Breast cancer bone metastases are attenuated in a Tgif1-deficient bone microenvironment

Abstract

Abstract Background Osteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases. Methods Transwell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1 −/− ) or control littermates (Tgif1 +/+ ). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry. Results Medium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1 −/− mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1 −/− osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1 −/− osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1 −/− mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1 −/− mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might be mediated by osteoblasts rather than by the bone marrow vasculature. Conclusion We propose that the lack of Tgif1 in osteoblasts increases Sema3E expression and attenuates breast cancer cell migration as well as metastases formation.

Keywords

• Breast cancer
• Bone metastases
• Osteoblasts
• Tgif1