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 that have been implicated in migration include activation of the Rho/ROCK/LIMK, Ras/MAPK, PI3K/Akt, FAK/paxillin/CAS pathways [30]. Although the kinome analyses identified possible ROCK activity after TRAIL exposure, we were not able to confirm ROCK activation in Western blots and co‐treatment with a ROCK inhibitor did not prevent TRAIL‐induced invasion (not shown). On the other hand, knockdown of Src in A549 cells inhibited TRAIL‐dependent FAK phosphorylation suggesting a possible role of this pathway as well. FAK is a non‐receptor protein tyrosine kinase known to stimulate migration/ invasion when in complex with Src [37]. Of note, in SW1573 cells TRAIL resulted in Src‐dependent FAK phosphorylation, but not STAT3 phosphorylation. This may be related to the smaller stimulatory effect of TRAIL on migration/ invasion in SW1573 cells (1.5 fold) when compared to A549 cells (2‐3 fold). Inhibition of ERK did partially suppress invasive behavior of A549 cells however TRAIL‐induced ERK phosphorylation was independent of Src as was also the case for Akt activation. Active Src has the ability to phosphorylate caspase‐8 at tyrosine 380 resulting in blocking of its pro‐apoptotic function [38]. However, we could not detect phosphorylated procaspase‐8 (not shown) and shRNA‐mediated knockdown or chemical inhibition of Src by dasatinib and saracatinib did prevent TRAIL‐induced invasion, but failed to effectively sensitize for apoptosis in A549 cells, pointing to other mechanisms of resistance. TRAIL stimulatory effects on tumor cell migration and invasion have been previously reported to be mediated by NF‐κB in apoptosis resistant cholangiocarcinoma cancer cells [39], and by TRAIL‐dependent up‐regulation of interleukin‐8 and chemoattractant protein 1 in pancreatic ductal adenocarcinoma cells [12]. A more recent study has shown that TRAIL‐induced migration in colon cancer cells involves oncogenic K‐Ras and Raf‐1 that convert death receptors into invasion‐inducing receptors by suppressing the ROCK/LIM kinase/cofilin pathway [11]. However, the K‐Ras status not always predicts the outcome of death receptor signaling, since for example H460 NSCLC cells in our study are highly sensitive for TRAIL‐induced apoptosis despite the presence of oncogenic K‐Ras. Thus, in NSCLC cells TRAIL‐induced Src activation appears to be the main route responsible for migration and invasion. RIP1 is a serine threonine kinase belonging to the RIP family involved in promoting pro‐ survival, inflammatory and pro‐apoptotic signals depending on the signal and tumor type [10]. RIP1 is part of the secondary complex and has been associated with non‐apoptotic functions of TRAIL [8]. To understand the role of RIP1 in our model shRNA‐mediated knockdown as well as RIP1 inhibition by necrostatin‐1 revealed prevention of TRAIL‐ induced Src‐STAT3 activation and migration/ invasion, corroborating the importance of this pathway. In contrast to a previous report we did not observe sensitization for TRAIL‐ induced apoptosis in A549 RIP1 knockdown cells [40]. Regarding Akt activation, in prostate adenocarcinoma cells TRAIL‐dependent Src activation has been reported to result in activation of PI3K‐Akt signaling [41]. We found in NSCLC cells TRAIL to activate a ‐ 77 ‐
Chapter 4 Src‐independent mechanism responsible for Akt activation, and moreover, Akt and also ERK activation was independent of RIP1. The activation of Akt and ERK may be a more indirect consequence of TRAIL pathway activation. For example, in colorectal cancer cells TRAIL was found to activate EGFR and HER2 through Src family kinases (SFK) that in turn activated the cell surface protein A Disintegrin And Metalloproteinase‐17 (ADAM‐17) also known as Tumor Necrosis Factor Converting Enzyme (TACE) leading to cleavage and shedding of TGF‐α. Subsequently, TGF‐α activated the EGFR/HER2 pro‐survival signaling pathways in an autocrine and paracrine manner [42]. Furthermore, we found TRAIL to be able to phosphorylate FAK in RIP1 knockdown NSCLC cells, in which TRAIL‐induced Src activation was prevented. Thus, FAK can be activated in a RIP1‐dependent and ‐ independent way, indicating more complex interactions at the level of RIP1, Src and FAK. In this study we identified