towards improved death receptor targeted therapy for ... - TI Pharma
towards improved death receptor targeted therapy for ... - TI Pharma towards improved death receptor targeted therapy for ... - TI Pharma
TRAIL‐induced migration and invasion TRAIL‐induced kinases activation and effects on migration, invasion and apoptosis Finally, we explored possible interactions between the identified TRAIL‐induced kinases cascades. Knockdown of Src in A549 cells prevented, and even decreased, the phosphorylation of STAT3 after TRAIL application. Also TRAIL‐induced FAK phosphorylation appeared to depend on Src, whereas p‐Akt and p‐ERK levels were not affected in A549‐shSrc cells (Fig. 8A). On the other hand, inhibition of Akt by LY294002 and ERK by PD098059 also reduced migration and invasion induced by TRAIL (Fig. 8B). Interestingly, inhibition of Akt converted TRAIL resistant A549 cells into apoptosis sensitive cells. In conclusion, we identified novel parallel branches of TRAIL‐induced kinase activation that mediate metastases‐prone and/ or pro‐survival effects (see also Fig. 8C). Figure 8. The role of Src in TRAIL‐induced kinase activation and effect of Akt and ERK inhibition on migration, invasion and cell death. (A) Western blots showing effect of Src knockdown on TRAIL‐ dependent activation of the indicated kinases. (B) Migration, invasion (at 8 h post‐treatment) and cell death (24 h after treatment) in A549 cells with or without PI3K/Akt inhibition by LY294002 and ERK inhibition by PD098059. *p
Chapter 4 DISCUSSION The TRAIL receptor pathway is currently therapeutically exploited for selective activation of apoptosis in tumor cells. However, stimulation of the TRAIL receptors in preclinical models can also activate unwanted non‐apoptotic/non‐canonical signaling leading to proliferative, pro‐survival and even pro‐invasive effects. In the present study using a panel of TRAIL sensitive and resistant NSCLC cell lines we demonstrated non‐canonical signaling in resistant cells that enhanced their migratory and invasive properties. Interestingly, whereas the NSCLC cells express both TRAIL‐R1 and TRAIL‐R2 on their cell surface we only found TRAIL‐R2/DR5 to mediate these effects, thus illustrating that the TRAIL receptors have different signaling properties. Intriguing in this respect is an earlier described correlation of high TRAIL‐R2 levels in advanced stage NSCLC patients with increased risk of death [27]. The underlying causes of differential signaling are still elusive and are subject of further studies. Using peptide arrays exhibiting 1,024 specific consensus sequences for protein kinases we identified substrates and corresponding kinases that are stimulated following TRAIL exposure in A549 cells contrasting findings in sensitive H460 cells. Subsequent confirmation of kinase phosphorylation by Western blotting allowed us to identify several kinases that are activated by TRAIL in resistant A549 cells (see also Fig. 8C for a schematic overview). In particular activation of the Src‐STAT3 axis was important for mediating TRAIL‐induced migration and invasion. The non‐receptor protein tyrosine kinase Src is a known activator of signal transduction pathways controlling various key cellular processes such as cell division, proliferation, survival and motility [28]. Elevated Src protein levels and/or kinase activity have been reported in 50‐80% of lung cancer cases and correlates with poor patient survival [29]. Src is a known mediator of tumor cell migration and invasion as it leads to reorganization of the cytoskeleton and modulation of the cell adhesion system [30]. Downstream of Src we identified STAT3 as a mediator of migration. This is not unprecedented since recently Src‐induced STAT3 activation has been implicated in the formation of podosome structures in primary murine cells facilitating cell migration [31]. STAT3 inhibitors have also been reported to inhibit the migration of prostate cancer cells [32]. Mechanisms described by which STAT3 can promote cell migration include binding of STAT3 to βPIX leading to Rac1 activation [33] and direct phosphorylation of fascin, an actin‐bundling protein, by STAT3 [34]. The precise downstream effectors of STAT3 signaling in TRAIL‐induced migration/ invasion in NSCLC cells remains to be explored. Another mechanism through which Src can stimulate invasion is via the activation of epithelial‐mesenchymal transition (EMT) [35]. The acquisition of mesenchymal properties by epithelial cells characterized by for example loss of E‐cadherin and gain of fibronectin expression has been found to stimulate tumor cell dispersion [36]. However, we did not detect clear changes in the expression of these markers in response to TRAIL in NSCLC cells (not shown). Other Src‐dependent signals ‐ 76 ‐
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TRAIL‐induced migration and invasion<br />
TRAIL‐induced kinases activation and effects on migration, invasion and apoptosis<br />
Finally, we explored possible interactions between the identified TRAIL‐induced kinases<br />
cascades. Knockdown of Src in A549 cells prevented, and even decreased, the<br />
phosphorylation of STAT3 after TRAIL application. Also TRAIL‐induced FAK<br />
phosphorylation appeared to depend on Src, whereas p‐Akt and p‐ERK levels were not<br />
affected in A549‐shSrc cells (Fig. 8A). On the other hand, inhibition of Akt by LY294002<br />
and ERK by PD098059 also reduced migration and invasion induced by TRAIL (Fig. 8B).<br />
Interestingly, inhibition of Akt converted TRAIL resistant A549 cells into apoptosis<br />
sensitive cells. In conclusion, we identified novel parallel branches of TRAIL‐induced<br />
kinase activation that mediate metastases‐prone and/ or pro‐survival effects (see also<br />
Fig. 8C).<br />
Figure 8. The role of Src in TRAIL‐induced kinase activation and effect of Akt and ERK inhibition on<br />
migration, invasion and cell <strong>death</strong>. (A) Western blots showing effect of Src knockdown on TRAIL‐<br />
dependent activation of the indicated kinases. (B) Migration, invasion (at 8 h post‐treatment) and<br />
cell <strong>death</strong> (24 h after treatment) in A549 cells with or without PI3K/Akt inhibition by LY294002 and<br />
ERK inhibition by PD098059. *p
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