Inhibition of STAT3 by 2-Methoxyestradiol suppresses M2 polarization and protumoral functions of macrophages in breast cancer

Abstract

Abstract Background Breast cancer metastasis remains the leading cause of cancer-related deaths in women worldwide. Infiltration of tumor-associated macrophages (TAMs) in the tumor stroma is known to be correlated with reduced overall survival, and inhibitors of TAMs are sought after for reprogramming the tumor microenvironment. 2-Methoxyestradiol (2ME2), a potent anticancer and antiangiogenic agent, has been in clinical trials for treatment of breast cancer. Here, we investigated the potential of 2ME2 in modulating the pro-tumoral effects of TAMs in breast cancer. Methods THP-1-derived macrophages were polarized to M2 macrophages with or without 2ME2. The effect of 2ME2 on M2 surface markers and anti-inflammatory genes was determined by Western blotting, flow cytometry, immunofluorescence, and qRT‒PCR. The effect of M2 macrophages on malignant properties of breast cancer cells was determined using colony formation, wound healing, Transwell, and gelatin zymography assays. An orthotopic model of breast cancer was used to determine the effect of 2ME2 on macrophage polarization and metastasis in vivo. Results First, our study found that polarization of THP-1 cells to alternatively activated M2 macrophages is associated with the reorganization of the microtubule cytoskeleton. 2ME2 depolymerized microtubules and reduced the expression of CD206 and CD163, suggesting that it inhibits the macrophages to attain pro-tumoral M2 phenotype. Concurrently, 2ME2 inhibited the expression of anti-inflammatory cytokines and growth factors, including CCL18, TGF-β, IL-10, FNT, arginase, CXCL12, MMP9, and VEGF-A, and hindered the metastasis-promoting effects of M2 macrophages. 2ME2 treatment reduced the expression of CD163 in tumors and inhibited tumor growth and lung metastasis in the orthotopic breast cancer model. Mechanistically, 2ME2 was found to decrease the phosphorylation and nuclear translocation of STAT3. Furthermore, we confirmed the STAT3 inhibition-mediated effects of 2ME2 by using colivelin (a STAT3 activator) which abrogated the effects of 2ME2 on STAT3 phosphorylation and nuclear translocation. Conclusions Our study presents novel finding on mechanism of 2ME2 from the perspective of its effects on TAMs via the STAT3 signaling in breast cancer. The data supports further clinical investigation of 2ME2 and its derivatives as therapeutic agents to modulate the tumor microenvironment and immune response in breast carcinoma.