Expanding the Scope of HER2-targeted Medicine Neratinib to Combat Triple- Negative Breast Cancer by Inhibiting the EGFR-ERK-STAT3 Signaling Axis

Background Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer (BC), for which targeted medicine options are still very limited. This study provides comprehensive experimental evidences on neratinib as a molecularly targetable inhibitor for treatment, at least for basal-like and mesenchymal-like TNBC subtypes, where both EGFR and STAT3 activation are pronounced. Methods Molecular docking was used for in silico evaluation of neratinib binding to STAT3 and EGFR. TCGA and METABRIC data were used for gene expression and pathway enrichment analysis. The phospho-STAT3 BRET assay was used to measure STAT3 dimerization and activation. Phospho-proteome array and immunoblotting were used for validation of Neratinib effect on the EGFR/ERK/STAT3 signaling axis. The effect of neratinib on tumor growth, metastasis, and angiogenesis was assessed in TNBC orthotopic xenografts using in vivo bioluminescence imaging, immunohistochemistry, and Western blot. Results By using in silico molecular docking, for the first time neratinib was found to directly bind on the STAT3 SH2 domain, in addition to its known binding on EGFR kinase domain. TCGA and METABRIC cohort analyses confirmed EGFR overexpression in TNBC linked with poor patient survival (P ≤ 0.0001) and upregulation of EGFR and the MAPK signaling cascade. Association between EGFR overexpression and increased non-canonical STAT3 activation was established in TNBC cell types. Neratinib showed substantial inhibition of EGF/ERK/STAT3 signaling axis in phospho-proteome arrays, thereby establishing its control mechanism over STAT3 activation. STAT3 inhibition showed reduced dimerization and nuclear translocation, thereby suppressing transcription of its downstream target genes. Consequently, neratinib treatment showed reduced survival and proliferation, induced apoptosis, and reduced migration/invasiveness of the TNBC cells tested. Further, in a preclinical orthotopic TNBC tumor xenograft model, neratinib treatment has shown significantly retarded tumor growth, organ metastasis, and angiogenesis. Conclusion Thus, this study provides staggered evidences showing that neratinib, the small molecule drug, can serve as a dual-target inhibitor for treatment of cancer with elevated activation of both EGFR and STAT3. The new evidences indicate that repurposing neratinib may extend significant benefit for treatment of certain subtypes of TNBCs.