Selective inhibition of transcription of the Ets2 gene in prostate cancer cells by a triplex-forming oligonucleotide.
The transcription factor Ets2 has a role in cancer development and represents an attractive therapeutic target. In this study, we designed a triplex-forming oligonucleotide (TFO) directed to a homopurine:homopyrimidine sequence in the Ets2 promoter. Transcription factors of the Sp family bound to this sequence and mutation of the Sp1 site reduced Ets2 promoter activity. The Ets2-TFO had high binding affinity for the target sequence and inhibited binding of Sp1/Sp3 to the overlapping site. This effect occurred with a high degree of sequence specificity. Mismatched oligonucleotides did not inhibit Sp1/Sp3 binding and mutations in the target sequence that abolished triplex formation prevented inhibition of Sp1/Sp3 binding by the TFO. The Ets2-TFO inhibited Ets2 promoter activity and expression of the endogenous gene in prostate cancer cells at nanomolar concentrations. The TFO did not affect reporter constructs with mutations in the TFO binding site and promoters of non-targeted genes. Expression of non-targeted genes was also not affected in TFO-treated cells. Collectively, these data demonstrated that the anti-transcriptional activity of the Ets2-TFO was sequence- and target-specific, and ruled out alternative, non-triplex mediated mechanisms of action. This anti-transcriptional approach may be useful to examine the effects of selective downregulation of Ets2 expression and may have therapeutic applications.
Carbone
Giuseppina M
GM
Laboratory of Cancer Genomics, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA. carbonep@musc.edu
McGuffie
Eileen M
EM
Collier
Angela
A
Catapano
Carlo V
CV
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008297:Male; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D012334:RNA, Neoplasm / Q000096:biosynthesis; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051704:Sp2 Transcription Factor; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; D009841:Oligonucleotides; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012334:RNA, Neoplasm; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; C051802:triplex DNA; D051704:Sp2 Transcription Factor; D004247:DNA