org/10.1007/s12094-016-1497-x
[2] Schnöller, L. E., Piehlmaier, D., Weber, P., Brix, N., Fleischmann, D. F., Nieto, A. E., Selmansberger, M., Heider, T., Hess, J., Niyazi, M., Belka, C., Lauber, K., Unger, K., & Orth, M. (2023). Systematic in vitro analysis of therapy resistance in glioblastoma cell lines by integration of clonogenic survival data with multi-level molecular data. Radiation Oncology, 18(1), 51. https://doi.org/10.1186/s13014-023-02241-4
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[5] Knoblauch, S. V., Desai, S. H., Dombroski, J. A., Sarna, N. S., Hope, J. M., & King, M. R. (2023). Chemical Activation and Mechanical Sensitization of Piezo1 Enhance TRAIL-Mediated Apoptosis in Glioblastoma Cells. ACS Omega, 8(19), 16975–16986. https://doi.org/10.1021/acsomega.3c00705