Characterization of a cytoskeletal signaling pathway underpinning CD44-initiated, integrin-mediated adhesion of breast cancer cells to bone marrow endothelium

Background

Bone metastasis is a frequent complication of breast cancer. It is estimated that up to 85% of breast cancers will metastasize to the bone. The selective metastasis of breast cancer to the bone is thought to result from the preferential adhesion of breast cancer cells to the bone marrow endothelial cells (BMECs) lining the bone marrow sinusoids. Our studies have shown that CD44 promotes the primary adhesion of breast cancer cells to bone marrow endothelium in vitro. The aim of the present study was to further explore the cascade of events underpinning CD44-initiated adhesion.

Methods

Experiments using parental and bone-homing (BO) clones of the MDA-MB-231 breast cancer cell line established the importance of CD44 to integrin-mediated adhesion to BMECs.

Results

MDA-MB-231BO cells displayed increased CD44 expression and adhesion to both BMECs and fibronectin, relative to parental cells. MDA-MB-231BO cells also displayed increased expression and activation of the β₁-integrin subunit. In addition, the bone-homing cells exhibited elevated constitutive phosphorylation of the kinases Src and FAK and the cytoskeletal proteins cortactin and paxillin relative to the parental cells. Stimulation of MDA-MB-231BO cells with the CD44 ligand hyaluronan (HA) induced an increase in the expression of the β₁-integrin chain, FAK and paxillin; and, furthermore, promoted a rapid increase in the activation status of the β₁-integrin subunits, Src, cortactin and paxillin in these cells. The HA-induced phosphorylation of paxillin was attenuated by depletion of CD44 and cortactin expression using selective RNAi strategies, suggesting that it is a downstream target of HA-CD44-cortactin signaling. MDA-MB-231BO cell adhesion to fibronectin or to hBMECs was attenuated by RNAi-mediated suppression of CD44, cortactin and paxillin expression or following administration of two neutralizing antibodies that inhibit β₁-integrin and α₄β₁-integrin receptor signaling. Antibody-based inhibition of integrin signaling also attenuated the HA-induced phosphorylation of cortactin and paxillin, suggesting that these proteins constitute a signaling cascade activated downstream of a CD44-initiated, integrin-dependent process.

Conclusion

Our results describe a molecular pathway promoting cytoskeletal reorganization that is activated downstream of a CD44-induced, integrin-dependent event and that is critical to efficient breast cancer cell adhesion to hBMECs.