Chemotherapeutic resistance of head and neck squamous cell carcinoma is mediated by EpCAM induction driven by IL-6/p62 associated Nrf2-antioxidant pathway activation.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been associated with chemotherapeutic resistance, leads to aggressive tumor behavior, and results in an adverse clinical outcome. The molecular mechanism by which EpCAM enrichment is linked to therapeutic resistance via Nrf2, a key regulator of antioxidant genes is unknown. We have investigated the link between EpCAM and the Nrf2 pathway in light of therapeutic resistance using head and neck squamous cell carcinoma (HNSCC) patient tumor samples and cell lines. We report that EpCAM was highly expressed in Nrf2-positive and HPV-negative HNSCC cells. In addition, cisplatin-resistant tumor cells consisted of a higher proportion of EpCAMhigh cells compared to the cisplatin sensitive counterpart. EpCAMhigh populations exhibited resistance to cisplatin, a higher efficiency in colony formation, sphere growth and invasion capacity, and demonstrated reduced reactive oxygen species (ROS) activity. Furthermore, Nrf2 expression was significantly higher in EpCAMhigh populations. Mechanistically, expression of Nrf2 and its target genes were most prominently observed in EpCAMhigh populations. Silencing of EpCAM expression resulted in the attenuation of expressions of Nrf2 and SOD1 concomitant with a reduction of Sox2 expression. On the other hand, silencing of Nrf2 expression rendered EpCAMhigh populations sensitive to cisplatin treatment accompanied by the inhibition of colony formation, sphere formation, and invasion efficiency and increased ROS activity. The molecular mechanistic link between EpCAM expression and activation of Nrf2 was found to be a concerted interaction of interleukin-6 (IL-6) and p62. Silencing of p62 expression in EpCAMhigh populations resulted in the attenuation of Nrf2 pathway activation suggesting that Nrf2 pathway activation promoted resistance to cisplatin in EpCAMhigh populations. We propose that therapeutic targeting the Nrf2-EpCAM axis might be an excellent approach to modulate stress resistance and thereby survival of HNSCC patients enriched in EpCAMhigh populations.

Widespread expression of Sonic hedgehog (Shh) and Nrf2 in patients treated with cisplatin predicts outcome in resected tumors and are potential therapeutic targets for HPV-negative head and neck cancer.

BACKGROUND: Sonic hedgehog (Shh) and Nrf2 play a critical role in chemotherapeutic resistance. These two genes have been found to be dysregulated in head and neck squamous cell carcinomas (HNSCC). The purpose of this study was to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). METHODS: We analyzed a cohort of patients with HNSCC to identify potential therapeutic biomarkers correlating with overall survival (OS) as well as disease-free survival (DFS) from our own data and validated these results using The Cancer Genome Atlas dataset. Expression of Shh and Nrf2 was knocked down by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. RESULTS: Widespread abundant expression of Shh and Nrf2 proteins were associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a significant predictor of patient DFS. The tumor stromal index was correlated with Shh expression and inversely associated with shorter OS and DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to the chemotherapeutic agents. Concomitant upregulation of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in patients with HNSCC. CONCLUSIONS: These findings suggest that Shh and Nrf2 could serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC.