Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, USA.
Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, USA.
Nucleic Acids Res. 2023 Apr 11;51(6):2655-2670. doi: 10.1093/nar/gkad043.
Overexpression of androgen receptor (AR) is the primary cause of castration-resistant prostate cancer, although mechanisms upregulating AR transcription in this context are not well understood. Our RNA-seq studies revealed that SMAD3 knockdown decreased levels of AR and AR target genes, whereas SMAD4 or SMAD2 knockdown had little or no effect. ChIP-seq analysis showed that SMAD3 knockdown decreased global binding of AR to chromatin. Mechanistically, we show that SMAD3 binds to intron 3 of the AR gene to promote AR expression. Targeting these binding sites by CRISPRi reduced transcript levels of AR and AR targets. In addition, ∼50% of AR and SMAD3 ChIP-seq peaks overlapped, and SMAD3 may also cooperate with or co-activate AR for AR target expression. Functionally, AR re-expression in SMAD3-knockdown cells partially rescued AR target expression and cell growth defects. The SMAD3 peak in AR intron 3 overlapped with H3K27ac ChIP-seq and ATAC-seq peaks in datasets of prostate cancer. AR and SMAD3 mRNAs were upregulated in datasets of metastatic prostate cancer and CRPC compared with primary prostate cancer. A SMAD3 PROTAC inhibitor reduced levels of AR, AR-V7 and AR targets in prostate cancer cells. This study suggests that SMAD3 could be targeted to inhibit AR in prostate cancer.
雄激素受体 (AR) 的过度表达是去势抵抗性前列腺癌的主要原因，尽管在这种情况下上调 AR 转录的机制尚未完全理解。我们的 RNA-seq 研究表明，SMAD3 敲低降低了 AR 和 AR 靶基因的水平，而 SMAD4 或 SMAD2 敲低几乎没有影响。ChIP-seq 分析表明，SMAD3 敲低降低了 AR 与染色质的整体结合。在机制上，我们表明 SMAD3 结合到 AR 基因的内含子 3 以促进 AR 表达。通过 CRISPRi 靶向这些结合位点可降低 AR 和 AR 靶基因的转录水平。此外，约 50%的 AR 和 SMAD3 ChIP-seq 峰重叠，SMAD3 可能还与 AR 合作或共同激活以表达 AR 靶基因。功能上，在 SMAD3 敲低细胞中重新表达 AR 部分挽救了 AR 靶基因表达和细胞生长缺陷。AR 内含子 3 中的 SMAD3 峰与前列腺癌数据集的 H3K27ac ChIP-seq 和 ATAC-seq 峰重叠。与原发性前列腺癌相比，转移性前列腺癌和 CRPC 的 AR 和 SMAD3 mRNA 上调。SMAD3 PROTAC 抑制剂降低了前列腺癌细胞中 AR、AR-V7 和 AR 靶基因的水平。这项研究表明，SMAD3 可能成为抑制前列腺癌中 AR 的靶点。
