PHF8 is a major upstream regulator of RIPK2 in prostate cancer cells

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.