the mechanism of ligand-independent activation of estrogen receptor in letrozole-resistant breast cancer cells

Objective　To investigate the functions and mechanisms of transcriptional regulation changes in estrogen receptor (ER) on letrozole resistance in breast cancer cells. Methods　Letrozole resistant cells (MCF-7-LR-1 and MCF-7¬LR-2) and control cells (MCF-7-Aro and MCF-7-T) were used in present study. The levels of ER phosphorylation at Ser-118 and expressions of ER in these two groups were determined by Western blotting. The conjugation of ER with XBP-1 enhancer and TFF-1 promoter with or without β-Estradiol (E2) was detected by Chromatin Immuno-coprecipitation (ChIP) assay. The mRNA expression levels of XBP-1 and TFF-1 were determined by quantitative reverse transcription PCR (qRT-PCR) analysis. Furthermore, the mRNA expressions of estrogen responsive genes (ERGs) including CA2, GJA1, PGR, and CTSD were assayed also by qRT-PCR analysis. Results　The level of ER phosphorylation at Ser-118 significantly increased in MCF-7-LR cells than in control cells. However, the ER expression showed no significant change. ChIP assay demonstrated that, under ligand-independent (without E2 treatment) or ligand-dependent (with E2 treatment 10nmol/L E2 treatment for 45min) condition, the conjugation of ER with XBP¬1 enhancer and TFF-1 promoter significantly increased in MCF-7-LR-2 cells than in control cells. qRT-PCR analysis showed that the mRNA expressions of XBP-1 and TFF-1 increased in MCF-7-LR-2 cells compared with those in MCF-7-Aro cells without E2 treatment. Furthermore, the mRNA expressions of CA2 and GJA1 increased, whereas the mRNA expressions of PGR and CTSD decreased in MCF-7-LR-2 cells versus MCF-7-T cells. Conclusion　The level of ER phosphorylation at Ser-118 increased in a ligand-independent manner, thereby promoting the transcription of target genes in letrozole resistant breast cancer cells, and it plays an important role in letrozole resistance.