Calprotectin Regulates EGFR Not VEGF-A/C or NGF Expression in HNSCC

Objectives: Calprotectin (S100A8/A9) regulates cell cycle progression by restoring the G2/M checkpoint. Down-regulated in head and neck squamous cell carcinoma (HNSCC), S100A8/A9 over-expression suppresses tumorigenesis in vitro and in vivo; patients survive longer with S100A8/A9-high than S100A8/A9-low HNSCCs. Poor clinical outcome is also associated with EGFR-mediated proliferation, VEGF-A and-C angiogenetic and lymphangiogenetic signaling and NGF-regulated perineural growth. We aimed to investigate whether S100A8/A9 regulates the expression of EGFR, VEGF-A and -C, and NGF in HNSCC
Methods: S100A8/A9-expressing, shRNA knockdown, and shRNA control TR146 HNSCC cells were analyzed in vitro and compared to S100A8/A9-negative KB carcinoma cells, KB cells co-transfected to express both S100A8 and S100A9, and a sham-control transfectant, KB-EGFP. Controls included S100A8/A9+ immortalized oral keratinocytes (TERT-2) and primary tonsillar epithelial cells. EGFR, VEGF-A and -C, and NGF protein expression was assessed in vitro using confocal immunofluorescence microscopy and western blotting, and ex vivo by immunohistochemistry in FFPE human HNSCCs (n=8).
Results: EGFR, VEGF-A and -C, and NGF were expressed in TR146 and KB mutant and WT cells. Interestingly, silencing calprotectin in TR146-S100A8/A9-shRNA cells increased expression of EGFR greater than WT and TR146-shRNA-control cells. S100A8/A9 status of the TR146 and KB cell lines did not appear to affect VEGF-A and-C, and NGF protein levels. TERT-2 and primary epithelial cells (S100A8/A9+) showed lower expression of EGFR than carcinoma cells, whereas EGFR expression was low in S100A8/A9-high HNSCCs. Conversely, S100A8/A9-low carcinomas appeared to upregulate EGFR. VEGF-A and -C, and NGF expression appeared to be independent of S100A8/A9 in tumor samples.
Conclusions: Calprotectin appears to regulate EGFR expression in HNSCC. Since decreased expression of EGFR is a positive prognostic indicator for HNSCC, S100A8/A9-mediated down-regulation of EGFR could explain better survival of patients with S100A8/A9-high tumors.