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We recently reported that multiple c-erbB ligands differentially modulate in vitro proliferation, invasion and expression of matrix metalloproteinases in human head and neck squamous carcinoma cells (HNSCC). In order to evaluate further the importance of c-erbB ligands in tumor progression, the expression and regulation of this growth factor family in HNSCC cells was studied. We demonstrate that mRNAs for the 6 major c-erbB ligands, namely, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR) and heregulin (HRG), are expressed in a large panel of HNSCC cell lines. In addition to TGF-α, other ligands (notably BTC and HRG-β1) are involved in the autocrine growth regulation of these cells. Each c-erbB ligand when applied exogenously, induced mRNA expression of both itself and the remaining family members and a differential response in the kinetics of induction was found. HB-EGF and HRG mRNAs were induced rapidly (within 1 hr) and to a greater extent (3.2–6.2- and 4.8–7.3-fold increase) than TGF-α, BTC and AR mRNAs (1.6–2.7, 1.8–3.6- and 1.6–4.2-fold, respectively). This pattern was observed for all inducing ligands tested. Analysis of mRNA stability, and concurrent treatment with BTC (as an inducing ligand) and cycloheximide (to inhibit protein synthesis) suggested both transcriptional and post-transcriptional regulatory mechanisms. These results support and extend previous observations of c-erbB receptor signaling as a critical element in the pathogenesis and progression of HNSCC, and emphasize the role of autocrine ligand production. Int. J. Cancer 88:759–765, 2000. © 2000 Wiley-Liss, Inc.

Numerous reports have shown an association between overexpression of the epidermal growth factor receptor (EGFR), and poor prognosis in head and neck squamous cell carcinomas (HNSCC), however, the underlying mechanisms are still unclear. In the present study, we set out to determine whether EGFR expression was associated with in vitro invasive capacity in a panel of four established and ten newly derived HNSCC lines. Ten of the cell lines expressed high levels of EGFR as determined by a ligand-binding assay and dot blot analysis, whereas the remaining four showed weak overexpression or normal levels of EGFR. The ability of cells to invade through Matrigel was found to be higher in the EGFR overexpressing cell lines (p < 0.0001). Expression levels of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MT1-MMP) and tissue inhibitors of MMP (TIMP-1, TIMP-2) were evaluated by semiquantitative RT-PCR, substrate zymography and western blot. We found a strong positive correlation between EGFR levels and the expression of MMP-9 mRNA (r² = 0.95; p < 0.0001), MMP-9 enzyme activity (r² = 0.8099; p < 0.0001) and an inverse correlation with TIMP-1 (r² = 0.48; p = 0.0059). In six selected HNSCC lines, in vitro invasion was assayed in the presence of an anti-EGFR monoclonal antibody, ICR62. A significant reduction of invasion in four selected EGFR-overexpressing cell lines was found with 30 nM ICR62 (from 50% to 70%; p < 0.001) but there was no effect in two cell lines with normal EGFR levels. Our results show that the in vitro invasive phenotype of HNSCC lines correlates with high EGFR and MMP-9 expression, and it is therefore suggested that the EGFR signaling pathway might play an important role in the invasive behavior of HNSCC via specific upregulation of MMP-9 and downregulation of TIMP-1. Int. J. Cancer 86:307–317, 2000. © 2000 Wiley-Liss, Inc.