INDUCTION OF TRANSFORMING-GROWTH-FACTOR-BETA RECEPTOR-TYPE-II EXPRESSION IN ESTROGEN RECEPTOR-POSITIVE BREAST-CANCER CELLS THROUGH SP1 ACTIVATION BY 5-AZA-2'-DEOXYCYTIDINE

Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-beta (`T GF-beta) because of reduced expression levels of TGF-beta receptor typ e II (RII). We now report that treatment of ER+ breast cancer cells wi th the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2 '-dC) leads to accumulation of RII transcript and protein in three dif ferent cell lines. RII induction restored TGF-beta response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-bet a responsive promoter-reporter construct (p3TP-Lux). A transiently tra nsfected RII promoter-reporter element (RII-chloramphenicol acetyltran sferase) showed an increase in activity in the 5-aza-2'-dC-treated MCF -7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding: of proteins from 5-aza-2'-dC-treat ed MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was de monstrated. An RII promoter-chloramphenicol acetyltransferase construc t containing a mutation in the Sp1 site was not expressed in the 5-aza -2'-dC-treated MCF-7L cells, further demonstrating that induction of S p1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII exp ression. Northern analysis indicated that 5-aza-2'-dC treatment did no t affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2'-dC-treated MCF-7L cells, but t here was no change in the c-Jun levels. Studies after cyclohexamide tr eatment suggested an increase in the Sp1 protein stability from the 5- aza-2'-dC-treated MCF-7L extracts compared with untreated control extr acts. These results indicate that the transcriptional repression of RI I in the ER+ breast cancer cells is caused by suboptimal activity of S p1, whereas treatment with 5-aza-2'-dC stabilizes the protein thus inc reasing steady-state Sp1 levels and thereby leads to enhanced RII tran scription and subsequent restoration of TGF-beta sensitivity.