Estrogen inducibility of c-Ha-ras transcription in breast cancer cells. Identification of functional estrogen-responsive transcriptional regulatory elements in exon 1/intron 1 of the c-Ha-ras gene.

Although mutation of ras gene is rare in human breast cancer, overexpression of normal c-Ha-ras gene is frequently observed. Using a mouse mammary metastasis model consisting of genetically related mammary tumor sublines with variant metastatic potential, we have previously (i) demonstrated a direct correlation between c-Ha-ras mRNA and protein levels and metastatic potential and (ii) identified a novel hormone-responsive transcriptional regulatory element in intron 1 of the mouse c-Ha-ras gene that contains the consensus half-site of a glucocorticoid response element and flanking consensus half-sites for estrogen response element. Here, we have examined the functionality of intron 1 sequence in context of upstream sequences by using transient transfection assays with plasmids expressing chloramphenicol acetyltransferase. Intron 1 sequence and sequences similar to intron 1 element located in exon 1 function as transcriptional regulatory elements that confer hormonal inducibility to chloramphenicol acetyltransferase gene expression both independently and in context of 5'-flanking sequences. Measurement of c-Ha-ras transcription rates and protein expression by nuclear run-on and metabolic labeling assays showed a 5-12-fold enhancement, respectively, following treatment with 17beta-estradiol that was blunted by ICI 182,780 in the nonmetastatic variant. In contrast, constitutive overexpression of c-Ha-ras transcripts and protein in the metastatic subline was unaffected by estrogen and ICI 182,780. Gel shift assays demonstrated specific interaction of c-Ha-ras exon 1 sequence with nuclear proteins of human breast cancer MCF-7 cells with formation of two complexes, one of which contains estrogen receptor. Our data demonstrate a direct (i) interaction of c-Ha-ras sequence with estrogen receptor and (ii) stimulatory effect of estrogen on c-Ha-ras gene transcription and suggest that alteration in transcriptional regulation of c-Ha-ras gene by estrogen may play an important role in progression of breast cancer.