Ateeg, Salim (2018) Investigation of the Biological Mechanisms Activated by CD40 in Prostate Cancer Cells. Doctoral thesis, University of Huddersfield.

CD40 is a prominent member of the TNFR family due to its ability to be expressed by and regulate the fate of not only immunocytes, but also non-lymphoid cells. Previous studies have demonstrated that CD40 ligation by cell-surface presented agonists, and in particular membrane CD40L (mCD40L), caused extensive apoptosis specifically in a variety of malignant epithelial cells (including bladder and colorectal). By contrast, soluble CD40 agonists are weakly pro-apoptotic and only become significantly pro-apoptotic by pharmacological intervention. Recent work from our laboratory has shed light onto the tumour-specificity of CD40 as well as the differences in soluble versus membrane-presented agonists in terms of pro-apoptotic capacity. As the role of CD40 in prostate cancer remains unknown, the main aim of this study was to investigate the hypothesis that the CD40/CD40L dyad regulates prostate carcinoma (PCa) cell fate and to explore the mechanisms of this in a panel of well-characterised human PCa lines.

In order to achieve CD40 ligation by mCD40L, a co-culture in vitro model was used, whereby target PCa cells were co-cultured with third-party (murine fibroblasts 3T3CD40L (engineered to express mCD40L). This mode of ligation was compared to agonistic CD40 antibody. Flow cytometry allowed detection of CD40 expression in a panel of PCa lines, comprising DU145, LNCaP and PC-3 cells. Apoptosis was detected using several assays, focusing on classical hallmarks of apoptosis (loss of cell membrane integrity, caspase activation, and DNA fragmentation). ELISA assays were employed for detection of pro-inflammatory cytokine secretion and spectrophotometry and flow cytometry were used for detection of ROS. Immunoblotting techniques were also standardised and utilised for the accurate and sensitive detection of intracellular proteins involved in CD40 signalling. Experiments using retroviruses were also employed to engineer CD40 expression in negative PCa cells.

Ligation of CD40 caused apoptosis in DU145 cells and LNCaP cells. By contrast, CD40-ve cells PC-3 were refractory to CD40 ligation. Restoration of CD40 expression restores susceptibility to CD40 apoptosis. Importantly, receptor ligation by mCD40L, and not soluble agonist, could cause cell death, as soluble agonist (cross-linked G28-5 mAb) was not pro-apoptotic. mCD40L, but not G28-5, induced rapid secretion of pro-inflammatory cytokines IL-6, IL-8 and GM-CSF, thus CD40 killing was pro-inflammatory. CD40 induced apoptosis as evident by membrane integrity loss and DNA fragmentation, both hallmarks of apoptotic death. Yet, it was found that CD40 triggers a death type that is caspase-independent. The work showed that CD40 in PCa cells triggers death that does not involve cross-talk with the extrinsic pathway, but via a direct signal that involved the mitochondrial pathway as indicated by the induction of Bak and Bax proteins. mCD40L triggered rapid induction of TRAF1 and TRAF3 whilst TRAF2 expression was downregulated. ASK1 was activated which was subsequently followed by MKK7 but not MKK4 activation and this was followed by JNK phosphorylation. Functional inhibition experiments showed that both JNK/AP-1 and p38 are important for death induction. ROS production could not be detected upon CD40 activation and functional inhibition experiments showed ROS is not critical for CD40 mediated death in PCa cells, observations raising the possibility of ROS-independent ASK1 activation. Finally, preliminary experiments using prostate cancer stem cells (CSC), well-established ‘drivers’ of PCa, showed that CSCs were CD40+ve, however, within the time constraints of this project, it was not possible to assess whether CD40 ligation could induce CSC-targeted cell death.

These findings have not only generated novel observations in terms of the ability of CD40 to induce PCa cell death, but have also added to our knowledge of the intriguingly multifaceted effects of CD40 in carcinoma cells. These fascinating observations imply that CD40, whilst engaging signalling pathways with some common intracellular mediators, its precise death pathways can differ both in their exact nature and their exact features. Moreover, in addition to providing biological evidence for the mechanisms of CD40 apoptosis, these observations may represent a promising targeted approach for PCa therapy as the ability to lead to extensive apoptosis in PCa cells. Equally importantly, by efficiently killing PCa cells and causing rapid pro-inflammatory cytokine secretion, whilst at the same time targeting what is potentially the cellular driver of carcinogenesis (CSCs), CD40-mediated killing represents a very promising potential therapeutic tool for PCa therapy in the near future.

Salim Ateeg FINAL THESIS.PDF - Accepted Version
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