Abstract
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.
Highlights
Bone metastasis is pathological process which occurs in about 70% of breast cancer patients with progression of their disease, and causes disturbing consequences including bone pain, pathologic fractures, hypercalcemia, alteration of hematopoiesis, and spinal cord compression
OPN is known as secreted phosphoprotein 1 (SPP1), which is assigned to the small integrin-binding ligand n-linked glycoprotein (SIBLING) family
In the absence of doxycycline, tTA activates this promoter, which stimulates the simultaneous expression of firefly luciferase, red fluorescent protein mCherry, and a highly efficient and specific miRNA targeting OPN mRNA
Summary
Bone metastasis is pathological process which occurs in about 70% of breast cancer patients with progression of their disease, and causes disturbing consequences including bone pain, pathologic fractures, hypercalcemia, alteration of hematopoiesis, and spinal cord compression. Alkylphosphocholines like miltefosine and erufosine have been found active in treating secondary and primary breast cancers [5,6] They are membrane-seeking agents and interfere with signal transduction pathways. OPN plays a role in bone remodeling and mediates the interaction between osteoclasts and mineral matrix in the skeleton [12] It binds to integrins and CD44 receptors, takes part in cell–matrix interactions, including cell signaling, and is related to tumor growth and development [11] as well as cancer skeletal metastasis [9]. A model was established which realized a conditional knockdown by using doxycycline-dependent miRNA expression In this approach, a tet-regulated expression gene cassette, containing an artificial miRNA targeting OPN mRNA, was integrated into a predetermined genomic locus of MDA-MB-231 breast cancer cells by Flp-recombinase-mediated cassette exchange (RMCE). The combination effect of conditional OPN knockdown and erufosine treatment was investigated in breast cancer cells
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