Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order torepurpose drugs as CD44 antagonists, we performed consensus-docking studiesusing the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifyingetoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; andreduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereasother to poisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreasedthe proportion of CD44⁺/CD24^- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the developmentof new CD44 antagonists.