Systems analysis of the prostate tumor suppressor NKX3.1 substantiates roles in DNA repair and a transcriptional program that drives luminal cell differentiation

NKX3.1 is an androgen-regulated, prostate-specific gene located on chromosome 8p21, a region frequently undergoing allelic loss in human prostate cancer. Mice deficient in NKX3.1 show signs of epithelial de-differentiation and develop dysplasia and prostatic intraepithelial neoplasia (PIN) that progresses to overt prostate cancer when combined with deletions of additional tumor suppressors such as PTEN or p27Kip1. Although NKX3.1 displays the typical features of an NK class homeobox transcription factor, mechanisms of NKX3.1-mediated tumor suppression remain insufficiently understood because neither the transcriptional program governed by NKX3.1 nor its interacting proteins have been comprehensively revealed. Adenoviruses were created that constitutively express either GFP alone, or GFP and NKX3.1 (Ad-GFP and Ad-GFP-NKX3.1 viruses, respectively). PrEC LH cells were infected with these viruses and were harvested at 7h and 10h after infection. Using affinity purification and tandem mass spectrometry, we have established an extensive NKX3.1 interactome. In particular, we found that the transcription factor forms stable complexes with the DNA repair proteins Ku70, Ku80, and PARP, interactions that provide a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of immortalized human prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human PIN devoid of NKX3.1. Pathway and network analyses suggested that NKX3.1 actuates a fundamental cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-Myc and interferon-STAT signaling and activation of tumor suppressor pathways.

NKX3.1是一种受雄激素调控的前列腺特异性基因，定位于8号染色体8p21区域，该区域在人类前列腺癌中常发生等位基因缺失。NKX3.1基因缺陷小鼠可出现上皮去分化征象，并发展出异型增生与前列腺上皮内瘤变（prostatic intraepithelial neoplasia，PIN）；当联合PTEN或p27Kip1等其他肿瘤抑制基因缺失时，此类病变可进展为显性前列腺癌。尽管NKX3.1具备NK类同源盒转录因子的典型特征，但其介导的肿瘤抑制机制仍未得到充分阐明——这是由于其调控的转录程序以及互作蛋白均未被全面揭示。本研究构建了两类组成型表达的腺病毒：仅表达绿色荧光蛋白（GFP）的腺病毒，以及同时表达GFP与NKX3.1的腺病毒（分别记为Ad-GFP与Ad-GFP-NKX3.1）。将上述病毒感染PrEC LH细胞，并于感染后7小时、10小时收集细胞样本。通过亲和纯化与串联质谱技术，本研究构建了全面的NKX3.1互作组图谱。尤为关键的是，本研究发现该转录因子可与DNA修复蛋白Ku70、Ku80及PARP形成稳定复合物，这一相互作用为此前关于NKX3.1参与DNA修复的研究报道提供了分子层面的依据。对瞬时表达NKX3.1的永生化人类前列腺上皮细胞进行转录组分析后发现，其呈现出快速且复杂的应答反应，该应答与缺失NKX3.1的人类PIN的基因表达特征近乎镜像对应。通路与网络分析结果表明，NKX3.1可驱动细胞向腔上皮细胞分化进行根本性重编程，其特征为抑制促癌基因c-Myc与干扰素-STAT信号通路，并激活肿瘤抑制通路。