ONECUT2 Drives Neuroendocrine Prostate Cancer Through Hypoxia Signaling

To determine the underlying mechanism of ONECUT2 in prostate cancer hypoxia, we conducted a series of RNA-Seq and ChIP-Seq experiments in LNCaP and PC3 cells under normoxia and hypoxia conditions. We did RNA-Seq in LNCaP cells with or without OC2 overexpression and in PC3 cells with or without OC2 knockdown. We used anti-Flag antibody to perform the ChIP-Seq experiment in PC3 cells with Flag and OC2 fusion protein overexpression. We also performed HIF1A ChIP-Seq in AR-negative prostate cancer cell line PC3 under hypoxia condition with or without ONECUT2 or SMAD3 siRNA knockdown. SMAD3 and HIF2A ChIP-Seq were conducted in PC3 cells under hypoxia condition. To confirm the interactions between transcription factors, we also performed ChIP-reChIP-seq. We did the primary ChIP experiment using anti-SMAD3 antibody and then we subjected the ChIPed chromatin by the primary ChIP to reChIP experiments using anti-HIF1A or anti-HIF2A antibody. The reChIPed DNA was submitted to next generation sequencing. Profiling transcription factor binding and gene expression related with OC2 function in hypoxia response of prostate cancer