Breast cancer (BC) patient prognosis is determined by estrogen-receptor (ER) status. ER+ (ERα66) cancers are treated with ER antagonists. ER- cancers are more aggressive and respond less to conventional treatments. However, ER splice variant ERα36 is present in ER+ and ER- BC cells. ERα36 increases proliferation and enhances metastatic factors in response to 17b-estradiol (E2). BC cells metastasize to bone, but how they regulate bone remodeling is unclear. We examined effects of ERα36 on osteolytic factors and their role in bone resorption.
Method:
ERα36 was examined in ER- HCC38 and MDA-MB-231 cells by PCR and Western blot. Protein kinase-C was measured in cells treated with E2 or BSA-conjugated-E2 (E2-BSA) ± ERα36-antibodies (AbERα36) for 15minutes. Secretion of pro-osteoclastogenic interleukin-6 (IL6), and mRNA for osteoclast activators (IL6, RANKL) and inhibitor (osteoprotegerin, OPG) were measured after treatment with E2-BSA±AbERα36. Osteoclast activation was examined using a fluorescent in-vitro assay in which osteoclast precursors are incubated with conditioned media after treatment with E2±AbERα36. ER- cells were implanted in femur marrow canals of female nude mice±E2 and osteolysis was examined after 8w. Data are mean±SEM (n=6 in-vitro, n=8 mice, ANOVA, post-hoc Bonferroni’s Student’s t-test).
Result:
HCC38 and MDA-MB-231 were negative for ERα66 but positive for ERα36. E2 and E2-BSA dose-dependently increased PKC, an effect abolished by AbERα36. IL6 secretion and IL6 and RANKL mRNA increased and OPG mRNA decreased after E2-BSA, but AbERα36 prevented this. Conditioned media from E2-treated BC cells dose-dependently increased osteoclast activation, which was inhibited by AbERα36. Systemic E2 significantly enhanced osteolysis induced by ERα36+ BC cells.
Conclusion:
ER- BC cells possess ERα36. Cells secrete osteoclastogenic factors that activate osteoclasts in-vitro and induce osteolysis in-vivo. These effects are mediated by ERα36, suggesting this receptor is an important target for therapies.