Many human diseases are caused by dysregulated gene expression. The oversupply or over activity of one or more transcription factors (TF) may be required for the survival, growth, and metastatic behavior of all human cancers, and therefore provide an attractive therapeutic target. The NCI estimates that in the US alone 217,730 men will be diagnosed with and 32,050 men will die of prostate cancer in 2010 (6). Androgen receptor (AR) is the aberrant TF responsible for expression changes leading to prostate cancer. Preliminary ChIP-Seq data on androgen treated LNCaP cells, a prostate cancer cell line, revealed a novel motif sequence, an androgen response element (ARE) plus a forkhead (Fkhd) response element (FHRE) separated by 4 base pairs (bps), enriched relative to background genomic data. This novel IS plus FHRE motif is suggestive of co-occupancy of DNA by AR and a forkhead TF to regulate specific genes in an unprecedented regime. Forkhead TFs are integral to a number of cancer related cellular functions, such as metabolism, development, proliferation, and apoptosis, and therefore may prove important to understanding prostate cancer (17). To demonstrate that co-occupancy of AR and a Fkhd is possible, an electrophoretic mobility shift assay (EMSA) will be employed. Recombinant human AR and human FoxA1, a candidate forkhead family member believed to interact with AR, will be incubated with a 30 bp DNA fragment containing the ARE plus FHRE motif (18). The cooperatively of the AR:Fkhd interaction will be quantitated using DNase I footprint titrations, and then repeated with truncated forms of the TFs in order to determine which domains are responsible for the protein-protein or allosteric interactions. The biophysical importance of the 4 bp spacer to the interaction will be assessed by repeating the EMSA experiments with DNA fragments containing spacers ranging from 0 to 8 bps. Pyrrole- imidazole (Py-Im) polyamides, sequence specific DNA minor-groove binding molecules, will be designed to disrupt the Fkhd:DNA interface to probe the in vivo role of the AR:Fkhd interaction. The effects of disrupting the Fkhd:DNA or AR:DNA interactions in genes containing the ARE plus FHRE motif in an enhancer or promoter region will be tested using qRT-PCR, ChIP-Seq, and RNA-Seq on LNCaP cells treated with an androgen, the designed Fkhd polyamides, and existing AR:DNA disrupting polyamides. qRT-PCR experiments will be used to test the ability of the polyamides to effectively disrupt the Fkhd:DNA and ARE:DNA interactions, as well as test the hypothesis that disruption of either AR or the Fkhd will restore mRNA expression to basal levels in genes containing the ARE plus FHRE motif. In order to understand the genome wide roles of the AR:Fkhd:DNA interaction on transcriptional programs, ChIP-Seq and RNA-Seq experiments will be performed on LNCaP cells treated with polyamides to disrupt each individual interaction. Biophysical characterization of this novel transcription factor complex combined with genome-wide characterization of genes regulated by AR:Fkhd using polyamides can reveal a new target for prostate cancer drug development.
This work offers fundamental data on a novel transcriptional regime implicated in prostate cancer. Biophysical studies of the interaction of two different transcription factors, androgen receptor and a forkhead family member, could help reveal a new critical target for prostate cancer therapeutics. Perturbation of this protein- protein-DNA ternary complex by small molecules will be explored.
|Hargrove, Amanda E; Martinez, Thomas F; Hare, Alissa A et al. (2015) Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide. PLoS One 10:e0143161|
|Martínez, Thomas F; Phillips, John W; Karanja, Kenneth K et al. (2014) Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response. Nucleic Acids Res 42:11546-59|