Abstract
Breast cancer-specific gene 1 (BCSG1) is not expressed in normal breast tissue but is highly expressed in the vast majority of invasive and metastatic breast carcinomas. When over-expressed, BCSG1 significantly stimulates the proliferation and invasion of breast cancer cells. The accumulated evidence suggests that the aberrant expression of BCSG1 in breast carcinomas is caused by transcriptional activation of the BCSG1 gene. However, the transcription factors that activate BCSG1 transcription have not been identified. In this study, we extensively investigated the role of AP1 in BCSG1 expression in breast cancer cells. We demonstrate that there are two closely located AP1 binding sites residing in the first intron of the BCSG1 gene. Mutation of either AP1 motif on the BCSG1 promoter constructs markedly reduces the promoter activity. We further show that 12-O-tetradecanoylphorbol-13-acetate (TPA) increases BCSG1 mRNA expression and up-regulates BCSG1 promoter activity through the intronic AP1 sites. The effect of TPA on BCSG1 transcription is also demonstrated under in vivo conditions in intact cells by using chromatin immunoprecipitation assays that show the TPA-induced binding of c-Jun to the chromatin region encompassing the intronic AP1 sites. Finally, to examine the direct effect of AP1 transactivation on BCSG1 expression, we established stable cell lines of T47D that express the dominant negative mutant of c-Jun, TAM67. RT-PCR and Western blot analyses demonstrated that levels of BCSG1 mRNA and protein in TAM67 transfectants were drastically reduced as compared with mock-transfected cells. Furthermore, inhibition of BCSG1 expression by blocking AP1 transactivation produced a similar repressive effect on cell growth as that by expressing BCSG1 antisense mRNA. We show that the anchorage-independent growth of T47D cells expressing either TAM67 or BCSG1 antisense mRNA is significantly inhibited. Taken together, we provide strong evidence that AP1 plays an overriding role in the transcription of the BCSG1 gene and that blockade of AP1 transactivation down-regulates BCSG1 expression and suppresses tumor phenotype.
Highlights
To elucidate the molecular and cellular mechanisms that control Breast cancer-specific gene 1 (BCSG1) transcription in breast cancer cells, we recently isolated a 2.2-kb fragment of human BCSG1 gene that includes 1 kb of the 5Ј-flanking region, exon 1, and intron 1 (7)
Mutation at either AP1 site markedly reduced the promoter activity of BCSG1967 in all three cell lines. It appeared that disruption of the consensus AP1 site (AP1-MU2) produced a stronger inhibitory effect on BCSG1 transcription than the mutation at the AP1 homologous sequence (AP1MU1)
These results demonstrate that AP1 motifs located on the sense strand and the antisense strand of intron 1 are both required for BCSG1 transcription
Summary
To elucidate the molecular and cellular mechanisms that control BCSG1 transcription in breast cancer cells, we recently isolated a 2.2-kb fragment of human BCSG1 gene that includes 1 kb of the 5Ј-flanking region, exon 1, and intron 1 (7). In vivo genomic bisulfite sequencing demonstrates that the BCSG1 CpG island is totally unmethylated in BCSG1positive SKBR-3 and T47D cells but partially methylated in BCSG1-negative MCF-7 and HepG2 cells (7). This suggests that DNA demethylation may be an important factor contributing to the aberrant expression of BCSG1 in breast cancer cells. Further deletion analysis localized a consensus AP1 binding site (TGACTCA) in the intron that was largely responsible for the increased promoter activity (7) These previous studies suggest that the activator protein AP1 may regulate BCSG1 transcription, possibly through the intronic AP1 binding site. Our studies clearly demonstrate that BCSG1 is a new target gene of AP1 and that AP1 plays an overriding role in BCSG1 transcription in breast cancer cells
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