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Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231.

ABSTRACT: Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGF?1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGF?1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.

SUBMITTER: Rustamov V

PROVIDER: S-EPMC6370714 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231.

Rustamov Valeh V Keller Florian F Klicks Julia J Hafner Mathias M Rudolf Rüdiger R

Frontiers in oncology 20190205

Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to r ...[more]

PMID: 30805306

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Project description:Vasculogenic mimicry (VM) with the pattern of endothelial independent tubular structure formation lined by aggressive tumor cells mimics regular tumor blood vessels to ensure robust blood supply and correlates with the proliferation, invasion, metastasis, and poor prognosis of malignant tumors, which was demonstrated to be a major obstacle for resistance to antiangiogenesis therapy. Therefore, it is urgent to discover methods to abrogate the VM formation of tumors, which possesses important practical significance for improving tumor therapy. Brucine is a traditional medicinal herb extracted from seeds of Strychnos nux-vomica L. (Loganiaceae) exhibiting antitumor activity in a variety of cancer models. In the present study, the effect of brucine on vasculogenic mimicry and the related mechanism are to be investigated. We demonstrated that, in a triple-negative breast cancer cell line MDA-MB-231, brucine induced a dose-dependent inhibitory effect on cell proliferation along with apoptosis induction at higher concentrations. The further study showed that brucine inhibited cell migration and invasion with a dose-dependent manner. Our results for the first time indicated that brucine could disrupt F-actin cytoskeleton and microtubule structure, thereby impairing hallmarks of aggressive tumors, like migration, invasion, and holding a possibility of suppressing vasculogenic mimicry. Hence, the inhibitory effect of brucine on vasculogenic mimicry was further verified. The results illustrated that brucine significantly suppressed vasculogenic mimicry tube formation with a dose-dependent effect indicated by the change of the number of tubules, intersections, and mean length of tubules. The in-depth molecular mechanism of vasculogenic mimicry suppression induced by brucine was finally suggested. It was demonstrated that brucine inhibited vasculogenic mimicry which might be through the downregulation of erythropoietin-producing hepatocellular carcinoma-A2 and matrix metalloproteinase-2 and metalloproteinase-9.

Dataset Information

Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231.

Publications

Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231.

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