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Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B,
Paula Lindner1,2, Søren Brøgger Christensen3, Poul Nissen2
1Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership for Molecular Medicine, University of Oslo, P.O. Box 1137, Blindern, N-0318, Oslo, Norway.
Background:
Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive.
Methods:
To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling.
Results:
In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg.
Conclusions:
Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. Video Abstract.
Insights
Thapsigargin (Tg) triggers cell death via the unfolded protein response (UPR) and death receptor 5, revealing new signaling pathways for cancer therapy. This research clarifies ER stress-induced apoptosis mechanisms.
Area of Science:
- Cellular Biology
- Molecular Biology
- Cancer Research
Background:
- Endoplasmic reticulum (ER) stress is implicated in disease, but its cell death mechanisms remain unclear.
- Thapsigargin (Tg) is a key tool for studying ER stress and a basis for cancer therapeutics.
- The roles of the unfolded protein response (UPR) and autophagy in Tg-induced apoptosis are not fully understood.
Purpose of the Study:
- To elucidate the signaling pathways and mediators involved in thapsigargin (Tg)-induced cell death.
- To investigate the integration of UPR components and the role of autophagy in ER stress-induced apoptosis.
- To compare the mechanisms of cell death induced by Tg and its therapeutic analogs.
Main Methods:
- Analyzed Tg and analog effects in LNCaP prostate and HCT116 colon cancer cells.
- Utilized RNA interference (RNAi) and inhibitory drugs targeting death receptors, UPR, and ATG proteins.
- Quantified cell death and monitored molecular changes via fluorescence imaging, RT-PCR, and Western blotting.
Main Results:
- Tg-induced cell death requires death receptor 5 and caspase-8, with MAP1LC3B acting non-autophagically upstream of caspase-8.
- PERK, ATF4, and CHOP are essential for Tg-induced cell death, acting in parallel pathways.
- ATF4 and CHOP independently upregulate death receptor 5 and MAP1LC3B, while IRE1 signaling is cell-type specific.
Conclusions:
- Provides a novel, integrated model of UPR signaling in ER stress-induced cell death.
- Identifies key mediators and parallel pathways contributing to thapsigargin-induced apoptosis.
- Confirms identical molecular requirements for cell death induction by Tg and its therapeutic analogs.
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