In our previous studies, by establishing and using trastuzumab-resistant breast cancer cell models, we initially found upregulation of C-X-C motif chemokine receptor 4 (CXCR4), a G protein-coupled receptor (GPCR) of stromal cell-derived factor-1 (SDF-1α CXCL12) in trastuzumab-resistant breast cancer. Thus, further understanding the underlying mechanism of resistance to targeting HER2 is critical to improve outcomes for this breast cancer subtype. While several newer therapies have been approved for progressive HER2 + breast cancer, all rely on targeting HER2. Although many molecules/pathways have been implicated in trastuzumab resistance, the associated mechanisms remain unclear, and no biomarker can reliably predict a lack of benefit from trastuzumab. In addition, variable HER2 C-terminal fragments are kinase-active but lack the trastuzumab-binding epitope. Reported underlying mechanisms of trastuzumab resistance include activation of the PI3K pathway, upregulation of the insulin-like growth factor-I receptor (IGF-1R) or an increase in IGF-1R/HER2 heterodimers, and upregulation of epidermal growth factor receptor (EGFR), HER3/4, or their ligands, given that trastuzumab is unable to block ligand-induced EGFR/HER2 and HER2/HER3 heterodimers. However, resistance to trastuzumab remains a clinical challenge many patients with advanced breast cancers do not respond or eventually develop clinical resistance. The humanized anti-HER2 monoclonal antibody, trastuzumab (Herceptin), was the first oncogene-targeted therapy, and its use over the last 25 years has improved disease-free and overall survival in patients with early and advanced-stage HER2 + breast cancer. Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.Īmplification of the human epidermal growth factor receptor 2 (HER2)/neu (ERBB2) gene and overexpression of the oncoprotein HER2 occur in around 20% of breast cancers, termed “HER2-positive (HER2+) breast cancer”. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. We were the first to report the role of CXCR4 in trastuzumab resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance.
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