Abstract
Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.
Highlights
In the present study, using cellular models of Inflammatory breast cancer (IBC) with high expression of either epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2), we demonstrate that X-linked inhibitor of apoptosis protein (XIAP) expression modulates IBC cell susceptibility to natural killer (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) when challenged with the anti-EGFR antibody cetuximab or the anti-HER2 antibody trastuzumab, respectively
We co-cultured these tumor cells with human peripheral blood mononuclear cells (PBMCs) with and without addition of the monoclonal antibodies, cetuximab, which binds to EGFR, or trastuzumab, which binds to HER2
Evaluation of the growth inhibitory effects of each antibody showed that cetuximab alone had little to no effect on SUM149 and rSUM149 cell proliferation and trastuzumab inhibited proliferation of SUM190 and rSUM190 only at higher concentrations than those used (10 μg/ml) for the ADCC assay (Supplementary Figure 2)
Summary
Induction of apoptotic signaling through both the intrinsic [cytotoxic granule (perforin, granzyme B) exocytosis] and extrinsic [engagement of death receptors (FAS, TNFR and TRAILR)] cell death pathways is key to both natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated lysis of tumor cells.[9,10] These pathways primarily converge at the point of activation of effector caspases 3 and 7, the chief executioners of apoptosis.[9,10,11,12] X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis protein (IAP) family, is considered the most potent caspase-binding protein and inhibitor of both the extrinsic and intrinsic death pathways.[13]. In the present study, using cellular models of IBC with high expression of either EGFR or HER2, we demonstrate that XIAP expression modulates IBC cell susceptibility to NK-mediated ADCC when challenged with the anti-EGFR antibody cetuximab or the anti-HER2 antibody trastuzumab, respectively. Our results reveal that cells with acquired therapeutic resistance are insensitive to ADCC, which can be reversed by specific downregulation of XIAP expression. We provide evidence for two distinct functions of XIAP in suppressing cell death in response to ADCC: inhibition of caspase activity and suppression of reactive oxygen species (ROS) accumulation. This study uncovers a unique mechanism for evasion of ADCC and highlights XIAP as a novel target for the enhancement of immunotherapy
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