Activation of the epidermal growth factor receptor by skin tumor promoters and in skin tumors from SENCAR mice.

The present study was designed to further investigate the role of the epidermal growth factor receptor (EGFr) in mouse skin tumor promotion by evaluating the status of the EGFr in tumor promoter-treated mouse epidermis and in mouse skin tumors. Female SENCAR mice received three topical treatments of either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or the nonphorbol esters okadaic acid and chrysarobin. Membrane proteins from SENCAR mouse epidermis were isolated 6 h after the last treatment, and the phosphotyrosine content of the EGFr and several potential substrates were examined by Western blot analysis. The results indicated that multiple applications of all three tumor promoters led to an increase in the phosphotyrosine content of the EGFr and also of several lower molecular weight proteins (M(r) approximately 80,000-85,000). Phosphorylation of PLC gamma 1 on tyrosine residues could not be detected in tumor promoter-treated mouse epidermis when the phosphotyrosine content of the EGFr was elevated or in cultured keratinocytes exposed to exogenous EGF. When two tyrosine kinase inhibitors (tyrphostins RG50864 and RG13022) were incorporated into the treatment regimens, the TPA-induced epidermal hyperplasia and cell proliferation were effectively blocked, and the TPA-stimulated EGFr tyrosine phosphorylation was significantly reduced. Examination of the phosphotyrosine content of epidermal membrane proteins isolated from skin papillomas revealed that the EGFr also had elevated phosphotyrosine levels. These results demonstrate that multiple topical treatments with both phorbol ester and nonphorbol ester tumor promoters lead to activation of the EGFr tyrosine kinase in mouse epidermis. In addition, these data suggest that signaling through the EGFr pathway plays an important role in the tumor promotion stage of multistage carcinogenesis in mouse skin.