Abstract

There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, which antagonize inhibitor of apoptosis proteins (IAPs). Recent studies have shown that combinations of Smac mimetics such as LBW242 or LCL161 in combination with chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with the Smac mimetic LBW242 induces cell death in two basal breast cancer cell lines (MDA-MB-468 and BT-549) that are resistant to Smac mimetic as single agent. Ten other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα, which can induce death in the presence of Smac mimetics. The cell death, coinciding with caspase-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with caspase-8, as seen in complex II formation. Smac mimetics, but not TPA, induced the non-canonical NF-κB pathway in both MDA-MB-231 and MDA-MB-468 cells. Blocking the canonical NF-κB pathway suppressed TPA induction of TNFα in MDA-MB-468 cells whereas isolated downregulation of either the canonical or non-canonical pathways did not abolish the Smac mimetic induction of the NF-κB driven genes TNFα and BIRC3 in MDA-MB-231 cells although the absolute levels were suppressed. A combined downregulation of the canonical and non-canonical pathways further suppressed TNFα levels and inhibited Smac mimetic-mediated cell death. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with a Smac mimetic induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers.

Highlights

  • Evasion of cell death is one important hallmark of cancer.[1,2] Cell death comprises different subroutines[3,4] with two main apoptotic pathways, the extrinsic and the intrinsic, as important examples.[5]The extrinsic pathway is induced by death receptors (DRs) leading to the activation of caspase-8 whereas the intrinsic apoptotic pathway is initiated by cellular stress resulting in release of cytochrome c and second mitochondria-derived activator of caspase (Smac) from the mitochondria leading to activation of caspase-9

  • We previously found that the pro-apoptotic protein Smac and the protein kinase C (PKC) isoform PKCδ form a complex that is dissociated during cell death induction.[20]

  • One way to promote cell death is by mimicking the function of the pro-apoptotic protein Smac with Smac mimetic (SM)

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Summary

INTRODUCTION

Evasion of cell death is one important hallmark of cancer.[1,2] Cell death comprises different subroutines[3,4] with two main apoptotic pathways, the extrinsic and the intrinsic, as important examples.[5]. The extrinsic pathway is induced by death receptors (DRs) leading to the activation of caspase-8 whereas the intrinsic apoptotic pathway is initiated by cellular stress resulting in release of cytochrome c and second mitochondria-derived activator of caspase (Smac) from the mitochondria leading to activation of caspase-9. Both pathways converge in the activation of executioner caspases-3 and 7.6,7. A Smac mimetic (SM) is thought to facilitate cell death by mimicking the antagonizing effect of Smac on inhibitor of apoptosis proteins (IAPs).[8] Two IAPs, cellular IAP1 (cIAP1) and cIAP2, regulate tumor necrosis factor receptor 1 (TNFR1) signaling.[9] TNFR1 activation can lead to extrinsic apoptotic signaling pathway. The effect of TPA is dependent on the canonical NF-κB pathway, whereas both the canonical and non-canonical pathways contribute to sensitivity to SM

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