Abstract
TNFR member CD40 plays a critical role in immune response when activated via crosslinking by its cognate ligand (CD40L). Yet, CD40 is also expressed on a variety of non-lymphoid origin cells which include fibroblast, endothelial and epithelial cells which respond in different ways to CD40 ligation. In fact, depending on the cell origin and tumorigenesis stage (malignant versus non-malignant), CD40 ligation can lead to cell proliferation, cytostasis or apoptosis, additionally to secretion of cytokine and chemokine. Moreover, the quality of the CD40 signal can have profound effects on functional outcome, as membrane-presented CD40L (mCD40L) but not soluble agonist, can induce extensive apoptosis in malignant cells. The main objective of this study was to investigate the outcome of the CD40 activation using either soluble agonist CD40L (cross-linked G28-5 mAb) or membrane-presented CD40L (mCD40L) in pancreatic carcinoma cells (PaC) and to determine if CD40 ligation can induce death in these cells, as well as understanding the underlying mechanisms of action.After confirmed that CD40 receptor was expressed by three (Panc-1, CFPAC-1 and Capan-2) out of five of the pancreatic cell lines. Activation of CD40 on the receptor-positive cell lines by mCD40L caused a remarkable degree of cell death; by contrast, soluble agonist did not, mCD40L-mediated apoptosis was confirmed by detecting DNA fragmentation and caspase 3/7 activity, indicating that the observed cell death appears to display classical features (hallmarks) of apoptosis. The detection of pro-inflammatory cytokine production was performed to detect intracellular proteins implicated in CD40 signalling.
In Panc-1, CFPAC-1, and Capan-2 cells, CD40 ligation resulted in apoptosis. CD40 ligation, on the other hand, had no effect on BxPC-3 cells (which are CD40 negative). Importantly, soluble agonist (G28-5 mAb) was not pro-apoptotic, whereas mCD40L receptor ligation could trigger cell death. CD40 killing was pro-inflammatory because mCD40L caused fast release of pro-inflammatory cytokines such as IL-6, IL-8, CCL5/RANTES, CCL2/MCP-1, ICAM-1/CD54, CD40Ligand/TNFSF5, and CXCL1/GRO. CD40 triggered apoptosis, which was evident by the loss of membrane integrity, DNA fragmentation as well as caspase activation, all of which are hallmarks of apoptosis. The study demonstrated that CD40 in PaC cells induces death directly, without utilising cross-talk with the extrinsic pathway, as revealed by the activation of Bak and Bax proteins and lack of any classical death ligands. TRAF1, TRAF2, and TRAF6 expression was induced by mCD40L in all CD40-postive PaC cell lines, while TRAF3 was expressed only in Panc-1 cells, which resulted in MKK4 but not MKK7 activation and subsequent phosphorylation of p38. Functional inhibition tests showed that p38 (mainly) and JNK both have a role in death induction, causing activation of caspase-9 and effector caspase-3/7 and intrinsic apoptosis.
These results not only provided unique insights into CD40's potential to promote PaC cell death, but also expanded our understanding of CD40's intriguingly complex impacts on carcinoma cells. These interesting discoveries show that, whereas CD40 interacts with a number of similar intracellular mediators, its precise death pathways may vary in their exact form and characteristics. Equally importantly, in addition to providing scientific evidence for the mechanisms behind CD40-mediated apoptosis, these findings may suggest a prospective target for PaC treatment via the capacity of CD40 to induce widespread death in PaC cells.
| Date of Award | 5 Sep 2022 |
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| Original language | English |
| Supervisor | Nikolaos Georgopoulos (Main Supervisor) & Patrick McHugh (Co-Supervisor) |