towards improved death receptor targeted therapy for ... - TI Pharma

DISCUSSION
p38 and JNK induction by TRAIL
Tumor cells, including NSCLC cells, display variable sensitivity to TRAIL receptor‐targeted
agents ranging from highly sensitive, intermediate sensitive to highly resistant [31;32]. In
this study, we aimed to examine the role of MAPK p38 and JNK in modulating the
apoptotic response in NSCLC cells and to elucidate the underlying mechanisms. JNK and
p38 were phosphorylated within a few hours in sensitive H460 cells, but not in resistant
A549 cells (Fig. 1). In H460 cells, co‐exposure of TRAIL with chemical inhibitors of JNK and
p38 or siRNAs against these kinases identified a proapoptotic function of p38, contrasting
a prosurvival effect of JNK (Fig. 2). In a broader NSCLC cell panel with varying TRAIL
sensitivity, JNK inhibition enhanced apoptosis particularly in the TRAIL intermediate
sensitive H1975 and H322 cells, confirming antiapoptotic activity of this kinase (Fig. 6).
Inhibition of p38 did not significantly affect TRAIL sensitivity.
The mechanism by which JNK and p38 affect apoptosis by TRAIL in H460 cells was studied
in more detail. We found that TRAIL‐induced p38 phosphorylation
preceded JNK
phosphorylation (Fig. 1). Interestingly, JNK activation occurred when RIP1 cleavage was
evident. Furthermore, comparison of caspase cleavage patterns in TRAIL and TRAIL/ p38
inhibitor‐treated H460 cells indicated a stimulatory effect of p38 on caspase‐9 and ‐3 and
PARP cleavage (Fig. 2e). Similar experiments with the JNK inhibitor revealed a suppressive
effect of JNK on the cleavage of caspase‐8, Bid, caspase‐9, ‐3 and PARP, and also RIP1
cleavage. These findings suggest a role of both p38 and JNK in affecting mitochondrial/
caspase‐9‐dependent apoptosis, whereas JNK inhibition has additive effects on the
activation of the DISC compounds RIP1 and caspase‐8, and subsequent engagement of the
mitochondrial route via Bid cleavage. Part of the prosurvival effect of JNK may be the
suppression of RIP1 cleavage, and RIP1 on its turn being able to prevent TRAIL‐dependent
JNK activation in some sort of feedback loop. However, thus far RIP1 is not a known
substrate of JNK [33] and the effect of JNK on RIP1 cleavage may be indirect requiring
another protein.
RIP kinases constitute a family of seven members, namely RIP1‐7. They are serine
threonine kinases being crucial regulators of cell survival and cell death. Depending on the
cellular context, RIP1 is implicated in the activation of NF‐κB, MAPKs, apoptosis or necrosis
[34]. RIP1 can be cleaved by caspase‐8, abrogating NF‐κB activation and together with the
cleaved product, RIPc, interacts with TRADD and FADD, leading to an increase of death
receptor‐induced apoptosis [35;36]. We found that reduction of RIP1 expression by a
specific shRNA or inhibition of its kinase activity with necrostatin‐1 enhanced TRAIL‐
induced apoptosis in H460 cells that could be augmented by JNK inhibition. This indicates
that the anti‐apoptotic activity of JNK was maintained in the absence of RIP1 activity (Fig.
3). Indeed, TRAIL‐induced phosphorylation of JNK remained detectable in RIP1 knockdown
cells. Interestingly, phosphorylation of p38 was enhanced but did not have pro‐apoptotic
activity under these conditions, since p38 inhibition did not alter the level of TRAIL‐
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