Loss of retinoic acid receptors in mouse skin and skin tumors is associated with activation of the ras(Ha) oncogene and high risk for premalignant progression

Retinoic acid receptor transcripts (RARalpha and RARgamma) are decreased in benign mouse epidermal tumors relative to normal skin and are almost absent in carcinomas. In this report, the expression of RARalpha and RARgamma proteins was analyzed by immunoblotting in benign skin tumors induced by two different promotion protocols designed to yield tumors at low or high risk for malignant conversion. RARalpha was slightly reduced in papillomas promoted with 12-O-tetradecanoylphorbol-13-acetate (low risk) and markedly decreased or absent in papillomas promoted by mezerein (high risk). However, mezerein also caused substantial reduction of RARalpha in nontumorous skin. RARgamma was not detected in tumors from either protocol and was greatly reduced in skin treated by either promoter. Both RARalpha and RARgamma proteins were decreased in keratinocytes overexpressing an oncogenic v-ras(Ha) gene, and RARalpha was underexpressed in a benign keratinocyte cell line carrying a mutated c-ras(Ha) gene. Introduction of a recombinant RARalpha expression vector into benign keratinocyte tumor cells reduced the S-phase population and inhibited [3H]thymidine incorporation in response to retinoic acid. Furthermore, transactivation of B-RARE-tk-LUC by retinoic acid was markedly decreased in keratinocytes transduced with the v-ras(Ha) oncogene (v-ras(Ha)-keratinocytes). Blocking protein kinase C function in v-ras(Ha)-keratinocytes with bryostatin restored RARalpha protein to near normal levels, reflecting the involvement of protein kinase C in RARalpha regulation. Both RARalpha and RARgamma are down-regulated in cultured keratinocytes by 12-O-tetradecanoylphorbol-13-acetate, further implicating PKC in the regulation of retinoid receptors. Our data suggest that modulation of RARs could contribute to the neoplastic phenotype in mouse skin carcinogenesis and may be involved in the differential promoting activity of mezerein and 12-O-tetradecanoylphorbol-13-acetate, particularly for selecting tumors at high risk for malignant conversion.