Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer

Abstract

Introduction: Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL.

Methods: IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining.

Results: Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells.

Conclusions: The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis and reduce the cell turnover index of breast cancers. We also show that combination therapy of IAP antagonists with some pro-apoptotic agents (for example, TRAIL) enhances apoptosis of breast cancer cells. In some cases (for example, MDAMB468 cells), the enhanced apoptosis is profound.

Figure 1

Inhibitor of apoptosis levels in breast cancer. Inhibitor of apoptosis (IAP) expression in a panel of normal and breast-cancer-derived cell lines was examined by immunoblotting with antibodies for (a) XIAP, (b) cIAP1 and cIAP2 (blotted sequentially) and (c) Survivin. Survivin and XIAP were detected on the same blot, which was stripped and reprobed for calnexin. (d) IAP expression was also determined in the MCF10 progression panel: (i) cIAP2, XIAP and Survivin and (ii) cIAP1. Representative blots from four experiments for the cell lines are shown. The receptor status of each cell line, determined by western blotting, is shown; there is no correlation with IAP expression. ER, oestrogen receptor; EGFR, epidermal growth factor receptor.

Figure 2

Inhibitor of apoptosis suppression sensitises cells to TRAIL-induced apoptosis. (a) BT20 and MDAMB468 cells were transfected with siRNA targeting XIAP, and 24 hours later cells were treated with TNF-related apoptosis-inducing ligand (TRAIL) (10 ng/ml) for 48 hours. Cells were then either (i) analysed by western blotting to confirm knockdown or (ii) cytospun and scored for apoptosis by staining for nuclear morphology. Data presented as fold changes in apoptosis (mean ± standard error of the mean (SEM)) from at least three independent experiments. (b) BT474 cells were either transfected with siRNA targeting XIAP and drug treated as above or treated with the Smac mimetic (10 nM) for 2 hours prior to TRAIL (10 ng/ml) addition for 48 hours: (i) knockdown was confirmed by western blotting (cIAP1 only, as cIAP2 was not detected in BT474 cells), and (ii) apoptosis was scored by examining nuclear morphology. Data presented as fold changes in apoptosis (mean ± SEM) from at least three independent experiments. (c) Effect of the Smac mimetic on MDAMB468 cells, examined as for BT474 cells in (b). Data presented as mean ± SEM from at least three experiments. nt, not treated; con, no IAP inhibitor.

Figure 3

ErbB antagonists inhibit growth factor-induced proliferation and signalling. (a) The anti-proliferative effect of the ErbB antagonists was confirmed in BT474 and MDAMB468 cells, using Ki67 staining as a proliferation marker (48 hours post drug treatment). Data presented as mean ± standard error of the mean from at least three experiments. (b) Cells were serum-starved for 4 hours prior to the addition of vehicle only (con), Trastuzumab (100 μg/ml), Lapatinib (100 nM) or Gefitinib (10 μM) for 24 hours. Cells were stimulated with epidermal growth factor (EGF) (100 ng/ml) for 15 minutes at 37°C. Activation of Erk was detected by immunoblotting (IB).

Figure 4

Targeting inhibitors of apoptosis increases sensitivity to ErbB antagonists. (a) BT474 cells and (b) MDAMB468 cells were transfected with siRNA targeting XIAP, and 24 hours later were treated with Trastuzumab (100 μg/ml), Lapatinib (100 nM), or Gefitinib (10 μM) for 48 hours. Apoptosis was scored, by examining nuclear morphology. Data presented as fold changes in apoptosis (mean ± standard error of the mean (SEM)) from at least three experiments. (c) BT474 cells were pretreated with the Smac mimetic for 2 hours before addition of Trastuzumab (100 μg/ml), Lapatinib (100 nM), or Gefitinib (10 μM). Cells were examined for nuclear morphology 48 hours later. Data presented as fold changes (mean ± SEM) from at least three experiments. (d) Cell turnover indexes (CTIs) for ErbB antagonist-treated BT474 cells in the presence or absence of XIAP depletion. Data presented as mean ± SEM from at least three experiments. *P values indicating significance. nt, not treated; con, no ErbB antagonist.

Figure 5

Inhibitor of apoptosis levels in patient samples. Inhibitor of apoptosis (IAP) levels in breast cancer biopsy samples and in samples of normal breast (N1 to N3) from reduction mammoplasties were examined by immunoblotting with the relevant antibodies. (a) XIAP was detected using enhanced chemiluminescence, blots were then stripped and reprobed for cytokeratin 18 (CK18). (b) cIAP1 and (c) cIAP2 and Survivin were detected using the Li-Cor Odyssey™ system. Blots were simultaneously probed for Desmoplakin. Prognostic indicators were determined in the clinic. ER, oestrogen receptor; EGFR, epidermal growth factor receptor; PR, progesterone receptor; +, positive; -, negative; nd, not determined.