CUL4B在前列腺癌恶性进展中的作用及其调控机制的研究

One of the greatest challenges of the prostate cancer (PCa) management is how to accurately distinguish indolent from aggressive tumors. Previously, We have investigated the roles and prognostic significance of the ERG and SOX4 genes in PCa progression. Most recently, we found that CUL4B, a scaffold protein in ubiquitin E3 ligase complex, is potentially regulated by ERG and involved in PCa development. Although CUL4B has been suggested to be anovel oncogene, little is known about its role in PCa. Our preliminary data indicated that CUL4B promtes invasive capacity and epithelial mesenchymal transition of PCa cells. A small cohort of clinical data suggested that the expression of CUL4B has significantly increased from benign prostatic hyperplasia to localized PCa, and from localized PCa to metastatic PCa. Additionally, CUL4B overexpression was positively correlated with the presence of clinical recurrence as well as distant metastasis. This project aims to characterize the expression and biological phenotype of CUL4B in PCa progression, evaluate the role of CUL4B in distinguishing indolent and aggressive PCa, analyze the regulatory mechanisms of CUL4B in PCa, and illustrate how CUL4B uses unique gene regulatory networks to promote PCa invasion and metastasis. This can provide a theoretical basis for understanding the biological mechanisms of CUL4B in PCa progression.

如何准确判定肿瘤的“惰性”与“侵袭性”，是前列腺癌(Prostate cancer, PCa)临床面临的重大挑战。我们前期探讨了ERG和SOX4等癌基因在PCa进展中的作用和预后判定价值，并于最近发现CUL4B可能作为ERG调控的靶基因参与PCa病变过程。CUL4B是泛素连接酶复合物的骨架蛋白，可能是一个新颖的癌基因，但在PCa中却知之甚少。我们的初步研究显示，CUL4B促进PCa细胞侵袭能力及上皮间质转化；小样本临床数据提示，CUL4B在前列腺增生、局限性PCa和转移性PCa组织中的表达依次明显升高；其过表达与PCa的临床复发和远处转移正相关。本课题拟明确CUL4B在PCa中的表达特征及生物学表型，评价其在区分“惰性”和“侵袭性”PCa中的价值；分析CUL4B参与PCa恶性进展的分子调控机制；阐明CUL4B如何影响侵袭性PCa的进展。为理解CUL4B在PCa进展中的作用机制提供理论依据。

如何准确判定肿瘤的“惰性”与“侵袭性”，是前列腺癌(Prostate cancer, PCa)临床面临的重大挑战。我们前期探讨了ERG和SOX4等癌基因在PCa进展中的作用和预后判定价值，并发现CUL4B作为ERG调控的靶基因参与PCa病变过程。CUL4B是泛素连接酶复合物的骨架蛋白，可能是一个新颖的癌基因，但在PCa中却知之甚少。本课题拟明确CUL4B在PCa中的表达特征及生物学表型，评价其在区分“惰性”和“侵袭性”PCa中的价值；分析CUL4B参与PCa恶性进展的分子调控机制；并阐明CUL4B如何影响侵袭性PCa的进展。我们的研究结果显示，CUL4B在前列腺癌中表达明显上调，其过表达与PCa的临床复发和远处转移正相关；体外实验证实，CUL4B促进PCa细胞的增殖、侵袭能力及上皮间质转化， CUL4B促进前列腺癌干细胞成干能力；CUL4B和SOX4、BMI1及C-MYC在PCa组织均表达上调并与恶性进展高度相关。机制方面：首先，染色质免疫共沉淀实验证实CUL4B与miR-204启动子结合，RT-qPCR结果发现CUL4B抑制miR-204表达，而miR-204靶向抑制SOX4。同时SOX4可通过转录激活机制上调CUL4B的表达，形成正反馈环路。此环路可进一步激活Wnt通路，进而促进PCa恶性进展；其次，对敲减CUL4B的基因表达谱芯片GSEA（Gene Set Enrichment Analysis）分析发现，CUL4B相关印记基因与干细胞表型相关。蛋白免疫印迹结果显示CUL4B促进BMI1的表达。结合miRNA芯片，靶基因预测软件发现CUL4B转录抑制miR200b/c的表达。PCa中CUL4B的高表达降低了miR200b/c对BMI1及干细胞标志物的抑制作用；最后，染色质免疫共沉淀实验证实CUL4B可通过结合miR-33b上游启动子区域抑制miR-33b的转录，而荧光素酶实验证实miR-33b-5p可直接靶向C-MYC 3’UTR区，生物学功能实验显示miR-33b-5p可通过下调C-MYC蛋白表达进而抑制CUL4B的促癌活性。综上，CUL4B可以通过转录抑制miR-204、miR200b/c及miR-33b-5p的表达，从而促进SOX4、BMI1及C-MYC的表达，促进PCa进展。本研究为理解CUL4B在PCa进展中的作用机制及寻找治疗靶点提供理论依据。