Akt as an important mediator of TRAIL resistance in NSCLC cells. This confirms earlier studies where the PI3K inhibitors Wortmannin or LY‐294002 and the Akt‐inhibitor perifosine were shown to cooperate with rhTRAIL to induce apoptosis in NSCLC cells involving increased TRAIL‐R2 expression and a reduction of c‐ FLIP levels [43;44]. Taken together, it can be concluded from our work that TRAIL non‐canonical signaling in NSCLC involves a parallel activation of RIP1‐dependent and ‐independent mechanisms that stimulate both pro‐survival (Akt) and migration/ invasion (Src, STAT3) mechanisms. Although more work is required to evaluate the unwanted non‐canonical effects in other models, our findings may indicate that care should be taken when using TRAIL receptor targeting agents for treating patients. Moreover, treatments with TRAIL agonistic agents in NSCLC may benefit from combined treatment with PI3K/Akt and Src inhibitors in order to potentiate anti‐tumor activity and to prevent unwanted side effects. Acknowledgements The authors would like to thank dr. Elisa Giovannetti for helpful discussions, Meyram Cil for technical assistance and Ingrid van Roosmalen for the receptor expression experiments. This research was performed within the framework of project T3‐112 and T3‐103 of the Dutch Top Institute Pharma. Conflict of interest Wim Quax receives commercial research support for valorization (STW valorization grant GBC.7660) ‐ 78 ‐
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Chapter 4<br />
Src‐independent mechanism responsible <strong>for</strong> Akt activation, and moreover, Akt and also<br />
ERK activation was independent of RIP1. The activation of Akt and ERK may be a more<br />
indirect consequence of TRAIL pathway activation. For example, in colorectal cancer cells<br />
TRAIL was found to activate EGFR and HER2 through Src family kinases (SFK) that in turn<br />
activated the cell surface protein A Disintegrin And Metalloproteinase‐17 (ADAM‐17) also<br />
known as Tumor Necrosis Factor Converting Enzyme (TACE) leading to cleavage and<br />
shedding of TGF‐α. Subsequently, TGF‐α activated the EGFR/HER2 pro‐survival signaling<br />
pathways in an autocrine and paracrine manner [42]. Furthermore, we found TRAIL to be<br />
able to phosphorylate FAK in RIP1 knockdown NSCLC cells, in which TRAIL‐induced Src<br />
activation was prevented. Thus, FAK can be activated in a RIP1‐dependent and ‐<br />
independent way, indicating more complex interactions at the level of RIP1, Src and FAK.<br />
In this study we identified Akt as an important mediator of TRAIL resistance in NSCLC<br />
cells. This confirms earlier studies where the PI3K inhibitors Wortmannin or LY‐294002<br />
and the Akt‐inhibitor perifosine were shown to cooperate with rhTRAIL to induce<br />
apoptosis in NSCLC cells involving increased TRAIL‐R2 expression and a reduction of c‐<br />
FLIP levels [43;44].<br />
Taken together, it can be concluded from our work that TRAIL non‐canonical signaling in<br />
NSCLC involves a parallel activation of RIP1‐dependent and ‐independent mechanisms<br />
that stimulate both pro‐survival (Akt) and migration/ invasion (Src, STAT3) mechanisms.<br />
Although more work is required to evaluate the unwanted non‐canonical effects in other<br />
models, our findings may indicate that care should be taken when using TRAIL <strong>receptor</strong><br />
targeting agents <strong>for</strong> treating patients. Moreover, treatments with TRAIL agonistic agents<br />
in NSCLC may benefit from combined treatment with PI3K/Akt and Src inhibitors in order<br />
to potentiate anti‐tumor activity and to prevent unwanted side effects.<br />
Acknowledgements<br />
The authors would like to thank dr. Elisa Giovannetti <strong>for</strong> helpful discussions, Meyram Cil<br />
<strong>for</strong> technical assistance and Ingrid van Roosmalen <strong>for</strong> the <strong>receptor</strong> expression<br />
experiments. This research was per<strong>for</strong>med within the framework of project T3‐112 and<br />
T3‐103 of the Dutch Top Institute <strong>Pharma</strong>.<br />
Conflict of interest<br />
Wim Quax receives commercial research support <strong>for</strong> valorization (STW valorization grant<br />
GBC.7660)<br />
‐ 78 ‐