Targeting S100A7/RAGE-driven Stat3/Serpin-E1 signaling for immunotherapy in metastatic breast cancer

Background: Triple-negative breast cancer (TNBC) is an aggressive type that lacks targeted therapies, with immunosuppressive variants resistant to checkpoint blockade. Elevated S100A7 expression is reported in these immune-ignored, anti-PD1-refractory tumors; yet S100A7 mechanisms, which shape the immune tumor microenvironment (iTME), remain unclear. Here, we investigated how S100A7 activates the RAGE/Stat3 axis to induce Serpin-E1, reshaping the iTME, and assessed therapeutic potential in immunosuppressive TNBC. Method: Expressions of S100A7, RAGE, Stat3, and Serpin-E1 were analyzed in TNBC cells by Western blotting, cytokine arrays, and ELISA. Functional assays evaluated the impact of S100A7 overexpression (OE) or knockdown (KD) and pharmacologic inhibition of RAGE and Stat3, alone or combined, on viability, migration, and colony formation. Pre-clinical models, including doxycycline (DOX)-inducible mammary gland-specific S100A7-OE bitransgenic mice treated with RAGE and Stat3 inhibitors ± Serpin-E1 neutralizing antibody (nAb) were utilized for in-vivo studies. Multi-color flow cytometry determined macrophage polarization and T-cell activation. CD4/CD8 depletion assays and in-silico analyses evaluated T-cell dependency and prognostic significance. Results: S100A7 upregulation enhanced phosphorylation of Stat3 (Ser727) and Serpin-E1 expression, whereas its downregulation suppressed pStat3/Serpin-E1. Dual inhibition of RAGE and Stat3 produced synergistic suppression of TNBC cell viability, migration, and colony formation and markedly inhibited downstream pStat3 signaling. Mechanistic studies revealed that S100A7/RAGE signaling activates Stat3, which binds to the Serpin-E1 promoter and enhances its transcription. In-vitro assays demonstrated a potential role for Serpin-E1 in modulating macrophage polarization. Next, in-vivo treatment of RAGE and Stat3 inhibitors ± Serpin E1 nAb led to significant reductions in primary tumor growth and distant metastasis in female NSG mice. Significantly decreased tumor burden in S100A7-OE mice treated with the combination of RAGE/Stat3 inhibitors was observed compared to alone treatment groups alone. Additional immune profiling within tumor tissues revealed a significant abundance of antitumor iNOS and MHCII high TAMs and increased activation of CD4+ and CD8+ T cells with elevated effector markers in combinatorial treatment compared to a single regimen. These effects were abrogated by CD4/CD8 depletion. Clinically, high co-expression of S100A7 and Serpin-E1 correlated with poorer outcomes, particularly in basal and immunomodulatory TNBC subtypes. Conclusions: This study identifies the S100A7/RAGE/Stat3/Serpin-E1 axis as a key regulator of tumor growth and iTME suppression in TNBC and its potential for a targeted therapeutic strategy for immune-ignored TNBC subtypes.