Abstract 2614: Bone sialoprotein is an essential target in breast cancer skeletal metastasis

Abstract

Abstract Breast cancer is a leading cause of cancer related death in women due to onset of metastasis. Bone metastasis is the most frequent complication occurring in patients with advanced breast cancer and bone sialoprotein (BSP) is related to this process. However, the underlying mechanisms are not clear yet. Therefore, the aim of the study was to analyze BSP functions in greater detail and to decipher its signaling pathways contributing to bone metastasis. To that purpose a combination of the tetracycline-controlled transcription activation system (“Tet-Off system”) and RNA interference was used to initiate and maintain the conditional knockdown of BSP for any intended period. This new technique was established in MDA-MB-231 subclones by recombinase-mediated cassette exchange. Additionally, the cell clones were equipped with the reporter genes mCherry and firefly luciferase for testing their regulative properties and following their fate. In absence of doxycycline, the expression of a miRNA targeting BSP was activated and after six days of BSP knockdown the ensuing cellular, metastatic or molecular properties were monitored by fluorescent microscopy, flow cytometry analysis, assays for proliferation, migration and colony formation, as well as expression profiling analysis. Furthermore, the cell clones were examined in a nude rat model for soft tissue and osteolytic lesions after 2 to 6 weeks of miRNA treatment. The clones revealed good regulative properties to doxycycline. Phenotypic changes indicating apoptosis were observed after 6 days of conditional knockdown which was characterized by up to 86% decreased BSP levels and resulted in significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro. Additionally, the effect of miRNA-mediated BSP knockdown was assessed in vivo. Significant decreases (p < 0.03) and even complete remissions of soft tissue and osteolytic lesions were found following 3 and 6 weeks of miRNA treatment by bioluminescence and magneting resonance imaging, as well as volume computed tomography. The microarray data showed modulated expression in 1.3% of all genes, thus hinting to specific effects in response to BSP knockdown. These genes included increased expression of endoplasmic reticulum stress and apoptosis related genes (ATF3, CHOP), of transcription factor c-FOS, of the gene related to breast epithelial differentiation (ID2) and the tumor suppressor gene EGR1. Conversely, there was suppression of metastasis associated genes (CD44, IL11). These findings were confirmed by western blot for induction of intrinsic and extrinsic apoptotic pathways as shown by cleavage of caspases 8, 9, 3 and 7, and of PARP, as well as the upregulation of ATF3, DDIT3 (CHOP), c-FOS, ID2 and CD44. In conclusion, the role of BSP in the development of skeletal metastasis has been defined more precisely and renders this protein an attractive target in the treatment of this disease. Citation Format: Marineta Kovacheva, Michael Zepp, Stefan Berger, Martin R. Berger. Bone sialoprotein is an essential target in breast cancer skeletal metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2614. doi:10.1158/1538-7445.AM2014-2614