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

Chapter 3
induced apoptosis in H460‐shRIP1 cells. The molecular mechanism underlying this finding
is yet unclear, but may point to a role for RIP1 in determining the substrate specificity of
p38 and thus the functional consequences.
TRAIL‐induced JNK phosphorylation required caspase‐8 cleavage and activation as it was
suppressed in H460‐CrmA cells, whereas TRAIL binding is sufficient to induce p38
phosphorylation (Fig. 4). Together, these findings reflect our observation that JNK
phosphorylation occurs at the time that RIP1 is cleaved and inactivated. P38
phosphorylation remains present also after RIP1 is cleaved, in accordance with the finding
that p38 can be activated both in the presence and absence of RIP1 (Fig. 3c). In an earlier
study it was reported that in HeLa cells and mouse fibroblasts RIP1 and TRAF2 are required
for TRAIL‐mediated JNK activation [37]. However, another study showed that in
fibrosarcoma cells RIP1 is not essential for TRAIL‐induced JNK stimulation and that on the
other hand p38 activation was RIP1‐dependent [22]. Thus, the role of RIP1 in TRAIL‐
induced JNK and p38 activation appears to vary in a cell‐specific manner.
We identified Mcl‐1 as a major mediator of both JNK and p38 activity leading to enhanced
or decreased Mcl‐1 expression, respectively. A role for Mcl‐1 as an apoptotic
modulator of
TRAIL‐induced p38 and JNK activation has been described earlier in TRAIL resistant
prostate cancer cells [29]. However, contrary to our findings in NSCLC, activation of both
kinases led to transcriptional upregulation of Mcl‐1 expression [29]. Mcl‐1 transcription
has been reported to be regulated by the transcription factors SRF/ETS, STAT3, CREB and
PU.1 [38]. The transcription factors involved in TRAIL/ MAPK‐dependent modulation of
Mcl‐1 in NSCLC remains to be identified. In addition, Mcl‐1 is also subject to post‐
translational control, as it is degraded by the 26S proteasome after ubiquitylation by the
E3 ligase MULE [39]. Mcl‐1 degradation was shown to be regulated through
phosphorylation at different sites by ERK, JNK, and GSK‐3β, and phosphorylation at Thr163
by ERK
prolonged the Mcl‐1 half‐life [40]. A previous study in hepatocytes showed that
JNK phosphorylation of Mcl‐1 at Ser121, in conjunction with Thr163, stabilizes Mcl‐1 and
protects against TNFα‐induced apoptosis [41]. Thus, in addition to transcriptional effects,
TRAIL‐induced JNK and p38 may also lead to posttranslational modification of Mcl‐1 in
NSCLC cells and affect protein stability. Furthermore, it was reported that the pro‐
apoptotic Bcl‐2 homolog Bim is involved in mediating the TRAIL‐dependent enhancement
of Fas‐induced apoptosis in hepatocytes that occurred through JNK activation [42].
However, in NSCLC cells we did not detect changes in the expression of Bim following
TRAIL treatment and inhibition of either JNK or p38 (data not shown).
Based on our findings in H460 cells we propose a model in which TRAIL induces p38
activation in a RIP1–dependent manner causing a reduction in the level of Mcl‐1, thus
enhancing apoptosis. The activation of caspase‐8 leads to RIP1 cleavage that is required
for JNK activation subsequently causing enhanced Mcl‐1 expression and suppression of
apoptosis (see Fig. 7). JNK on its turn appears to suppress RIP1 cleavage in a direct or
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