525 - Salinomycin affects Golgi apparatus function in cancer stem-like cells

Abstract

Background: Cancer stem cells (CSC) are an immortal tumor-initiating population that can self-renew and has pluripotent capacity. Despite their small number, CSCs are believed to be critical for tumor initiation, progression and metastasis, as well as for the treatment failure and disease relapse. CSCs have been confirmed to exist in various types of tumors which include leukemia as well as various solid cancers. There are several obstacles involved with CSC research among which are their low number within tumor cell population and their relative instability in culture. Recently, a breast CSC model was established by experimental induction of epithelial to mesenchymal transition (EMT) in immortalized human mammary epithelial cells (HMLE). Using this model salinomycin was identified to be selectively toxic towards epithelial CSCs. Salinomycin is a K+/H+ exchanger that can affect cation transport across different membranes present in the cell. However the exact mechanism of the observed selectivity remains largely unknown. We have noticed that salinomycin affects posttranslational modifications of membrane proteins in the fore mentioned CSC model. This lead us to the hypothesis that salinomycin induces Golgi apparatus stress that affects secretory pathway which may represent a key vulnerability of EMT cells. Of note, monensin, another ionophore with chemical structure similar to that of salinomycin, is a well described inhibitor of Golgi function. Materials and Methods: We used HMLE cells with knocked-down E-cadherin (HMLEshEcad) that went through EMT and display characteristics of stem cells. To test our hypothesis we performed western blotting, confocal microscopy and flow cytometry after staining with fluorescently labeled Golgirelated antibodies. Results: The treatment of stem-like HMLEshEcad cells with submicromolar concentrations of salinomycin causes dysfunctional maturation of N-cadherin. In addition, we observed similar changes in processing of other plasma membrane proteins that could be accounted for aberrant Golgi apparatus function. The same effect was not observed in control cells. Immunostaining confirmed changes of Golgi apparatus morphology and function. Conclusion: Our results indeed point that salinomycin differentially affects mesenchymal and epithelial cells due to their different dependence on the Golgi apparatus function. We believe that agents that promote endoplasmic reticulum and Golgi apparatus malfunction should be further explored for their potential to selectively eradicate cancer cells that have undergone an EMT.