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Identification of DR5 as a critical, NF-?B-regulated mediator of Smac-induced apoptosis.

ABSTRACT: Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-? (TNF?) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNF?-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNF?/TNFR1, as the TNF?-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-?B (NF-?B) activation is critically required for apoptosis, as inhibition of NF-?B by overexpression of dominant-negative I?B? superrepressor (I?B?-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in I?B?-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-?B-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.

SUBMITTER: Eckhardt I

PROVIDER: S-EPMC3847333 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis.

Eckhardt I I Roesler S S Fulda S S

Cell death & disease 20131128

Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although s ...[more]

PMID: 24287697

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Project description:As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-?B (NF-?B) signaling and a tumor necrosis factor-? (TNF?)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-?B DNA binding and enhanced NF-?B transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-?B DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-?B pathway. BV6-stimulated NF-?B activation leads to elevated mRNA levels of TNF? and additional NF-?B target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-?B by overexpression of dominant-negative I?B? superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-?B signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-?B target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNF? autocrine/paracrine loop by the TNF?-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-?B signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.

Dataset Information

Identification of DR5 as a critical, NF-?B-regulated mediator of Smac-induced apoptosis.

Publications

Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis.

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