Estradiol induces bone osteolysis in triple-negative breast cancer via its membrane-associated receptor ER36

Triple-negative breast cancer (TNBC) is thought to be an estradiol-independent, hormone therapy-resistant cancer because of lack of estrogen receptor alpha 66 (ER alpha 66). We identified a membrane-bound splice variant, ER alpha 36, in TNBC cells that responds to estrogen (E-2) and may contribute to bone osteolysis. We demonstrated that the MDA-MB-231 TNBC cell line, which expresses ER alpha 36 similarly to MCF7 cells, is responsive to E-2, forming osteolytic tumors in vivo. MDA-MB-231 cells activate osteoclasts in a paracrine manner. Conditioned media (CM) from MDA-MB-231 cells treated with bovine serum albumin-bound E-2 (E-2-BSA) increased activation of human osteoclast precursor cells; this was blocked by addition of anti-ER alpha 36 antibody to the MDA-MB-231 cultures. Osteoclast activation and bone resorption genes were elevated in RAW 264.7 murine macrophages following treatment with E-2-BSA-stimulated MDA-MB-231 CM. E-2 and E-2-BSA increased phospholipase C (PLC) and protein kinase C (PKC) activity in MDA-MB-231 cells. To examine the role of ER alpha 36 signaling in bone osteolysis in TNBC, we used our bone-cancer interface mouse model in female athymic homozygous Foxn1(nu) mice. Mice with MDA-MB-231 tumors and treated with tamoxifen (TAM), E-2, or TAM/E-2 exhibited increased osteolysis, cortical bone breakdown, pathologic fracture, and tumor volume; the combined E-2/TAM group also had reduced bone volume. These results suggest that E-2 increased osteolytic lesions in TNBC through a membrane-mediated PLC/PKC pathway involving ER alpha 36, which was enhanced by TAM, demonstrating the role of ER alpha 36 and its membrane-associated signaling pathway in bone tumors. This work suggests that ER alpha 36 may be a potential therapeutic target in patients with TNBC.