Conditional knockdown of integrin beta-3 reveals its involvement in osteolytic and soft tissue lesions of breast cancer skeletal metastasis.

Integrin ß3 (ITGB3) is probably related to skeletal metastasis, which is the most frequent complication in breast cancer progression. We aimed to define its role and suitability as target for anti-metastatic therapy. We generated two MDA-MB-231 cell clones with conditional miRNA-mediated ITGB3 knockdown for analyzing the resulting effects in vitro regarding mRNA expression, proliferation and migration, as well the impact on skeletal metastasis in a nude rat model. Furthermore, ITGB3 levels were analyzed in exosomes from plasma of rats with skeletal metastases, and from MDA-MB-231 cells incubated with these vesicles, as well as from exosomes secreted by cells with conditional ITGB3 knockdown. This inhibition of ITGB3 expression decreased cellular proliferation and more distinctly inhibited cellular migration. Reduction and even complete remissions of respective soft tissue and osteolytic lesions were detected after ITGB3 knockdown in vivo. Furthermore, ITGB3 levels were increased in exosomes isolated from plasma of rats harboring MDA-MB-231 lesions as well as in respective cells incubated with these vesicles in vitro. ITGB3 was distinctly decreased in exosomes from cells with ITGB3 knockdown. The observed in vitro and in vivo anti-ITGB3 effects can be explained by downregulation of specific genes, which have 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). ITGB3 has a role in breast cancer skeletal metastasis via gene expression modulation, as mirrored for ITGB3 in exosomes, thus it could serve as target for anti-metastatic therapy.

Conditional Knockdown of Osteopontin Inhibits Breast Cancer Skeletal Metastasis.

High osteopontin (OPN) expression is linked to breast cancer bone metastasis. In this study we modulated osteopontin levels conditionally and investigated any related antineoplastic effects. Therefore, we established cell clones from human breast cancer MDA-MB-231 cells, in which the expression of OPN is regulated by the Tet-Off tet-off system. These cells, which conditionally express a specific miRNA targeting OPN, were used for in vitro studies as well as for a bone metastasis model in nude rats. Changes in whole-genome expression elicited by conditional OPN knockdown and vesicle formation were also analyzed. The alkylphosphocholine erufosine was used for combination therapy. Conditional OPN knockdown caused mild anti-proliferative, but more intensive anti-migratory and anti clonogenic effects, as well as partial and complete remissions of soft tissue and osteolytic lesions. These effects were associated with specific gene and protein expression modulations following miRNA-mediated OPN knockdown. Furthermore, high levels of OPN were detected in vesicles derived from rats harboring breast cancer skeletal metastases. Finally, the combination of OPN inhibition and erufosine treatment caused an additive reduction of OPN levels in the investigated breast cancer cells. Thus, knockdown of OPN alone or in combination with erufosine is a promising strategy in breast cancer skeletal metastasis treatment.

IDK1 is a rat monoclonal antibody against hypoglycosylated bone sialoprotein with application as biomarker and therapeutic agent in breast cancer skeletal metastasis.

Changes in glycosylation are salient features of cancer cells. Here, we report on the diagnostic and therapeutic properties of IDK1, an antibody against tumour associated, hypoglycosylated bone sialoprotein (hypo-BSP). The affinity of the rat monoclonal antibody IDK1 for hypo-BSP, as determined by microscale thermophoresis, was three orders of magnitude higher than for mature BSP, whereas the mouse monoclonal antibody used had similar affinity for both BSP forms. IDK1 showed no activity against the proliferation or migration of normal or cancer cells growing in vitro. In vivo, however, IDK1 caused dose-dependent regression of soft tissue and skeletal lesions in nude rats harbouring human MDA-MB-231 cells. At optimal dose, 80% of the treated rats showed complete remission of all tumour lesions. Analysis of BSP expression in vitro by fluorescence-activated cell sorting (FACS) and immunocytochemistry showed basal levels of this protein, which were visible only in a fraction of these cells. Cells of the metastatic cell lines MDA-MB-231 and PC-3 were more often positive for hypo-BSP. In addition, there was co-expression of both forms in some cells, but almost no co-localization; rather, hypo-BSP was present in the nucleus, and mature BSP was detected extra-cellularly. Normal osteoblasts and osteoclasts were negative for hypo-BSP. Breast cancer tissue, however, showed strong expression of mature BSP, which was present intra-cellularly as well as in vesicles outside cells. Hypo-BSP was present mainly in lesions from skeletal sites, thus explaining the antineoplastic activity of IDK1, which was high in lesions growing in the vicinity of the skeleton but low in lesions growing subcutaneously. Finally, hypo-BSP was detected in specimens from breast cancer patients, with a significantly greater intensity in skeletal metastases as compared to the respective primary cancers. In conclusion, IDK-1 is an antibody with diagnostic and therapeutic applications in skeletal metastases of breast cancer.

Sustained conditional knockdown reveals intracellular bone sialoprotein as essential for breast cancer skeletal metastasis.

Increased bone sialoprotein (BSP) serum levels are related to breast cancer skeletal metastasis, but their relevance is unknown. We elucidated novel intracellular BSP functions by a conditional knockdown of BSP. Conditional MDA-MB-231 subclones were equipped with a novel gene expression cassette containing a tet-reg-ulated miRNA providing knockdown of BSP production. These clones were used to assess the effect of BSP on morphology, proliferation, migration, colony formation and gene expression in vitro, and on soft tissue and osteolytic le-sions in a xenograft model by three imaging methods. BSP knockdown caused significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro (p<0.001). In vivo, significant de-creases of soft tissue and osteolytic lesions (p<0.03) were recorded after 3 weeks of miRNA treatment, leading to complete remission within 6 weeks. Microarray data revealed that 0.3% of genes were modulated in response to BSP knockdown. Upregulated genes included the endoplasmic reticulum stress genes ATF3 and DDIT3, the tumor suppressor gene EGR1, ID2 (related to breast epithelial differentiation), c-FOS and SERPINB2, whereas the metastasis associated genes CD44 and IL11 were downregulated. Also, activation of apoptotic pathways was demonstrated. These results implicate that intracellular BSP is essential for breast cancer skeletal metastasis and a target for treating these lesions.