Inflammatory mediators drive metastasis and drug resistance in head and neck squamous cell carcinoma

Objectives/hypothesis: The presence of regional metastases in head and neck squamous cell carcinoma (HNSCC) patients is a common and adverse event associated with poor prognosis. Understanding the molecular mechanisms that mediate HNSCC metastasis may enable identification of novel therapeutic targets. E-cadherin plays a key role in epithelial intercellular adhesion; its downregulation is a hallmark of the epithelial-to-mesenchymal transition (EMT) (an essential process during tumor progression); and it is associated with invasion, metastasis, and decreased survival. Inflammatory cytokines have been implicated in the progression of HNSCC. Herein, the mechanisms by which the inflammatory mediator, Interleukin-1β (IL-1β), might contribute to EMT in HNSCC is investigated. The pathways involved in E-cadherin regulation in HNSCC had not previously been defined. It is hypothesized that 1) inflammatory mediators upregulate cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2), which then in turn regulate E-cadherin expression in HNSCC; and 2) PGE2 downregulates E-cadherin via transcriptional repressors of E-cadherin (such as Snail) in HNSCC. The outcome of the proposed research will allow us to define how resistance to epidermal growth factor receptor (EGFR)-selective tyrosine kinase inhibitors is mediated and whether the benefits of combination therapy are due to the capacity of COX-2 inhibitors to increase E-cadherin expression and thus create a more sensitive target for EGFR TK inhibition.

Study design: Basic science, molecular biology, animal model, immunohistochemistry.

Methods: We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a severely compromised immunodeficient (SCID) xenograft model was used to assess the effects in vivo.

Results: COX-2-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was demonstrated in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 small hairpin RNA (shRNA). IL-1β -treated HNSCC cell lines demonstrated a significant decrease in E-cadherin messenger RNA (mRNA) and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. ShRNA-mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. Snail overexpression in normal oral keratinocytes and HNSCC cells is sufficient to drive EMT and confers resistance to erlotinib. In a SCID xenograft model, HNSCC Snail overexpressing cells demonstrated significantly increased primary and metastatic tumor burdens.

Conclusions: The inflammatory mediator IL-1β modulates Snail and thereby regulates COX-2-dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy.

Level of evidence: N/A.