Transient receptor potential melastatin member 4 is the executioner protein for anticancer drug-induced necrotic cell death

Ghosh, Santanu

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https://hdl.handle.net/2142/117657 Copy

Description

Title

Transient receptor potential melastatin member 4 is the executioner protein for anticancer drug-induced necrotic cell death

Author(s)

Ghosh, Santanu

Issue Date

2022-11-29

Director of Research (if dissertation) or Advisor (if thesis)

Shapiro, David J

Doctoral Committee Chair(s)

Shapiro, David J

Committee Member(s)

• Nelson, Erik R
• Procko, Erik
• Christian-Hinman, Catherine A

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• cancer
• UPR
• necrosis
• ion channel
• osmotic stress

Abstract

Classical cell death pathways like apoptosis, autophagy and, more recently, necroptosis have well-established protein markers and pathways. In contrast, protein mediators of anticancer therapy-induced necrotic cell death were poorly defined. Necrosis has been primarily characterized by external, morphological changes as seen by electron microscopy images and the release of immune cell-activating damage-associated molecular patterns, or DAMPs. To explore necrosis, we used our necrosis-inducing anticancer agents, the anticipatory unfolded protein response hyperactivators, BHPI and ErSO. From genome wide CRISPR-Cas9 screens with negative selection against BHPI and follow-on experiments with ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy-induced necrosis. Cancer cells selected for resistance to BHPI and ErSO exhibit robust down-regulation of TRPM4. ErSO works by initiating activation of the anticipatory unfolded protein response (a-UPR), thereby increasing intracellular calcium and decreasing ATP. We propose the increased calcium and decreased ATP opens the TRPM4 channel. Since the extracellular Na+ concentration is ~10x higher than the concentration in cells, this leads to an influx of extracellular Na+, accompanying Cl- to balance the charge, and water to maintain osmolality. This leads to cell swelling, membrane rupture and necrotic cell death. In multiple breast and ovarian cancer cell models, TRPM4 knockout abolished ErSO-induced cell swelling, ATP depletion, sustained UPR activation, necrotic cell death and the ability of medium from ErSO-treated cells to induce activation and increased migration of human macrophage. Notably, knockout of TRPM4 completely abolished ErSO-induced regression of breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy-induced necrosis, TRPM4 knockout strongly reversed rapid cancer cell death induced by four unrelated necrosis-inducing cancer therapies, all with diverse modes of action. We show that although ErSO initiates activation of the a-UPR, it is TRPM4-mediated Na+ influx and cell swelling that sustains and propagates lethal UPR hyperactivation. The cellular oncosis induced by ErSO causes osmotic stress, resulting in sustained activation of the UPR arms and robust ATP depletion. Consistent with this, combining cell swelling by ionomycin and ATP depletion by 2-deoxyglucose, partially replicates the necrotic cell death induced by ErSO. Thus, this work identified both a protein and mechanism for sustained lethal hyperactivation of the UPR in cancer cells and a key protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. In endometrial cancer cells, this pathway is slightly altered. Upon TRPM4 activation by ErSO, endometrial cancer cells attempt to restore the disrupted Na+ ion homeostasis across the plasma membrane using the Na+/K+-ATPase pump to drive out excess Na+ ions. However, due to the sustained influx of Na+ ions through the open TRPM4 channel, this creates a futile cycle leading to significant ATP depletion and cell death. Thus, we show that ErSO-induced necrosis in endometrial cancer cells follows a TRPM4-dependent pathway with a modified ATP depletion mechanism. From a second CRISPR-Cas9 screen with negative selection against ErSO, we also identified FYVE, RhoGEF and PH domain-containing protein 3 (FGD3). FGD3 is a poorly studied paralog of FGD1, a known guanyl-nucleotide exchange factor (GEF) and activator of Cdc42. FGD3 has been predicted to play a role in the organization of the actin cytoskeleton and regulation of cell shape, but has never been connected to necrotic cell death. Cell selected for resistance to BHPI downregulate FGD3, indicating that FGD3 enables BHPI-like necrosis-inducing compounds to effectively induce cell death. Additionally, we also identified the ERα regulator LMTK3, and the TRPM4 regulators, KCTD5 and SATL1, indicating that the screen is recognizing components of a single pathway with multiple components. We also identified a Ca2+-activated phospholipase C, PLCη2, whose activity in a cellular context is largely uncharacterized. This protein can be activated by Ca2+ released from the endoplasmic reticulum (EnR), and would produce more inositol triphosphate (IP3) to keep IP3R channels on the EnR open and help maintain elevated intracellular calcium. This may constitute a feed-forward loop which would explain help the highly sustained nature of the ErSO-induced necrosis pathway.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Santanu Ghosh

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