AbstractCD40, a non-classical TNFR family member, is expressed by a variety of cancer cells. Previously, our laboratory has shown that membrane-presented CD40L (mCD40L) causes extensive apoptosis in renal, bladder and colon cancer-derived cells but not in normal epithelial cells, rendering CD40 a promising target for cancer therapy. Although the apoptotic outcome of mCD40L is well-documented in epithelial cells, the role of CD40 and the effects of CD40 ligation in human bone cancer-derived cells remain largely unknown. This thesis aimed to investigate for the first time the functional consequences of CD40 activation, and to underline the intracellular signalling pathways responsible for the mCD40L-mediated effects in human osteosarcoma and Ewing’s Sarcoma-derived cell lines.
The first part of the experimental work in this thesis focused on the optimisation of a cell death assay (based on membrane permeability) utilising an in vitro co-culture model for the delivery of the mCD40L signal to the osteosarcoma and Ewing’s sarcoma cells. Moreover, additional cell death assays were utilised for the detection of apoptotic cell death hallmarks including DNA fragmentation and activation of effector caspases-3/7. Immunoblotting techniques were also adapted for the detection of intracellular CD40 signalling mediators, followed by functional inhibition experiments of intracellular mediators and caspases for the validation of signalling molecules involved in CD40-mediated cell death. A significant part of the thesis involved the detection of cytokine/chemokine secretion following the delivery of mCD40L signal.
Expression of CD40 by osteosarcoma and Ewing’s sarcoma cells was detected at variable expression levels and treatment with pro-inflammatory cytokines induced up-regulation of CD40 in the already CD40 positive cell lines in a cytokine-dependent type and cell line-specific manner. Cell death assays showed for the first time that mCD40L induces apoptosis in osteosarcoma cells whereas Ewing’s sarcoma cells are less susceptible. By contrast, anti-CD40 agonistic antibody G28.5 mAb failed to cause cell death in osteosarcoma cells. Additionally, mCD40L-mediated cell death in osteosarcoma cells was accompanied by activation of effector caspases-3/7 and DNA fragmentation, whereas in Ewing’s sarcoma cells cell death was supplemented with limited DNA fragmentation indicating that mCD40L triggered cell death in a tumour cell-specific manner and probably is dependent on the level of CD40 expression and degree of ligation.
mCD40L triggered the rapid and sustained induction of TRAF1 and TRAF3 as early as 1.5h CD40 post ligation in osteosarcoma cells, whilst G28.5 mAb caused a more gradual up-regulation of TRAF1 and TRAF3 following 3h receptor post ligation and also triggered the rapid induction of TRAF2 and TRAF6 as early as 1.5h post ligation. Strikingly, mCD40L triggered differential regulation of TRAFs in Ewing’s sarcoma cells compared to osteosarcoma, with the protagonist TRAF3 not being as highly expressed and sustainably up-regulated as it was in osteosarcoma. Additionally, mCD40L-CD40 engagement in osteosarcoma cells caused the activation of ASK-1, MKK4 and downstream JNK1 and p38 critical for the induction of CD40-mediated cell death in osteosarcoma cells, whereas treatment with anti-CD40 agonistic G28.5 antibody did not cause sustained activation of p38 and caused phosphorylation of the JNK2 isoform. By contrast, inhibition of MEK/ERK did not influence the CD40-mediated apoptotic cell death in osteosarcoma cells, whereas in Ewing’s sarcoma cells, MEK/ERK blockade caused a dramatic increase in the levels of CD40-mediated cells death; indicating that ERK1/2 signalling interferes with the pro-apoptotic signalling.
mCD40L triggered the induction of BAX and BAK pro-apoptotic proteins following 12h post-ligation and low induction of PUMA in osteosarcoma cells. Additionally, inhibition of caspase-9 nearly abrogated CD40-mediated apoptosis in osteosarcoma cells, while caspase-8 and caspase-10 inhibition did not cause any significant alteration at the levels of CD40-mediated cell death, indicating that CD40-mediated apoptosis in osteosarcoma cells occurs directly via the intrinsic apoptotic pathway. By contrast, inhibition of individual initiator caspases did not significantly alter the levels of CD40-mediated cell death in Ewing’s sarcoma cells, suggesting that the cell death observed is caspase-independent. Additionally, this study showed that blockade of ROS did not alter the levels of cell death in osteosarcoma cells, suggesting that further experiments should be conducted to investigate in more detail the role of ROS in CD40-mediated cells death in osteosarcoma and Ewing’s sarcoma cells. To note upon CD40-mCD40L interactionTrx-1 is downregulated in osteosarcoma and Ewing’s sarcoma cells, suggesting the possible involvement of ROS in CD40-mediated cell death.
Cytokine array assay showed that mCD40L induced marked induction of IL-8, GM-CSF, and other pro-inflammatory chemokines in osteosarcoma cells whereas in Ewing’s sarcoma cells it caused the induction of differential cytokine pattern at extremely low levels, indicating that cytokine/chemokine secretion highly depends on the signal quality and cell type and is in line with the level of apoptosis observed in each case.
Collectively, this study has for the first time demonstrated that mCD40L triggers apoptosis in osteosarcoma cells whereas in Ewing’s sarcoma cells it induces lower levels of cell death which is caspase-independent. Although additional experiments would be necessary to fully optimise the combinatorial treatment in osteosarcoma cells it was shown that anti-CD40 agonistic antibody G28.5 did not induce cell death in osteosarcoma cells, however it showed to be promising when combined with PX-12 (TRX-1 inhibitor). Furthermore, this study revealed that CD40-mediated killing in osteosarcoma cells occurs via a TRAF1/3-ASK-1-MKK4-JNK/p-38-AP-1 signalling pathway leading to the activation of caspase-9, and caspase-3/7, thus intrinsic apoptosis. Importantly, it was shown that ERK1/2 signalling pathway interferes with the induction of optimum cell death in Ewing’s sarcoma cells. These findings have supplied novel observations on the role of CD40 in affecting the fate of osteosarcoma and Ewing’s sarcoma cells, emphasising the significance of cell type, quality of signal and the expression levels of receptor in determining the outcome. Equally important are the findings showing the resistance mechanisms of Ewing’s sarcoma cells to CD40-mediated cell death. In addition, the findings are assisting in the improvement and formulation of novel therapeutic ways that might apply CD40 for anticancer therapies against different types of bone cancers.
|Date of Award
|5 Jul 2022
|Nikolaos Georgopoulos (Main Supervisor) & Roger Phillips (Co-Supervisor)