title: 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.
abstract: Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-beta (TGF-beta) because of reduced expression levels of TGF-beta receptor type II (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2'-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-beta response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-beta responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII-chloramphenicol acetyltransferase) 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-treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct 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 Sp1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2'-dC treatment did not 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 there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2'-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2'-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-beta sensitivity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ammanamanchi
authors.forename: S J
authors.initials: SJ
authors.lastname: Kim
authors.forename: L Z
authors.initials: LZ
authors.lastname: Sun
authors.forename: M G
authors.initials: MG
authors.lastname: Brattain
mesh_terms: D030201:Activin Receptors, Type I; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism*; D000077209:Decitabine; D005260:Female; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D011960:Receptors, Estrogen / Q000032:analysis*; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D011960:Receptors, Estrogen; D018125:Receptors, Transforming Growth Factor beta; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; D007335:Insulin-Like Growth Factor II; D000077209:Decitabine; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D001374:Azacitidine