LAUSR

Integrin β3 (ITGB3) is expressed by breast cancer cells and its heterodimer αvβ3 is related to skeletal metastasis. Here we aimed to investigate this mechanism for defining its role as target in anti-metastatic therapy. Therefore, we generated two MDA-MB-231 breast cancer cell clones (I3, I5) with conditional doxycycline-dependent miRNA-mediated ITGB3 knockdown. In the absence of doxycycline the bi-directional Ptet promoter drives the simultaneous expression of firefly luciferase, red fluorescent protein mCherry and a specific miRNA targeting ITGB3 mRNA. In the presence of doxycycline, their expression is switched off. This model allowed monitoring the effects of ITGB3 inhibition at defined time points on cellular and molecular properties by flow cytometry analysis, proliferation and migration assays, as well as expression profiling analysis. Furthermore, the I5 clone was examined in a nude rat model for breast cancer skeletal metastasis for up to 5 weeks of miRNA treatment. Finally, ITGB3 was analyzed in exosomes secreted from breast cancer (MDA-MB-231, MCF7) and from breast epithelial cells (MCF10), as well as from plasma of healthy and MDA-MB-231 implanted rats with skeletal metastasis. MDA-MB-231 cells were incubated with these ex vivo exosomes for 72h and ITGB3 production was analyzed. Also, the levels of ITGB3 were investigated in exosomes isolated from cell clone I5. ITGB3 was well regulated in cell clones I3 and I5, as shown by knockdown at mRNA (78% and 73%) and protein (22% and 40%) levels after 6 days in medium without doxycycline. After this period the proliferation was decreased only in I5 cells (22%), whereas the migration was inhibited in both cell clones, again more pronounced in I5 (87%) than in I3 cells (20%). Furthermore, decreased tumor sizes and even complete remissions were detected in rats bearing the I5 clone after 4 to 5 weeks of miRNA treatment by bioluminescence and magnetic resonance imaging, as well as volume computed tomography. MDA-MB-231 cells secreted higher levels of ITGB3 in exosomes than MCF7 or MCF10 cells. Also, exosomes from rats bearing MDA-MB-231 induced skeletal metastases contained higher levels of ITGB3 than exosomes from healthy controls. MDA-MB-231 cells incubated with these ex vivo exosomes showed increased ITGB3 levels, too. In line, decreased ITGB3 levels were detected in exosomes from cells with conditional knockdown of this protein. The microarray data of cells with ITGB3 knockdown for 3 or 6 days showed a downregulation of genes, which have specific roles in angiogenesis (NPTN, RRM2), tumor growth (NPTN), energy metabolism (ISCA1), cytokinesis (SEPT11), migration (RRM2, STX6), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) and vesicle trafficking (SEPT11, STX6). In conclusion, ITGB3 has a function related to skeletal metastasis of breast cancer cells and mediates its distant effects via exosomes, which points to this protein as target for treating the disease. Citation Format: Marineta Kovacheva, Michael Zepp, Martin R. Berger. Integrin beta3 is a target for treating breast cancer skeletal metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6086.