THE ER-POSITIVE PGR-NEGATIVE BREAST-CANCER PHENOTYPE IS NOT ASSOCIATED WITH MUTATIONS WITHIN THE DNA-BINDING DOMAIN

We have used in vitro DNA binding assays as a measure of estrogen rece ptor (ER) function in human breast tumors. We found that the majority of ER+ (25 ER+/progesterone receptor [PgR]+, and 25 ER+/PgR-) tumors w e examined were capable of binding consensus estrogen response element (ERE) oligonucleotides in this assay system. We found significant pro teolytic activity in many of the tumors such that protease inhibitors were found to be essential during the preparation of tumor extracts. W e next applied direct sequence analysis of the ER DNA binding domain o f several of these tumors, and determined that the ER+/PgR- breast tum ors did not contain mutations within the DNA binding domain which migh t explain their apparent discordant receptor phenotype. We did identif y an alternatively spliced ER variant missing exon 3 of the DNA bindin g domain. This variant was unable to function as a transcriptional ind ucer of an estrogen-responsive reporter in a yeast assay system. Furth ermore, the exon 3 ER deletion variant was expressed at equivalent lev els in all of the ER+ breast tumors, so that it does not appear to be involved in the evolution of the ER+/PgR- breast cancer phenotype.