METHODS: We applied integrated genomic and clinical data, experimental models and bioinformatic analysis to identify GATA2 as a highly prevalent metastasis-associated genomic amplification in PCa. Biological roles of GATA2 in PCa metastasis was determined in vitro and in vivo. Global chromatin co-occupancy and co-regulation of GATA2 and SMAD4 was investigated by coimmunoprecipitation, ChIP-seq and RNA-seq assays. Tumor cellular assays, qRT-PCR, western blot, ChIP, luciferase assays and CRISPR-Cas9 editing methods were performed to mechanistically understand the cooperation of GATA2 with SMAD4 in promoting TGFβ1 and AR signaling and mediating inherited PCa risk and progression.
RESULTS: In this study, by integrated genomics and experimental analysis, we identified GATA2 as a prevalent metastasis-associated genomic amplification to transcriptionally augment its own expression in PCa. Functional experiments demonstrated that GATA2 physically interacted and cooperated with SMAD4 for genome-wide chromatin co-occupancy and co-regulation of PCa genes and metastasis pathways like TGFβ signaling. Mechanistically, GATA2 was cooperative with SMAD4 to enhance TGFβ and AR signaling pathways, and activated the expression of TGFβ1 via directly binding to a distal enhancer of TGFβ1. Strinkingly, GATA2 and SMAD4 globally mediated inherited PCa risk and formed a transcriptional complex with HOXB13 at the PCa risk-associated rs339331/6q22 enhancer, leading to increased expression of the PCa susceptibility gene RFX6.
CONCLUSIONS: Our study prioritizes causal genomic amplification genes with prognostic values in PCa and reveals the pivotal roles of GATA2 in transcriptionally activating the expression of its own and TGFβ1, thereby co-opting to TGFβ1/SMAD4 signaling and RFX6 at 6q22 to modulate PCa predisposition and progression.
方法:我们应用了整合的基因组和临床数据,实验模型和生物信息学分析,以确定GATA2是PCa中高度普遍的转移相关基因组扩增。在体外和体内确定了GATA2在PCa转移中的生物学作用。通过共免疫沉淀研究了GATA2和SMAD4的全局染色质共占据和共调节,ChIP-seq和RNA-seq测定。肿瘤细胞检测,qRT-PCR,westernblot,ChIP,进行了荧光素酶测定和CRISPR-Cas9编辑方法,以机械地了解GATA2与SMAD4在促进TGFβ1和AR信号传导以及介导遗传PCa风险和进展方面的合作。
结果:在这项研究中,通过整合基因组学和实验分析,我们确定GATA2是一种普遍的转移相关基因组扩增,可在转录上增强其在PCa中的表达.功能实验表明,GATA2与SMAD4物理相互作用并合作,以实现全基因组染色质共占据和共调节PCa基因和转移途径,如TGFβ信号传导。机械上,GATA2与SMAD4协同增强TGFβ和AR信号通路,并通过直接与TGFβ1的远端增强子结合来激活TGFβ1的表达。令人发指的是,GATA2和SMAD4全球介导遗传PCa风险,并在PCa风险相关的rs339331/6q22增强子上与HOXB13形成转录复合物,导致PCa易感基因RFX6的表达增加。
结论:我们的研究优先考虑了在PCa中具有预后价值的因果基因组扩增基因,并揭示了GATA2在转录激活其自身和TGFβ1表达中的关键作用,从而共同选择TGFβ1/SMAD4信号和RFX6在6q22调节PCa易感性和进展。