Detection of Genome-Wide IGF-1R Recruitment to Enhancer and Promoter Regions of Chromatin in Clinical Prostate Cancers.
Mills JV., Taylor A., Singh R., Kim J., Engledow S., Colling R., Verrill C., Mills IG., Macaulay VM.
IntroductionNuclear insulin-like growth factor-1 receptor (IGF-1R) undergoes IGF-induced recruitment to cancer cell chromatin in vitro and associates with advanced prostate cancer (PCa) stage in clinical tissue, prompting this investigation of IGF-1R chromatin recruitment in vivo.MethodsHuman tissues surplus to diagnostic need were obtained from consenting patients undergoing transurethral resection of the prostate (TURP) or radical prostatectomy (RP). Initial tissue samples were processed for H3K4me1-positive control ChIP to optimise homogenisation, fixation and ChIP conditions. Following successful method optimization, IGF-1R and H3K4me1 ChIP-seq was performed on six treatment-naïve localized PCa samples, along with parallel IGF-1R immunohistochemistry analysis. MACS2 and LanceOtron peak callers were used to identify binding sites from ChIP-seq data and MEME Suite was used to identify an IGF-1R binding motif. In vitro chromatin immunoprecipitation qPCR (ChIP-qPCR) was used for ChIP-seq data validation.ResultsWe identified 5743 unique IGF-1R binding sites, with 37% within 3 kb of gene transcription start sites (TSSs). Of these sites, 72.3% coincided with enhancer mark H3K4me1, suggesting regulatory function. Motif analysis identified an IGF-1R consensus binding motif for the first time, with a sequence resembling that of the insulin receptor and PITX2 transcription factor binding motifs, supporting functional similarities. In vitro ChIP-qPCR confirmed IGF-1R recruitment to a site identified in vivo in the RRM2 TSS, a gene involved in DNA replication and repair and regulated by the IGF-axis, highlighting potential regulatory function of nuclear IGF-1R.ConclusionOverall, these data represent the first characterization of genome-wide IGF-1R recruitment in PCa tissue and are consistent with a transcriptional regulatory role, further elucidating the contribution of nuclear IGF-1R to advanced clinical stage.