Abstract 1397: BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells.

Abstract

Abstract Bone morphogenetic protein 4 (BMP4) is an extracellular signaling molecule that belongs to the bone morphogenetic protein (BMP) family, which in turn is part of the transforming growth factor β (TGF-β) -superfamily. BMPs regulate cell proliferation, differentiation and motility, and have also been shown to be involved in cancer pathogenesis. We have previously shown that BMP4 is able to consistently reduce breast cancer cell proliferation through G1 cell cycle arrest and simultaneously to induce the migration in a subset of these cell lines. In order to study the effects of BMP4 in a more physiological setting, we used two different 3D culture systems: Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel with matrix metalloproteinase (MMP) degradable cross-links and RGD peptides. MCF-10A normal breast epithelial cells formed round acini with a hollow lumen and correct apicobasal localization of α-6 integrin in Matrigel whereas irregular structures without lumen formation or apicobasal integrin localization were seen in PEG gel. Thus the two matrices do not provide identical 3D environments. In Matrigel, BMP4 treatment reduced the proliferation of MCF-10A cells and T-47D breast cancer cells. In PEG gel, reduced growth was also seen in two other breast cancer cell lines, MDA-MB-231 and MDA-MB-361. To clarify the mechanisms behind the growth suppressive effects of BMP4, we first examined the expression levels of 11 known cell cycle regulators in T-47D and MDA-MB-361cells grown in 2D culture using Western blot. Based on its consistent upregulation in both cell lines, p21 was selected for detailed analyses. In 2D culture, BMP4 induced the expression of p21 in all three breast cancer cell lines but not in MCF-10A cells. Unexpectedly, there was no correlation between p21 expression and the BMP4 induced growth suppression in 3D. These data seem to indicate that different mechanisms are involved in BMP4 mediated cell growth regulation in 2D and 3D environments. Finally, in response to BMP4 treatment MDA-MB-231 cells formed large branching, stellate structures in Matrigel, suggestive of increased cell migration. This effect was not seen in PEG gel. Gremlin, a BMP antagonist, reversed the stellate phenotype, confirming that the effect was indeed dependent on BMP4. Similarly, a broad-spectrum MMP inhibitor, Batimastat, was also able to reverse the stellate appearance. These results imply that the actions of MMPs are required for this migratory phenotype. Taken together, our results show that BMP4 reduced the growth of breast epithelial cells and breast cancer cells in 3D culture. In addition, the highly metastatic breast cancer cell line MDA-MB-231 formed stellate, branching structures upon BMP4 treatment in Matrigel. Further research is needed to elucidate the molecular mechanisms behind these BMP4 induced phenotypes and their clinical relevance. Citation Format: Minna E. Ampuja, Riikka Jokimäki, Emma-Leena Alarmo, Kati Juuti-Uusitalo, Anne Kallioniemi. BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2013-1397