Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma

Since inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the Smac mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical NF-κB signaling and a TNFα/TNFR1 autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations and also increase infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cIAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. EMSA supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e. IL-8, MCP-1, CXCR4) and invasion (i.e. MMP9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6- stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates the 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-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.