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In prostate cancer, castration resistance is a factor that frequently leads to death in individuals with this disease. Recent studies have suggested that prostate cancer stem cells (PCSCs) are pivotal regulators in the establishment of castration resistance. The nanog homeobox (NANOG) and the transforming growth factor (TGF)-β1/drosophila mothers against decapentaplegic protein (SMAD) signaling pathways are involved in several cancer stem cells but are not involved in PCSCs. The purpose of this study is to investigate the effect of NANOG on the proliferation of PCSCs regulated by the TGF-β1/SMAD signaling pathway.