GSK3 regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer.
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BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3 inhibitors were identified as EMT inhibitors. The effects of GSK3 inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3 correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3 inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3 inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3 reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3 inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3 inhibitors. We found that GSK3 inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3 inhibitors and found that GSK3 inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3 correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3 is a potential target for TNBCs and suggest that GSK3 inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3 inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.
