Function of the exon 7 deletion variant estrogen receptor alpha protein in an estradiol-resistant, tamoxifen-sensitive human endometrial adenocarcinoma grown in nude mice.

Background: In addition to hormone and DNA binding, interactions, including competition with other proteins, appear to be a critical component of transcriptional regulation by the estrogen receptor alpha (ER(alpha)). In vitro studies suggest that exon deletion (Delta exon) variant forms of ER(alpha) may also play an important role in determining the progression from hormone dependence to hormone independence in receptor positive tumors.

Methods: We investigated the presence of ERalpha mRNA and protein variants and their possible role in a moderately differentiated human endometrial adenocarcinoma grown in nude mice. In addition to wild-type (wt), RT-PCR assay of the tumor revealed the presence of two mRNA variants, a low concentration of Delta5 and a high concentration of Delta7 ER(alpha). We detected wt, Delta7, and Delta5,7 mRNA by sequencing the transcripts after stable transfection of three HeLa cells with either splice variant. The linked in vitro translation/transcription assay of the transfected cells and the Western blot analysis of the original tumor generated both wt (66 kDa) and Delta7 (52 kDa), Delta5,7 (46 kDa) ER(alpha) proteins.

Results: Tumor growth was characterized as estradiol and progesterone resistant but tamoxifen sensitive, i.e., neither estradiol nor progesterone treatment altered the growth rate, whereas tamoxifen treatment significantly increased the tumor volume doubling time. Estradiol treatment decreased the wt and increased the Delta7 variant ER(alpha) protein expression significantly in a dose-dependent manner. Tamoxifen treatment, however, increased the expression of both proteins whereas progesterone had no effect. Estradiol treatment did not influence expression of the Delta5,7 variant protein, which increased significantly in the tamoxifen-treated tumors. Gel mobility shift assays revealed that both wt and Delta7 ER(alpha) proteins bind to the consensus DNA sequence, whereas the Delta5,7 variant protein did not.

Conclusions: We conclude that estradiol, tamoxifen, and progesterone regulate wt and variant ER(alpha) mRNA and protein expression separately and differently and that this hormonal regulation probably occurs, via different mechanisms, at the transcriptional or posttranscriptional level. The Delta7 variant ER(alpha) may play a crucial role in the determination of hormone sensitivity and thus in the outcome of hormone treatment of human endometrial adenocarcinomas.