Abstract
Autophagy (or type II programmed cell death, PCD), as opposed to apoptosis (or PCD type I), is characterized by excessive accumulation of autophagic vacuoles in the cytoplasm. This mediates destruction of cellular components and produces building blocks essential for cell survival. Yet, autophagy can also participate in the process of cell death, acting in parallel with apoptosis, or synergise with apoptosis by activating or amplifying death signals. Thus, autophagy appears to be able to have both cytoprotective properties but can also facilitate the process of cell death.TRAIL receptors (TRAIL-Rs) activated (‘cross-linked’) by TRAIL agonists have been considered as potential targets for cancer therapy due to their ability to induce tumour cell death; yet they can cause toxicity to normal cells, whilst, importantly, several cancer cell types are more refractory to TRAIL agonists than others, or acquire resistance to
TRAIL toxicity. Some reports have suggested that autophagy may be cytoprotective during TRAIL-mediated signalling. Moreover, other TNF receptors (TNFRs) such as CD40 or Lymphotoxin receptors (LT-Rs) are non-death domain-containing TNFRs that can trigger cell death via recruitment of TNFR-associated factors (TRAF) via common downstream apoptotic signalling cascades; however such receptors appear to induce tumour cell-specific death and the ability of these TNFRs to activate autophagy remains unknown. We have therefore investigated using well established models of apoptosis whether autophagy can enhance TRAIL-mediated death (i.e. is a proapoptotic signal) or oppose apoptotic death (i.e. is cytoprotective). Moreover, for the first time, this study examined the hypothesis that autophagy is activated by non-death domain TNFRs, in particular CD40 and the LT-Rs LTβR and HVEM, the biological responses of which have been extensively characterised previously in our laboratory.
Activation of TRAIL-Rs on EJ, RT112, RT4, HCT116, PC-3 and NHU cells was mediated using TRAIL-R agonists (particularly HERA-TRAIL and Killer-TRAIL) and cells showed differential responses when cell viability and cell death were tested, with the extent of toxicity being both agonist dose- and cell line-dependent. By contrast, normal cells demonstrated limited biological responses to the agonists. Immunoblotting showed that TRAIL-R ligation caused rapid induction of autophagy in all cancer cell lines tested within 30mins of treatment, based on upregulation of marker LC3 and reduction in p62 expression; by contrast no such responses were evident in normal (NHU) cells, as in these cells we found evidence of sustained autophagy induction. Autophagy induction in carcinoma cells was confirmed by autophagic vesicle detection using flow cytometry and fluorescence microscopy. Combination of pharmacological inhibitors of autophagy (such as 3-MA and Wortmannin) with TRAIL-R agonist enhanced growth suppression and cell death in carcinoma cells. By contrast, in normal cells such combinations only caused growth inhibition but not apoptosis, whilst we found evidence that autophagy inhibition alone inhibited growth, indicating the possible importance of autophagy in sustaining the highly proliferative response of NHU cultures.
mCD40L activated autophagy signalling in EJ and HCT116 cells as confirmed by immunoblotting and fluorescence microscopy, unlike the non-apoptotic soluble CD40 agonist (G28-5 mAb), which only activated autophagy in EJ but not HCT116 cells, thus the strongly pro-apoptotic CD40 signal mCD40L is a more efficient activator of autophagy. Moreover, although mLIGHT clearly induced autophagy, it was not superior to soluble LIGHT as neither activated autophagy in HCT116 cells, an intriguing similarity to CD40 ligation by soluble agonist. Inclusion of inhibitors 3-MA, Chloroquine (CQ) and particularly Wortmannin in co-cultures for mCD40L-mediated apoptosis induction provided
evidence for a pro-apoptotic role for autophagy triggered by mCD40L. Caspase-3/7 activation was attenuated in EJ and even more so in HCT116 cells using 3-MA and Wortmannin, yet interestingly blockade of autophagy by CQ potentiated caspase activity in these cells. DNA fragmentation results agreed partially with results from cell death (Cytotox-Glo) and caspase 3/7 (SensoLyte) assays.
These findings have confirmed that TRAIL agonists (such as HERA-TRAIL) induce cytoprotective autophagy rapidly post-receptor ligation. The agonists cause differential cell death activation in cancer cells, but little death in normal (NHU) cells, responses that appear to be (at least partly) attributable to autophagy. In parallel, the results of this work provided evidence for the first time that CD40 and LT-Rs can induce autophagy and that the extent of autophagy activation is dependent on receptor cross-linking capacity of the agonist. More importantly, unlike TRAIL-R mediated autophagy that is cytoprotective, autophagy induced by CD40 appears to be pro-apoptotic, as blockade of autophagy reduced CD40-mediated death. Therefore, this work has provided the novel and extremely interesting observation that autophagy might either protect from or participate in apoptosis activated by different TNFR family members, death receptors versus non classical apoptosis-inducing TNFRs. These observations are not only biologically fundamental but may also have important implications for therapeutic interventions aiming to shift TNFR member-specific responses from cytoprotective autophagy towards efficient cell death.
| Date of Award | 18 Jun 2019 |
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| Original language | English |
| Supervisor | Nik Georgopoulos (Main Supervisor) & Georgios Psakis (Co-Supervisor) |