BONE-METASTATIC BREAST CANCER CELLS INHIBIT OSTEOBLAST FUNCTION
Open Access
- Author:
- Mercer, Robyn R.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 28, 2004
- Committee Members:
- Andrea Marie Mastro, Committee Chair/Co-Chair
Andrew Thomas Henderson, Committee Member
Richard John Frisque, Committee Member
John Patrick Vanden Heuvel, Committee Member
Carol V Gay, Committee Member - Keywords:
- breast cancer
osteoblast
metastasis
bone - Abstract:
- ABSTRACT Breast cancer frequently metastasizes to bone, resulting in osteolytic lesions. These lesions, formed by stimulated osteoclasts, cause pain, an increased susceptibility to fractures, and hypercalcemia. It has been shown that breast cancer cells communicate with osteoblasts and subsequently stimulate osteoclast activity; however, little research has focused on understanding the interaction between breast cancer cells and osteoblasts. To study how cancer cells affect osteoblasts, MC3T3-E1 cells, an immature osteoblast cell line that differentiates in vitro, were cultured with conditioned medium from MDA-MB-231 cells, a bone-metastatic breast cancer cell line. We determined that alkaline phosphatase activity and mineralization, two defining characteristics of a mature osteoblast, were blocked. Moreover, mRNA expression for both bone sialoprotein and osteocalcin, two genes upregulated during osteoblast differentiation, were not expressed even after 25 days of culture. Together, these data suggested that when cultured with MDA-MB-231 conditioned medium, MC3T3-E1 cells did not differentiate into mature osteoblasts. We speculated that the conditioned medium factor causing this inhibition in differentiation was transforming growth factor beta (TGFbeta). To test this, MDA-MB-231 conditioned medium was pretreated with a neutralizing antibody to TGFbeta. The neutralized conditioned medium was then added to MC3T3-E1 osteoblasts. RNA expression of alkaline phosphatase, bone sialoprotein, and osteocalcin were all restored in the presence of TGFbeta-neutralized conditioned medium. While trying to understand how breast cancer conditioned medium affected osteoblast differentiation, we made another key observation: MDA-MB-231 conditioned medium altered MC3T3-E1 morphology and adhesion. Examination with interference reflection microscopy revealed that MC3T3-E1 osteoblasts had fewer focal adhesion plaques when cultured with MDA-MB-231 conditioned medium. Further scrutiny revealed a substantial alteration in actin stress fibers. Instead of forming normal stress fibers, cells cultured with MDA-MB-231 conditioned medium demonstrated thick cortical filaments, as well as areas of large, punctate staining. MDA-MB-231 conditioned medium contains many factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and TGFbeta. Because each of these factors are capable of reorganizing actin stress fibers and altering cell adhesion, MDA-MB-231 conditioned medium was treated with neutralizing antibodies to them. MC3T3-E1 osteoblasts were then cultured with the neutralized conditioned medium and assayed for changes in adhesion. Only when all three cytokines were neutralized was there a restoration in actin stress fiber formation and in focal adhesion plaques. To date, research in the field has focused on finding ways to inhibit osteoclast activity in an effort to curb bone loss. While this is an important approach, the information obtained from our study indicates that the osteoblast is also affected by breast cancer tumors. A decrease in bone formation tethered with an increase in bone degradation will exacerbate bone loss associated with breast cancer metastasis. Focusing solely on osteoclast activity will not cure bone loss. Exploring the possibility of stimulating osteoblast activity is also needed and must be considered as an important step for therapeutic intervention.