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The acquisition of stemness is a hallmark of aggressive human hepatocellular carcinoma (hHCC). The stem cell marker OCT4 is frequently expressed in HCCs, and its expression correlates with those of putative cancer stem cell (CSC) markers and CSC properties. Here, we describe a novel mechanism of CSC maintenance by SRY through OCT4. We previously reported that Sry is involved in tumor malignancy in rodent HCCs. However, the oncogenic function of SRY in hHCCs is poorly understood. Ectopic expression of SRY increased multiple stem cell factors, including  OCT4  and  CD13 . The  OCT4  promoter contained SRY‐binding sites that were directly activated by SRY. In HCC‐derived cells, SRY knockdown decreased OCT4 expression and cancer stem‐like phenotypes such as self‐renewal, chemoresistance, and tumorigenicity. Conversely, OCT4 and SRY overexpression promoted cancer stem‐like phenotypes. OCT4 knockdown in SRY clones downregulated the self‐renewal capacity and chemoresistance. These data suggest that SRY is involved in the maintenance of cancer stem‐like characteristics through OCT4. Moreover, CSCs of HCC‐derived cells differentiated into Tuj1‐positive neuron‐like cells by retinoic acid. Noteworthily, SRY was highly expressed in some hHCC patients. Taken together, our findings imply a novel therapeutic strategy against CSCs of hHCCs. S tem  C ells   2015;33:2652–2663

SRY and OCT4 Are Required for the Acquisition of Cancer Stem Cell‐Like Properties and Are Potential Differentiation Therapy Targets