PKC activation sensitizes basal-like breast cancer cell lines to Smac mimetics

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.

Smac mimetics as novel promising modulators of apoptosis in the treatment of breast cancer

“…It has been shown that combination treatment of Smac mimetic‐resistant basal breast cancer cell lines, including MDA‐MB‐468 and BT‐549 enhanced sensitivity to Smac mimetic in a TNFα‐dependent manner. However, non‐triple negative cells did not respond to this treatment strategy, which may be related to the intact production of TNF‐α following treatment or resistance to TNF‐α‐induced apoptosis . In another attempt to increase the efficacy of therapeutic approaches using Smac mimetics, addition of Smac mimetic TL32711 to combination of gemcitabine and the CHK1 inhibitor (AZD7762) led to significant cell cycle arrest and apoptosis induction in triple‐negative breast cancer cell lines .…”

“…However, non-triple negative cells did not respond to this treatment strategy, which may be related to the intact production of TNF-α following treatment or resistance to TNF-α-induced apoptosis. 82 In another attempt to increase the efficacy of therapeutic approaches using Smac mimetics, addition of Smac mimetic TL32711 to combination of gemcitabine and the CHK1 inhibitor (AZD7762) led to significant cell cycle arrest and apoptosis induction in triple-negative breast cancer cell lines. 83 Moreover, treatment of breast cancer cell lines MDA-MB-231 and MCF-7 with Smac mimetic LCL161 led to significant cell cycle arrest and induction of both apoptosis and necrosis, which was associated with cIAP1 degradation and accumulation of active RIP1.…”

Induction of Breast Cancer Cell Apoptosis by TRAIL and Smac Mimetics: Involvement of RIP1 and cFLIP

“…For siRNA transfections, 500,000 cells per a 6 cm cell culture dish were seeded. Transfections were thereafter performed as described previously [ 21 ].…”

“…Western blot was performed as described [ 21 ]. Primary antibodies used were anti-actin (1:2000 MP Biomedicals C4), anti-caspase-8 (1:300 Cell Signaling #9746 and 1:300, Abcam ab32125), anti-caspase-3 (1:1000 Cell Signaling #9662), anti-caspase-7 (1:400 Cell Signaling #12827), anti-RIP1 (1:500 BD Biosciences 610458), anti-XIAP (1:400 BD Biosciences 610762), anti-cIAP1 (1:200 R&D Systems AF8181), anti-p100/p52 (1:500 Cell Signaling #4882) and anti-c-FLIP (1:400 Cell Signaling #5634).…”

The Proliferative and Apoptotic Landscape of Basal-like Breast Cancer

“…Interestingly, Smac and Protein Kinase C delta (PKCδ) interact in basal-like and luminal breast cancer cells, but are dissociated after taxane cytotoxic treatment [183]. Further, activation of PKC synergizes with the Smac mimetic LBW242 in BLBC cell lines [184]. TRAIL agonists have been also shown to induce apoptosis of BLBC cells.…”