ABSTRACT: Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. Our study showed that MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition (EMT), and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells. Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice. Our findings indicate that MELK supports metastasis by promoting EMT and the CSC phenotype in TNBC. In our microarray analysis, we identified potential downstream targets of MELK, including STAT5 and NF-kB target genes, as well as genes involved in tumor progression and metastasis (i.e., EMT, angiogenesis, hypoxia, and apical junction). EMT was the most strongly enriched hallmark among genes highly expressed in Cas9-p15 control cells, further confirming that EMT is a major factor contributing to MELK-induced metastasis in TNBC. We also identified a direct physical interaction partner (PRKAB2) of MELK and a set of intermediate proteins (CDC25B, EZH2, FOXM1, JUN, MAP3K5, PRKAB1, PRKAB2, and SMAD2), suggesting that these proteins are key components of MELK-induced signal transduction.
