Breast cancer frequently metastasizes to bone, resulting in osteolytic lesions formed by stimulated osteoclasts. It has been shown that breast cancer cells communicate with osteoblasts to stimulate osteoclast activity; however, little research has focused on the interactions between cancer cells and osteoblasts. To study how cancer cells affect osteoblasts, MC3T3-E1 cells, an immature osteoblast line that differentiates in vitro, were cultured with conditioned medium from MDA-MB-231 cells, a bone-metastatic breast cancer line. We determined that alkaline phosphatase activity and mineralization, two defining characteristics of mature osteoblasts, were inhibited. Moreover, mRNA expression for both bone sialoprotein and osteocalcin, two genes upregulated during osteoblast differentiation, were not expressed. Together, these data suggested that when cultured with MDA-MB-231 conditioned medium, MC3T3-E1 cells did not differentiate into mature osteoblasts. To determine if TGFβ, an inhibitor of osteoblast differentiation, was responsible, MC3T3-E1 cells were cultured with conditioned medium pre-treated with a neutralizing TGFβ antibody. RNA expression of alkaline phosphatase, bone sialoprotein, and osteocalcin were restored. In addition, MDA-MB-231 conditioned medium also altered MC3T3-E1 morphology and adhesion, as determined by interference reflection microscopy and phalloidin staining. MC3T3-E1 osteoblasts had fewer focal adhesion plaques and a substantial alteration in actin stress fibers when cultured with MDA-MB-231 conditioned medium. Actin appeared as thick cortical filaments with areas of large, punctate staining, instead of normal stress fibers. The active factors causing the alterations in adhesion were identified by pretreating MDA-MB-231 conditioned medium with neutralizing antibodies to platelet derived growth factor, insulin-like growth factor, and TGFβ. Only when all three cytokines were neutralized was there a restoration in actin stress fiber formation and in focal adhesion plaques. Furthermore, the alteration in adhesion was found to be due to signaling through phosphatidyl inositol 3’phosphate and the small G-protein, rac. In conclusion, this study reveals a paramount role for osteoblasts in breast cancer metastasis. A decrease in bone formation tethered with an increase in bone degradation will exacerbate bone loss associated with lytic metastasis. Exploring the possibility of stimulating osteoblasts, in addition to inhibiting osteoclasts, must be considered as an important step for therapeutic intervention. R.R.M. is supported by a pre-doctoral grant from the US Army Medical Research Materiel Command DAMD 17-02-1-0358. Research supported by a Biotechnology Grant, Penn State University; PA Tobacco Settlement Fund; and the National Foundation for Cancer Research.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]