Src Family Kinases Mediate Epidermal Growth Factor Receptor Ligand Cleavage, Proliferation, and Invasion of Head and Neck Cancer Cells

Abstract

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor ␣ (TGF-␣) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRPinduced proliferation or invasion of HNSCC cells. In addition to TGF-␣ release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-␣ and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-␣ and amphiregulin in HNSCC.

Phosphorylation of TNF-α converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation

“…Phosphorylation of TACE may result in its translocation to its targets or the formation of a functional complex of Src and TACE on the membrane. We previously reported that Src family kinases contribute to GRP-induced EGFR phosphorylation in SCCHN (15). Both c-Src and TACE are located in punctuate foci in the cytosol compatible with the association of c-Src and TACE with intracellular membranes through myristoylation of the Src N-terminal domain and TACE transmembrane domain, respectively.…”

“…For immunoprecipitation, 200 g of total protein was incubated for 2 h at 4°C with 3 g of anti-TACE antibody (QED Biosciences, San Diego), anti-EGFR antibody (Upstate Biotechnology), or 2 g of anti-c-Src antibody (Santa Cruz Biotechnology) with gentle agitation, followed by protocols reported in ref. 15.…”

“…EGFR activation by GPCRs has been proposed to be mediated by Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and͞or PKC signaling (11). Src family kinases have been reported to regulate GPCR ligand-induced EGFR phosphorylation in the colon cancer cell line Caco-2, gastric epithelial cells RGM1, Cos-7 cells, GT1-7 neuronal cells, lung cancer cell 201T, and head and neck cancer cells (12)(13)(14)(15)(16). PI3-K also has been implicated in GPCR and EGFR crosstalk (17)(18)(19).…”

“…We previously reported that Src family kinases regulate gastrinreleasing peptide (GRP)-induced EGFR proligand cleavage leading to downstream EGFR and MAPK activation in SCCHN, which contributes to cell proliferation and invasion (15,27). In addition, blockade of endogenous GRP by using the 2A11 antibody decreased SCCHN cell proliferation and tumor growth in vivo (28).…”

Interactions of STAP-2 with BRK and STAT3/5 in Breast Cancer Cells

“…These kinases might be activated by cytokines or by structural alterations. For instance, EGFR over-expression and activation may underlie STAT activation in breast, lung and head and neck cancer (Zhang et al, 2004). Constitutive activation or dysregulated expression of STATs is detected in primary tumors and cancer cell lines, including leukemia, multiple myeloma, lymphomas, melanoma and cancers of the breast, skin, lung, ovaries, pancreas, prostate, kidney, thyroid, and head and neck (Desrivieres et al, 2006).…”