CD44-mediated activation of α5ß1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and fibronectin-enriched matrices.

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of ß1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the ß1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with ß1-integrin and repressed HA-induced, CD44-mediated activation of ß1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and ß1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

CD44 increases the efficiency of distant metastasis of breast cancer.

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients.

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity.

INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.