Ibraheem, K. (2018) The role of CD40 in regulating renal cell carcinoma cell fate. Doctoral thesis, University of Huddersfield.

CD40 is a member of the TNF receptor (TNFR) superfamily and its expression by a variety of cell types including tumour cells has suggested a possible role for CD40 in epithelial homeostasis and potentially in the pathogenesis of cancer. CD40 ligation by membrane-presented CD40L (mCD40L), but not soluble agonists, causes extensive apoptosis in malignant epithelial cells, including bladder and colorectal cancer cells, while sparing their normal counterparts. However, the role of CD40 in renal cell carcinoma (RCC) is relatively unknown and the effect of CD40 ligation in RCC cells has not been studied previously. This thesis aimed to investigate the effect of CD40 ligation in RCC cells, compare this to their normal counterparts (HRPT cells), and identify the mechanisms of CD40-mediated effects.

The experimental work described in this thesis involved optimisation of assays for the detection of cell death (based on loss of plasma membrane integrity, DNA fragmentation and caspase activation) and for the detection of pro-inflammatory cytokine secretion. Immunoblotting techniques were adapted for a co-culture system to deliver mCD40L for detection of key intracellular CD40 signalling-associated mediators. Optimisation was also carried out for functional experiments using pharmacological inhibitors of intracellular mediators and caspases and for the detection of reactive oxygen species (ROS).

Expression of CD40 by RCC cells was detected in RCC lines and in their normal counterparts HRPT cells and treatment with IFN-ɣ up-regulated CD40 expression in RCC cells. Cytotoxicity assays showed for the first time that mCD40L induced massive apoptosis in human RCC cells which further increased in the presence of IFN-ɣ, whereas it caused no cytotoxic effect in their normal counterparts (HRPT cells). By contrast, the G28-5 mAb did not cause death in RCC cells, and combination of IFN-ɣ with cross-linked G28-5 antibody did not render the G28-5 antibody significantly pro-apoptotic. Moreover, induction of cell death by mCD40L was accompanied by caspase-3/7 activation and DNA fragmentation in RCC cells, while mCD40L did not induce detectable DNA fragmentation in normal HRPT cells indicating that mCD40L triggered “apoptotic” cell death in RCC cells and in a tumour cell-specific fashion.

ELISA assays showed that mCD40L induced marked secretion of IL-8 and IL-6 in RCC cells, which was stronger than that triggered by soluble agonist. More importantly, mCD40L induced GM-CSF secretion in RCC cells, but soluble agonist caused little GM-CSF release. In normal HRPT cells mCD40L caused secretion of IL-8 and IL-6 and a more pronounced secretion of GM-CSF, when it was compared to agonistic G28-5 mAb, confirming that CD40 on HRPT cells was functional despite being non-apoptotic.
mCD40L triggered rapid induction of TRAFs 1, 2, 3 and 6 as early as 1.5h post CD40 ligation in RCC cells. By contrast, despite up-regulation of TRAF1 at 6h post CD40 ligation, in normal HRPT cells mCD40L down-regulated TRAF2 expression and caused no induction in TRAF3 expression. In addition, CD40-mCD40L interactions in RCC cells triggered MKK4/7 activation and downstream phosphorylation of both JNK and p38. Functional inhibition experiments demonstrated that JNK and p38 phosphorylation was essential in CD40-mediated apoptosis in RCC cells, and suggested that activation of p38 may be dependent on JNK activity. By contrast, inhibition of MEK1/2 and NF-кB did not alter CD40-mediated apoptosis in RCC cells, whilst inhibition of AP-1 caused moderate (not complete) reduction in apoptosis.

This study demonstrated that induction of Bak and Bax occurred by 6h post CD40 ligation in RCC cells. Inhibition of caspase-9 significantly attenuated CD40-mediated apoptosis in RCC cells, while caspase-8 and 10 inhibition caused non-significant effects whilst no induction of death ligands was detectable, suggesting that CD40-induced apoptosis in RCC cells occurs via a direct, intrinsic apoptotic pathway. mCD40L triggered ROS production in RCC cells within 1h post CD40 ligation and ROS production were critical in the induction of death, as apoptosis was inhibited by the antioxidant NAC. Moreover, mCD40L triggered phosphorylation of the NOX subunit p40phox and the NOX inhibitor DPI attenuated apoptosis suggesting that a ROS-dependent NOX-triggered pathway may occur in RCC cells. By contrast, non-apoptotic CD40 agonist (G28-5 mAb) did not induce ROS production in RCC cells. Equally importantly, mCD40L caused rapid ASK-1 phosphorylation and down-regulated Trx-1 expression in all RCC lines.

Collectively, this study has for the first time reported that mCD40L induced extensive apoptosis in RCC cells while sparing their normal cell counterparts. However, agonistic anti-CD40 antibody G28-5 did not cause cell death in RCC cells. Although additional functional experiments would be necessary to fully elucidate the functional mechanisms of apoptosis, it appears that CD40-mediated killing in RCC cells occurs via a TRAF3-p40phox-ASK-1-MKK4/7-p38/JNK pathway leading to caspase-9 and effector caspase-3/7 activation and intrinsic apoptosis. Importantly, whilst increasing ROS levels in RCC cells, mCD40L actively down-regulated Trx-1 expression. These findings have provided novel observations on the role of CD40 in regulating human RCC cell fate, and have also reinforced the importance of the quality of CD40 signal in determining functional outcome. Equally importantly, the findings have also assisted in the formulation of novel therapeutic avenues that may exploit CD40 for anticancer therapy and specifically for renal cell carcinoma.

Khalideh Ibraheem FINAL THESIS.PDF - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.

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