Comprehensive bioinformatics and experimental analysis of PPOX reveals its carcinogenic effect in clear cell renal cell carcinoma.

Background: Dysregulated heme biosynthesis is a hallmark of metabolic reprogramming in cancer; however, the role of protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme synthesis pathway, remains poorly characterized in clear cell renal cell carcinoma (ccRCC). This study aims to elucidate the regulatory mechanism of PPOX on the malignant phenotype of ccRCC to evaluate its potential as a precision biomarker. Methods: To investigate the role of PPOX in ccRCC, we integrated multi-omics bioinformatic analysis with experimental validation. First, PPOX expression and subcellular localization were assessed across multiple databases, including Tumor IMmune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA), and the Human Protein Atlas (HPA). These findings were subsequently validated via immunohistochemistry (IHC) on ccRCC tissues and confirmed by RT-qPCR/Western blot across a panel of cell models (HK2, 786-O, 769-P, and A498). Utilizing the TCGA-KIRC cohort, Kaplan-Meier curves and univariate/multivariate Cox proportional hazards models were employed to determine the independent prognostic value of PPOX. Second, Mendelian Randomization (MR) using the inverse-variance weighted (IVW) method was performed to explore potential causal links between PPOX and ccRCC. Third, single-cell RNA sequencing (scRNA-seq) analysis mapped PPOX distribution within the tumor microenvironment (TME), while the CIBERSORT algorithm explored associations between PPOX expression, immune cell infiltration, and immune checkpoints. Fourth, potential therapeutic agents targeting PPOX were identified via drug sensitivity analysis and molecular docking. Finally, Results: PPOX was significantly upregulated in ccRCC and served as an independent risk factor for poor patient prognosis. A nomogram was initially constructed to illustrate its potential prognostic utility. MR analysis supported a potential causal relationship between PPOX levels and ccRCC risk. ScRNA-seq results revealed significant PPOX enrichment in malignant cell clusters, and its expression correlated with various immune cells and checkpoints, suggesting predictive value for immunotherapy. Furthermore, PPOX expression was associated with increased sensitivity to therapeutic agents such as 5-fluorouracil and doxorubicin. Mechanistic studies confirmed that PPOX expression positively correlated with cell proliferation, migration, and invasion, and regulated the Wnt/β-catenin signaling pathway. Conclusions: This study demonstrates that PPOX is a critical regulatory factor driving the malignant progression of ccRCC. The discovery of the PPOX-Wnt/β-catenin axis not only explains the biological basis of PPOX as a prognostic biomarker but also provides a potential metabolic therapeutic target for patients with advanced ccRCC.