Loss of an estrogen receptor isoform (ER(α)Δ3) in breast cancer and the consequences of its reexpression: Interference with estrogen-stimulated properties of malignant transformation

Abstract

Comparison of mRNA ratios of a non-DNA-binding estrogen receptor (ER(α)) isoform, missing exon 3 (ER(α)Δ3), to the full-length ER(α1) in normal breast epithelium to that in primary breast cancers and breast cancer cell lines revealed a 30-fold reduction of this ratio in cancer cells (P < 0.0001). To test what functions may have been affected by the loss of ER(α)Δ3, stable clones of MCF-7 cells expressing ectopic ER(α)Δ3 protein, at the range of physiological ER(α), were generated. In vector-transfected controls the ER)α)Δ3-mRNA and protein were less than 10% while in the ER(α)Δ3-expressing clones, ER(α)Δ3-mRNA and protein ranged from 36-76% of the total ER(α). Estrogen (E2) stimulated the expression of pS2-mRNA in pMV7 vector control cells, but the stimulation was reduced by up to 93% in ER(α)Δ3-expressing clones. In addition, several properties associated with the transformed phenotype wars also strongly affected when ER(α)Δ3 protein was reexpressed. Compared with vector-transfected control cells, the set, ration density of the ER(α)Δ3-expressing clones was reduced by 50-68%, while their exponential growth rate was only slightly (14.5 ± 5%) lower. The in vivo invasiveness of the ER(α)Δ3-expressing cells was significantly reduced (P = 0.007) by up to 79%. E2 stimulated anchorage-independent growth of the pMV7 vector control cells, but reduced it to below baseline levels in ER(α3)Δ3 clones. The reduction of the pS2 response to E2 in the ER(α)Δ3- expressing clones and the E2 block of anchorage-independent growth to below baseline were more pronounced than expected from the dominant negative function of ER(α)Δ3. These observations suggest that E2 may activate an additional ER(α)Δ3-dependent inhibitory pathway. The drastic reduction of ER(α)Δ3 to ER(α) ratio in breast cancer, and the fact that when present in breast cancer cells this isoform leads to a suppression, rather than enhancement, of the transformed phenotype by E2 suggests that the regulation of ER(α)-mRNA splicing may need to be altered for the breast carcinogenesis to proceed.