Identification of the biochemical signalling mechanisms underlying CD40 killing in colorectal cancer cells

CD40 is a member of the tumour necrosis factor receptor (TNFR) superfamily and ligation by
membrane- presented CD40 ligand (mCD40L), but not soluble agonists, causes extensive apoptosis in
malignant epithelial cells, including colorectal carcinoma (CRC) cells. This thesis aimed to
unravel the precise cell signalling pathways responsible for mCD40L-mediated apoptosis in CRC
cells.

This study has provided evidence that CRC cell death by mCD40L is rapid. mCD40L activated MOMP,
cytochrome c release from mitochondria and induction of Bak/Bax within <6 hours post ligation. The
pro- apoptotic role of Bax was confirmed by shRNA-mediated Bax knockdown as this attenuated
apoptosis and decreased caspase 3/7 activity. mCD40L triggered rapid TRAIL induction and
a caspase-dependent pathway that involved caspase-10 (but not caspase-8) and caspase-9 to cause
CRC cell death. Thus CD40 cross-talks with the extrinsic pathway by inducing TRAIL-mediated,
caspase-10 activation, mitochondrial disruption, tBid activation, Bak/Bax induction, and activation
of caspase-9 and -3/7 to cause CRC cell death.

When the signalling pathways triggered by CD40 were studied further, we found that CD40 induced
both p- JNK and p-p38 in CRC cells which is necessary for apoptosis, and that JNK might be acting
downstream of p38. p38 and JNK directly regulated Bak/Bax and TRAIL induction at the
transcriptional level. We also showed that TRAF1, -3, and 6 were induced in CRC cells as early as
1.5 hours post ligation. Our studies not only demonstrated a novel pattern of TRAF regulation in
CRC cells but revealed for the first time that TRAF3 has an essential role in CD40-mediated CRC
cells death. TRAF3 is central in the induction of apoptosis as its knockdown attenuates apoptosis,
by abrogating p38 and JNK activation, induction of Bak/Bax and caspase-3/7 activation. Therefore,
despite the existence to a dual apoptotic pathway being engaged in CRC cells, TRAF3 appears to be
central in both signalling axes.

ROS are rapidly induced in CRC cells by CD40 in a Nox-dependent fashion and this plays an important
role in CD40-mediated killing. More specifically, CD40 activation appears to result in
TRAF3-dependent p40phox activation. CD40 also regulates directly ROS scavenging mediators, as we
detected reduction in Trx-1 expression. Moreover, CD40 triggered activation of the Trx-regulated
pro-apoptotic kinase ASK-1, which provided direct molecular explanation for the importance of ROS
in CD40 signalling and downstream activation of MKKs and p38/JNK. Thus, the mCD40L-CD40-TRAF3-NOX
axis utilises ROS for the activation of ASK-1/MKK/p38/JNK pro-apoptotic pathways in CRC cells.

Based on observations in this thesis and more recent findings following completion of this work, we
hypothesise that at some point the MAPK/p38/JNK pathway diverges to drive on one hand
transcriptional upregulation of TRAIL, activation of tBid and cross talk to the mitochondria,
whilst the other p38/JNK pathway directly induces Bak/Bax to also induce MOMP and mitochondrial
death, the latter being more reminiscent of CD40-mediated cell death in UCC cells. However, unlike
UCC cells were the operation of only the latter pathway takes place means apoptosis requires a
minimum of 24-36 hours to occur, in CRC cells there is rapid amplification of the apoptotic signal
and quick induction of death. To our knowledge, this is the first demonstration of such extensive
and rapid carcinoma cell apoptosis triggered by CD40 ligation.

Overall, this study has identified the intracellular signalling cascade triggered by CD40 ligation
and results in extensive apoptosis in CRC cells. It has identified a TRAF3-Nox-ROS-ASK1-MKK-p38/JNK
pathway (that activates caspase-10 and caspase-9) as the driving force that triggers both a
TRAIL-associated extrinsic as well as the intrinsic apoptotic pathways. Thus, in CRC cells CD40
induces apoptosis by pathway cross talk which permits strikingly rapid apoptosis. These findings
not only provided novel observations on the mechanisms of apoptosis triggered by the TNSRF member
CD40, and also reinforced the importance of the quality of CD40 signal in determining functional
outcome, but they have also raised interesting hypotheses for further biological studies. Equally
importantly, the findings have also assisted in the formulation of a novel combinatorial
therapeutic approach that may exploit CD40 for anticancer therapy.