Targeting integrin beta-1 to reduce attachment and migration of breast cancer cells

摘要

Purpose: The attachment of breast cancer cells with the aid of integrins to extracellular proteins such as fibronectin and vitronectin present on bone marrow environment plays a critical role in the metastasis of tumor cells to bone. This attachment acts as a primary site of interaction between the two populations of cells and allows the tumors cells to remain dormant in bone for a longer period of time and resist chemo and radiation therapy. Blocking this primary site of attachment could be beneficial in reducing metastasis which could be achieved by silencing the cell surface integrins on transformed cells. In this study, siRNA targeting integrin β-1 was delivered using modified polyethylenimine polymers, anticipating the breast cancer cells to attach less to the bone cells as well as reduce their ability to migrate. Methods: Integrin β-1 silencing experiments were carried out in MDA MB-231 breast cancer cells through Immunostaining, qRT-PCR, fibronectin-binding, human Bone Marrow Stromal Cell (hBMSC) Adhesion and scratch (migration) assays. Results: Three specific lipopolymers with different lipid substitutions displayed effective silencing at 40 nM siRNA concentration based on immunostaining of cell-surface Integrin β-1, but 1.2PEI-LA6 (with linoleic acid substitution) exhibited the best silencing among them. The surface level of integrin β-1 was significantly silenced after treating 1.2PEI-LA6/siRNA complexes, and similarly the mRNA levels were also strongly silenced till day 9 {{Figure.1}). There was a small (but significant) reduction in viable cell numbers as a result of Integrin β-1 silencing {{Figure.2}}. The functionality of this effective silencing was assessed by studying its ability to attach to fibronectin and human bone marrow stem cells as integrin β-1 is a primary receptor for fibronectin. In both these assays, significant reduction in the cell attachment was observed in 1.2PEI-LA6 treated cells (not shown). Finally, the well-established scratch assay revealed strong inhibition of cell migration of MDA-231 cells at 24 hours of specific siRNA treatment{{Figure.3}}. Conclusions: Silencing integrin β-1 gene in invasive MDA-231 cells was shown to be feasible with a practical dose of specific siRNA, which was effectively delivered by non-viral (polymeric) carriers. Both the fibronectin binding and hBMSC adhesion assay showed that the extent of integrin silencing at cell surface was adequate to reduce its binding ability thereby revealing the functional benefit of integrin β-1 silencing. In addition to silencing integrin β-1, identifying other targets could be beneficial to fully reduce the attachment and migration of breast cancer cells. Nevertheless, this study provides a viable method to specifically abolish an important mechanism behind breast cancer metastasis.