The role of CD40 in epithelial cell fate

CD40 belongs to the tumour necrosis factor receptor (TNFR) family and its ligation by membrane- presented CD40 ligand (mCD40L) causes extensive apoptosis in malignant cells, while sparing their normal epithelial counterparts which, can respond to CD40 ligation by proliferation, thus representing an intriguing paradox in TNFR biology. Although the apoptotic potential of mCD40L is well-documented, the precise intracellular signalling pathways of cell death remain largely unknown. Moreover, due to the practical constraints imposed by the necessity to deliver surface-CD40L for therapeutic purposes, deciphering the molecular nature of the CD40 signalling ‘black-box’ may permit the design of novel therapeutic approaches. This thesis aimed to unravel for the first time the precise cell signalling pathways responsible for mCD40L-mediated apoptosis and attempted to determine the mechanisms underpinning the tumour-specificity of CD40 ligation.

The first part of this thesis focused on optimisation of cell models for the delivery of the mCD40L signal to epithelial cells (via co-culture with third party cells and based on the urothelial cell culture system). A number of assays were optimised for the detection of several apoptosis hallmarks (membrane integrity loss, caspase activation, DNA fragmentation and mitochondrial cytochrome c release). Immunoblotting techniques were improved for the sensitive detection of intracellular CD40-signalling mediators, followed by the optimisation of methodologies for retrovirus transduction-mediated delivery of short hairpin RNA (shRNA) molecules for functional (knockdown) experiments.

Expression studies and knockdown experiments reinforced the role of TRAF3 as a key mediator of apoptosis and cemented its essential role in both JNK/AP-1 activation and subsequent induction of pro-apoptotic Bak and Bax, with Bak and particularly Bax loss alone demonstrating as essential for apoptosis. The project also revealed for the first time the proximal events in the CD40 pathway that provided a link between TRAF3 induction and JNK/AP-1 activation followed by Bak/Bax induction. A series of functional experiments involving RNAi-mediated knockdown and/or pharmacological inhibition of critical target proteins suggested that mitogen activated protein kinases (MAPKs) ASK1 and MKK4 (but not MKK7) drive the activation of JNK/AP-1 and CD40-mediated death. Because of the well-documented sensitivity of ASK1 towards oxidative stress and its direct regulation by reactive oxygen species (ROS), a CD40-ROS paradigm was explored. It was found that oxidative stress is essential in CD40-mediated apoptosis as pharmacological inhibitors of ROS attenuated cytotoxicity. Additionally, the work provided novel evidence for a functional role for the NADPH oxidase (Nox) enzyme in apoptosis, as pharmacological inactivation of Nox attenuated ROS induction and blocked apoptosis, signifying that ROS-mediated oxidative stress is Nox-dependant. More specifically, it was found that CD40 ligation caused phosphorylation of p40phox, a Nox-2 subunit previously reported to interact with TRAF3, but not in the context of apoptosis induction. These observations implied that TRAF3-mediated CD40-Nox interactions may be crucial for CD40-induced apoptosis. Importantly, the observation that adequate ROS elevation, to permit efficient thioredoxin (Trx) down-regulation and ASK1 activation, was only observed following receptor ligation by pro-apoptotic mCD40L, but not by non-apoptotic soluble agonist, led to the hypothesis that pharmacological interference with Trx may compensate for the lack of signal strength and sensitise cells to apoptosis. Strikingly, combinatorial treatment of carcinoma cells with a specific Trx-1 inhibitor and agonistic anti-CD40 antibody showed efficient synergy and resulted in extensive apoptosis. The combinatorial treatment appeared functionally equivalent to mCD40L and employed ASK-1/JNK signalling to induce apoptosis.

Finally, to enhance our understanding of the role of malignant transformation in CD40 susceptibility, the effect of over-expression of the telomerase catalytic sub-unit (hTERT) in normal epithelial cells was assessed. It was found that, unlike normal human urothelial (NHU) cells where CD40 ligation did not cause apoptosis (but appeared to be cyto-protective), para-malignant hTERT NHU expressers were highly susceptible to CD40-killing and to an extent equivalent to that observed in carcinoma-derived cells. By comparing normal, para-malignant and tumour-derived cells, the study added to increasing evidence that during malignant transformation cells exhibit higher basal ROS levels, which functions as ‘double-edged sword’ that renders them more susceptible to signals that elevate ROS past a lethal pro-apoptotic threshold, such as that triggered by CD40.

Collectively, this thesis has unravelled for the first time the molecular nature of pro-apoptotic CD40 ligation revealing a key signalling axis triggered CD40 ligation that involves TRAF3, Nox-2 (p40phox), ROS, ASK1, MKK4, JNK, Bak/Bax and intrinsic apoptosis, whilst the new knowledge of the signalling pathway has now provided novel avenues for exploiting CD40 as a target for anti-cancer therapy.