Secretion of BMP-2 by TAMs up-regulates the expression of RUNX2 and TWIST1 to promote the microcalcifications and tumor metastasis in breast cancer

Abstract

Abstract INTRODUCTION: Microcalcifications is an important indicator of breast cancer yet the mechanism of its formation remains to be elucidated. Tumor-associated macrophages (TAMs) can secrete bone morphogenetic protein 2 (BMP-2), which is associated with presence of microcalcifications. BMP-2 is also known to up-regulate RUNX2, a regulator of the differentiation of osteoblasts, which could be the potential mechanism underlying microcalcification formation. METHODS:Immunohistochemical staining of CD163 (marker of the M2-like macrophages), BMP-2, RUNX2 and TWIST1 were performed on tissue microarray (TMA) sections of 272 primary invasive breast cancer. Following induction, M2‐like TAMs were co-cultured with breast cancer cells with or without adding BMP receptor Ⅰ inhibitor (LDN). The level of BMP-2 in culture solution was measured by ELISA. The expression of RUNX2, TWIST1 and their downstream factors were measured by western blot. The deposition of microcalcifications was analyzed by Alizarin Red S staining. Orthotopic metastasis in mice models were established to further determine the role of TAMs in microcalcifications and Epithelial Mesenchymal Transition (EMT). PET-CT was used to detect the metastasis in mice. RESULTS:Immunohistochemical analysis showed that the expression of RUNX2 and TWIST1 was significantly correlated with microcalcifications. Expression of RUNX2 was also correlated with infiltration of TAMs and the expression of BMP-2. Results from co-culturing of TAMs with breast cancer cells showed that the secretion of BMP-2 from TAMs could induce microcalcifications and EMT by upregulating the expression of RUNX2 and TWIST1 respectively, while these effects could be reversed by LDN. Similar results were observed in the mouse model of orthotopic metastasis. CONCLUSIONS:These findings support the hypothesis that the secretion of BMP-2 by TAMs could up-regulate the expression of RUNX2 and TWIST1 to promote microcalcifications and induce EMT in breast cancer.