<em>In Vitro</em> and <em>In Vivo</em> Characterization of ENOX2 (tNOX) Stemness Properties in Oral Cancer

Cancer remains one of the most common causes of death worldwide and imposes enormous social and economic burdens. Human tumor-associated NADH oxidase (ENOX2, also known as tNOX) is a cancer cell-specialized NADH oxidase that is expressed on the membranes of cancer cells. Here, we report in vitro and in vivo results demonstrating that ENOX2 is associated with the stemness of oral cancer cells. We found that ENOX2 interacted with the stem cell transcription factor, SOX2, in co-immunoprecipitation experiments. The expression and activity of ENOX2 were higher in p53-functional SAS and p53-mutated HSC-3 oral cancer cell spheroids compared with their monolayer counterparts. Consistently, the protein expression of SIRT1, which is modulated by ENOX2 via NAD+ generation, was also upregulated in oral cancer cell spheroids. Functional studies further established the role of ENOX 2 in stemness regulation. ENOX2 overexpression in human oral cancer cells significantly enhanced spheroid formation, self-renewal properties, stem cell marker expression, and PKCδ expression in vitro. In contrast, knockdown of ENOX2 by RNA interference significantly reduced spheroid formation, stem cell marker expression, and PKCδ expression in the same cell lines. In vivo studies performed in tumor xenograft models also showed that ENOX2-knockdown oral cancer cell spheroids yielded significantly less efficient tumor formation, while ENOX2-overexpression oral cancer cell spheroids yielded significantly more tumor and stem cell formation. Bioinformatics database searches and Western blot analyses also showed that the protein expression of ENOX2 was elevated in human oral tumor tissues compared with adjacent normal tissues. Taken together, these results suggest that ENOX2, through its interplay with SIRT1 and SOX2, promotes the acquisition and maintenance of stem-like properties of oral cancer cells.