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Published on: August 2, 2021
Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis
M Rytömaa1, L M Martins, J Downward
1Signal Transduction Laboratory Imperial Cancer Research Fund 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Abstract:
Detachment of most untransformed adherent cells from the extracellular matrix promotes apoptosis, in a process termed anoikis [1] [2]. The death signalling mechanisms involved in this process are not known, although adhesion or transformation by ras oncogenes have been shown to protect epithelial cells from apoptosis through activation of phosphatidylinositol 3-kinase and protein kinase B (PKB/Akt) [3]. Here we show that detachment-induced apoptosis (anoikis) is blocked by the expression of a dominant-negative form of FAS-associated death domain protein (FADD) in a number of untransformed epithelial cell lines. Because the soluble extracellular domains of the death receptors CD95, DR4 and DR5 failed to block anoikis, we conclude that ligand-dependent activation of these death receptors is not involved in this process. Detachment induced strong activation of caspase 8 and caspase 3. Detachment-induced caspase-8 activation did not require the function of downstream caspases but was blocked by overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). We propose that caspase-8 activation is the initiating event in anoikis, which is subsequently subject to a positive-feedback loop involving mitochondrial events.
Insights
Anoikis, or cell death upon detachment, is blocked by inhibiting FAS-associated death domain protein (FADD). This study reveals caspase-8 activation as a key initiator of anoikis, independent of death receptor signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Detachment from the extracellular matrix typically induces apoptosis, a process known as anoikis.
- The precise molecular mechanisms driving anoikis remain largely unknown.
- Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling can protect cells from anoikis.
Purpose of the Study:
- To elucidate the death signaling pathways involved in anoikis.
- To identify the initiating events and key mediators of detachment-induced apoptosis.
Main Methods:
- Expression of dominant-negative FAS-associated death domain protein (FADD) in untransformed epithelial cell lines.
- Assessment of anoikis inhibition by soluble death receptor extracellular domains.
- Analysis of caspase activation (caspase 8, caspase 3) following cell detachment.
- Evaluation of the impact of anti-apoptotic proteins (Bcl-2, Bcl-X(L)) on detachment-induced apoptosis.
Main Results:
- Dominant-negative FADD expression effectively blocked anoikis in multiple epithelial cell lines.
- Soluble death receptors (CD95, DR4, DR5) did not prevent anoikis, indicating their ligand-dependent activation is not involved.
- Cell detachment strongly activated caspase 8 and caspase 3.
- Caspase-8 activation during anoikis was independent of downstream caspases but was inhibited by Bcl-2 and Bcl-X(L) overexpression.
Conclusions:
- Ligand-independent activation of FADD is crucial for initiating anoikis.
- Caspase-8 activation is an early, critical event in the anoikis pathway.
- Mitochondrial events may be involved in a positive-feedback loop amplifying anoikis signaling.
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