SRC Phosphorylation of EPS8 Regulates SOX2 Expression and HNSCC Stemness

OBJECTIVES: Epidermal growth factor receptor pathway substrate 8 (EPS8) is a key driver of head and neck squamous cell carcinogenesis (HNSCC). SOX2, a stem cell transcription factor, is overexpressed in high-grade HNSCC and is correlated with poor prognosis. We discovered that overexpression of EPS8 in HNSCC cells led to an increase in SOX2 expression because of increased transcription. EPS8 is phosphorylated by multiple upstream kinases, including SRC, and we found that the Y602 target site is key in regulating multiple oncogenic properties. However, the impact of these posttranslational modifications on cancer stemness is unknown. In this study, we investigated the effect of SRC phosphorylation of EPS8 on SOX2 expression and cancer stem cell properties. METHODS: HNSCC cells expressing EPS8 mutants harboring phenylalanine substitutions at four SRC target sites (485/525/602/774; EPS8-F4), three sites (485/525/774; EPS8-F3) or at only one site (602; EPS8-Y602F) were used, with cells expressing low endogenous EPS8 or overexpressing wild-type EPS8 as controls. The effect of blocking SRC phosphorylation sites of EPS8 on SOX2 expression was determined by western blotting and RNA sequencing. RESULTS: Blocking the four SRC phosphorylation sites (EPS8-F4), but not in EPS8-F3, resulted in significantly reduced SOX2 expression as compared to cells overexpressing wild-type EPS8. However, pharmacological inhibition of SRC activity increased SOX2 expression in the presence of wild-type EPS8 but not in its absence. RNA sequencing studies indicated that EPS8-F4 or EPS8-Y602F cells expressed significantly less SOX2 compared to cells overexpressing wild-type EPS8. CONCLUSIONS: EPS8 expression and its phosphorylation by SRC regulate SOX2 expression. Studies also indicate a role for EPS8 and SOX2 in regulating HNSCC stemness. IMPLICATIONS: HNSCC, one of the most prevalent cancers worldwide, is associated with a high mortality and recurrence rate, in part due to the presence of cancer stem cells (CSCs). Understanding the interplay between key molecules (EPS8, SRC and SOX2) involved in the development and maintenance of the HNSCC CSC phenotype will be pivotal in identifying effective therapeutic strategies.