Steele Thomas, Sam Anhao, Evans Shawna, Browning Elizabeth, Krig Sheryl, Macias Katelyn, Konda Adarsh, Siddiqui Salma, Durbin-Johnson Blythe, Ghosh Paramita, Vinall Ruth
Department of Pharmaceutical & Biomedical Sciences, California Northstate University College of Pharmacy, Elk Grove, CA 95757, USA.
Department of Urological Surgery, School of Medicine, University of California Davis, 4860 Y Street, Sacramento, CA 95817, USA.
Cancers (Basel). 2021 Sep 2;13(17):4425. doi: 10.3390/cancers13174425.
To our knowledge, our group is the first to demonstrate that NRDP1 is located in the nucleus as well as the cytoplasm of CaP cells. Subcellular fractionation, immunohistochemistry, and immunofluorescence analysis combined with confocal microscopy were used to validate this finding. Subcellular fractionation followed by western blot analysis revealed a strong association between AR and NRDP1 localization when AR expression and/or cellular localization was manipulated via treatment with R1881, AR-specific siRNA, or enzalutamide. Transfection of LNCaP with various NRDP1 and AR constructs followed by immunoprecipitation confirmed binding of NRDP1 to AR is possible and determined that binding requires the hinge region of AR. Co-transfection with NRDP1 constructs and HA-ubiquitin followed by subcellular fractionation confirmed that nuclear NRDP1 retains its ubiquitin ligase activity. We also show that increased nuclear NRDP1 is associated with PSA recurrence in CaP patients (n = 162, odds ratio; 1.238, = 0.007) and that higher levels of nuclear NRDP1 are found in castration resistant cell lines (CWR22Rv1 and PC3) compared to androgen sensitive cell lines (LNCaP and MDA-PCa-3B). The combined data indicate that NRDP1 plays a role in mediating CaP progression and supports further investigation of both the mechanism by which nuclear transport occurs and the identification of specific nuclear targets.
据我们所知，我们的研究小组是首个证明NRDP1定位于前列腺癌细胞的细胞核和细胞质中的团队。采用亚细胞分级分离、免疫组织化学和免疫荧光分析并结合共聚焦显微镜来验证这一发现。通过用R1881、AR特异性小干扰RNA（siRNA）或恩杂鲁胺处理来操控AR表达和/或细胞定位后，进行亚细胞分级分离，随后的蛋白质免疫印迹分析显示AR与NRDP1定位之间存在强关联。用各种NRDP1和AR构建体转染LNCaP细胞，随后进行免疫沉淀，证实NRDP1与AR之间可能存在结合，并确定这种结合需要AR的铰链区。用NRDP1构建体和HA-泛素共转染，随后进行亚细胞分级分离，证实细胞核中的NRDP1保留其泛素连接酶活性。我们还表明，细胞核中NRDP1增加与前列腺癌患者（n = 162，比值比；1.238，P = 0.007）的PSA复发相关，并且与雄激素敏感细胞系（LNCaP和MDA-PCa-3B）相比，在去势抵抗细胞系（CWR22Rv1和PC3）中发现细胞核NRDP1水平更高。综合数据表明，NRDP1在介导前列腺癌进展中起作用，并支持对核转运发生机制以及特定核靶点鉴定的进一步研究。
