PO.CL01.22 · 临床研究
PHF8是前列腺癌细胞中RIPK2的主要上游调控因子
PHF8 is a major upstream regulator of RIPK2 in prostate cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：尽管已取得重大进展，前列腺癌（PC）仍是男性癌症相关死亡的主要原因之一，凸显了对新治疗策略的迫切需求。我们此前的研究表明，RIPK2在晚期PC中过表达，并通过激活一条非经典的RIPK2/MKK7/c-Myc信号通路促进疾病进展和转移（《Nature Communications》，2022）。然而，除基因扩增外，PC细胞中RIPK2上调的机制仍不清楚。本研究旨在鉴定调控PC细胞中RIPK2表达的关键转录因子。
方法：用三种NF-κB调节剂处理PC细胞22Rv1和PC3，并通过RT-qPCR检测RIPK2和NF-κB靶基因的表达。通过整合CHEA、ENCODE、JASPAR和DoRothEA数据库鉴定候选RIPK2转录因子，并利用PCTA和TCGA相关性以及ENCODE ChIP-Seq数据进行排序。在八种前列腺细胞系中通过RT-qPCR和western blotting评估PHF8的表达及其与RIPK2的相关性。通过CRISPR/Cas9在两种PHF8高表达细胞系（22Rv1、PC3）中敲除PHF8，并使用慢病毒表达野生型（WT）、去甲基化酶失活型（H283A）进行回补表达。采用双荧光素酶报告基因实验评估PHF8及其酶活性对RIPK2启动子活性的影响。建立多重荧光免疫组织化学实验，以评估临床组织标本中肿瘤细胞内PHF8与RIPK2的相关性。
结果：NF-κB降低RIPK2 mRNA水平，表明NF-κB在PC细胞中作为RIPK2的转录抑制因子而非转录激活因子发挥作用。整合分析鉴定出156个候选RIPK2转录因子，其中七个在PCTA和TCGA两个队列中均与RIPK2 mRNA持续相关（平均rho > 0.25）。其中，PHF8在ENCODE ChIP-Seq数据中显示出强烈的RIPK2启动子结合。Western blotting证实，在八种PC细胞系中PHF8与RIPK2蛋白高度相关，其中PHF8在22Rv1和PC3中含量最高。对这些细胞进行遗传学或药理学PHF8抑制显著降低了RIPK2前体mRNA、mRNA和蛋白水平，而在RWPE-2和LNCaP中过表达PHF8则以酶活性依赖和剂量依赖的方式增加RIPK2。在临床组织标本中，PHF8与RIPK2蛋白水平在前列腺肿瘤细胞中显著相关。
结论：我们的研究结果鉴定出PHF8（而非NF-κB）是PC细胞中RIPK2的主要转录调控因子，并证明PHF8的去甲基化酶活性对其转录调控RIPK2至关重要。
查看英文原文 English abstract
Background: Despite significant progress, prostate cancer (PC) remains a leading cause of cancer-related death in men, underscoring the urgent need for new therapeutic strategies. Our previous study demonstrated that RIPK2 is overexpressed in advanced PC and promotes disease progression and metastasis by activating a noncanonical RIPK2/MKK7/c-Myc signaling pathway ( Nature Communications , 2022). However, beyond gene amplification, the mechanisms underlying RIPK2 upregulation in PC cells remain elusive. The present study aims to identify the key transcription factor regulating RIPK2 expression in PC cells.
Methods: PC 22Rv1 and PC3 cells were treated with three NF-κB modulators, and RIPK2 and NF-κB target gene expression was measured by RT-qPCR. Candidate RIPK2 transcription factors were identified by integrating CHEA, ENCODE, JASPAR, and EoRothEA databases, and ranked using PCTA and TCGA correlations plus ENCODE ChIP-Seq data. PHF8 expression and its correlation with RIPK2 were assessed by RT-qPCR and western blotting in eight prostate cell lines. PHF8 was knocked out in two PHF8-high lines (22Rv1, PC3) via CRISPR/Cas9, and re-expressed using lentiviral wild-type (WT), demethylase-inactive (H283A. A dual-luciferase reporter assay was performed to evaluate the effect of PHF8 and its enzymatic activity on RIPK2 promoter activity. A multiplexed fluorescence immunohistochemistry assay was developed to assess the correlation between PHF8 and RIPK2 in tumor cells within clinical tissue specimens.
Results: NF-κB decreased RIPK2 mRNA levels, indicating that NF-κB acts as a transcriptional repressor rather than a transcriptional activator of RIPK2 in PC cells. Integrative analysis identified 156 candidate RIPK2 transcription factors, with seven consistently correlated with RIPK2 mRNA (average rho > 0.25) in both PCTA and TCGA cohorts. Among these, PHF8 showed strong RIPK2 promoter binding in ENCODE ChIP-Seq data. Western blotting confirmed a strong PHF8-RIPK2 protein correlation across eight PC cell lines, with PHF8 most abundant in 22Rv1 and PC3. Genetic or pharmacologic PHF8 inhibition in these cells markedly reduced RIPK2 pre-mRNA, mRNA, and protein levels, whereas PHF8 overexpression in RWPE-2 and LNCaP increased RIPK2 in an enzymatic activity- and dose-dependent manner. In clinical tissue specimens, PHF8 and RIPK2 protein levels were significantly correlated in prostate tumor cells.
Conclusion: Our findings identify PHF8, rather than NF-κB, as a major transcriptional regulator of RIPK2 in PC cells and demonstrate that PHF8's demethylase activity is essential for its transcriptional regulation of RIPK2.
利益披露 Disclosure
J. P. Sanches, None..
A. Elgehama, None..
L. Guerra, None..
W. Yang, None.