Resistance to TRAIL in non-transformed cells is due to multiple redundant pathways

M Van Dijk,T Sessler,A Halpin-Mccormick,A Samali,E Szegezdi

doi:10.1038/cddis.2013.214

Abstract

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine and a selective inducer of apoptosis in a range of tumour cells, but not in normal, untransformed cells. A large number of chemotherapeutics as well as biological agents are being tested for their potential to sensitise resistant tumour cells to TRAIL as a means to broaden the range of tumours treatable with TRAIL. However, because of the incomplete understanding of the mechanism(s) underlying TRAIL resistance in non-malignant cells, it is unpredictable whether the effect of these sensitisers will be restricted to tumour cells or they would also sensitise non-transformed cells causing unwanted toxicity. In this study, we carried out a systematic analysis of the mechanisms driving TRAIL resistance in non-transformed cells. We found that cellular FLICE-like inhibitory protein, anti-apoptotic B-cell lymphoma 2 proteins, and X-linked inhibitor of apoptosis protein were independently able to provide resistance to TRAIL. Deficiency of only one of these proteins was not sufficient to elicit TRAIL sensitivity, demonstrating that in non-transformed cells multiple pathways control TRAIL resistance and they act in a redundant manner. This is contrary to the resistance mechanisms found in tumour cell types, many of them tend to rely on a single mechanism of resistance. Supporting this notion we found that 76% of TRAIL-resistant cell lines (13 out of 17) expressed only one of the above-identified anti-apoptotic proteins at a high level (≥1.2-fold higher than the mean expression across all cell lines). Furthermore, inhibition or knockdown of the single overexpressed protein in these tumour cells was sufficient to trigger TRAIL sensitivity. Therefore, the redundancy in resistance pathways in non-transformed cells may offer a safe therapeutic window for TRAIL-based combination therapies where selective sensitisation of the tumour to TRAIL can be achieved by targeting the single non-redundant resistance pathway.

Highlights

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is attracting attention as a potential anti-cancer agent because of its property of inducing apoptosis selectively in tumour cells, but not in healthy, non-transformed cells.[6]
In order to determine non-toxic TRAIL-based drug combinations for clinical use, a better understanding of the mechanisms protecting non-transformed cells from TRAIL is necessary. This study addressed this question using two non-transformed cell types abundant in the body, fibroblasts and smooth muscle cells. These cell types were chosen despite the drawbacks and limitations of the in vitro culture system over an in vivo rodent model, because the TRAIL receptor family is significantly different between rodents and humans.[28,29]
Treatment of have been chosen: dermal fibroblasts (hFb) with TRAIL could induce a low level of pro-caspase-8 processing resulting in processing of pro-caspase-3 to its p20 fragment but no associated cleavage of cellular caspase-3 substrate proteins, such as PARP, could be found, indicating that there is more than one point where the apoptotic pathway is inhibited

Summary

Introduction

TRAIL is attracting attention as a potential anti-cancer agent because of its property of inducing apoptosis selectively in tumour cells, but not in healthy, non-transformed cells.[6]. Activation of DR4 and DR5 can be regulated by the decoy receptors, DcR1 and DcR2, which are able to sequester TRAIL from the DRs as well as to form inactive, heteromeric complexes with them.[10,11,12] Activation of caspase-8 (or -10) is another target for regulation by at least three different proteins: the caspase-8 homologue cellular FLICE-like inhibitory protein (cFLIP), phosphorylated MAPK-activating death domain protein or the complex of glycogen synthase kinase-3, DDX3 and cellular inhibitor of apoptosis protein-1 All these proteins act by binding to DR4/DR5 and prevent Fas-associated protein with death domain and/or caspase-8 recruitment.[13,14,15,16] In addition to the inhibitors that act at the Received 20.12.12; revised 03.4.13; accepted 05.4.13; Edited by D Aberdam level of the receptor, anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins can block Bax/Bak activation induced by caspase-8processed tBid and block the activation of the mitochondrial amplification loop.[17,18] in a number of tumour cells TRAIL-mediated apoptosis has been shown to be blocked by the caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP) that can directly bind to caspase-9 and -3 and block their activation or activity.[19]. This sensitivity increases by transforming the keratinocytes, which has been linked to reduced cFLIP or XIAP expression in response to the transformation.[23,26,27] our knowledge about the inherent resistance mechanisms in non-transformed cells is currently incomplete

Methods

Results

Conclusion