Department of Pathology, Division of Human Pathology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan.
Department of Pathology, Division of Human Pathology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan.
J Steroid Biochem Mol Biol. 2023 Dec;235:106407. doi: 10.1016/j.jsbmb.2023.106407. Epub 2023 Oct 6.
Castration-resistant prostate cancer (CRPC) is a big challenge in managing prostate cancer patients. The androgen receptor (AR) pathway is a major driver even in CRPC under androgen deprivation. The mechanism in maintaining of the AR pathway under androgen deprivation remains elusive. The recent discovery of biomolecular condensate, a membrane-less intracellular construct formed by liquid-liquid phase separation (LLPS), that facilitate molecular assembly, encouraged the re-screening of our previous microarray data list. We selected Rbm14 as a target molecule for further analysis because it works as a coactivator of nuclear receptors as well as it facilitates formation of biomolecular condensates via its intrinsically disordered region. GFP-tagged Rbm14 transfected into HEK293T cells formed droplet-like puncta, which diminished following treatment with 1,6-hexanediol. Droplet-like structures were also observed in immunofluorescence for endogenous RBM14 of PC-3 and DU145 cells. Luciferase assay revealed that Rbm14 enhanced androgen-responsive element (ARE)-mediated reporter activity in all conditions with or without testosterone and AR. Co-immunoprecipitation confirmed the Rbm14-AR interaction. Long non-coding RNAs, including NEAT1, SRA1, and HOTAIR, were also interacted with Rbm14. Small interfering RNAs of NEAT1 reduced ARE-mediated reporter activity, while transfection of SRA1 and HOTAIR enhance the reporter activity. Treatment with 1,6-hexanediol as well as transfection with a dominant-negative splice variant of Rbm14 reduced expression of prostate specific antigen (PSA), a prototype of androgen-regulated gene, in LNCaP, PC-3, and DU145 cells under androgen deprivation. Immunohistochemically, RBM14 expression was substantially upregulated in prostate cancer tissues after androgen deprivation therapy than in untreated tumors. In conclusion, RBM14 is a novel factor involved in maintenance of PSA expression via phase separation under androgen deprivation in prostate cancer.
去势抵抗性前列腺癌（CRPC）是管理前列腺癌患者的一大挑战。即使在雄激素剥夺的情况下，雄激素受体（AR）通路也是主要驱动因素。在雄激素剥夺下维持 AR 通路的机制仍然难以捉摸。最近发现的生物分子凝聚物是一种由液-液相分离（LLPS）形成的无膜细胞内结构，促进分子组装，这鼓励我们重新筛选以前的微阵列数据列表。我们选择 Rbm14 作为进一步分析的靶分子，因为它作为核受体的共激活因子起作用，并且通过其无规卷曲区域促进生物分子凝聚物的形成。转染 GFP 标记的 Rbm14 的 HEK293T 细胞形成类似液滴的点状结构，在用 1,6-己二醇处理后减少。PC-3 和 DU145 细胞的内源性 RBM14 的免疫荧光也观察到类似液滴的结构。荧光素酶测定显示，Rbm14 增强了所有条件下雄激素反应元件（ARE）介导的报告基因活性，无论是否有睾酮和 AR。共免疫沉淀证实了 Rbm14-AR 相互作用。长非编码 RNA，包括 NEAT1、SRA1 和 HOTAIR，也与 Rbm14 相互作用。NEAT1 的小干扰 RNA 降低了 ARE 介导的报告基因活性，而 SRA1 和 HOTAIR 的转染则增强了报告基因活性。用 1,6-己二醇处理以及转染 Rbm14 的显性负剪接变体降低了雄激素剥夺下 LNCaP、PC-3 和 DU145 细胞中前列腺特异性抗原（PSA）的表达，PSA 是雄激素调节基因的原型。免疫组织化学分析显示，与未经治疗的肿瘤相比，去势抵抗性前列腺癌组织中 RBM14 的表达在去势抵抗性前列腺癌治疗后显著上调。总之，RBM14 是一种通过雄激素剥夺下的相分离维持 PSA 表达的新型因子。
