P3H1 promotes malignant progression of esophageal squamous cell carcinoma (ESCC) and modulates the immune...

Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer (EC) in China and carries high lethality, in part because early disease is typically asymptomatic and thus diagnosed at advanced stages. To address the need for new biomarkers and targets, we investigated prolyl 3‑hydroxylase 1 (P3H1), a collagen‑modifying enzyme with emerging roles in cancer, aiming to clarify its contribution to ESCC progression and its potential as a prognostic biomarker and therapeutic target.

We integrated clinical and transcriptional profiles of ESCC from public datasets [The Cancer Genome Atlas (TCGA); Gene Expression Omnibus (GEO) cohorts GSE53623 and GSE53624] and single‑cell RNA‑seq data (GSE154763) to evaluate P3H1 expression, clinicopathologic associations, survival, and diagnostic performance. In vitro, we selected the high‑expressing ESCC line KYSE‑180 and performed lentiviral shRNA-mediated knockdown with validation by reverse transcription quantitative polymerase chain reaction (PCR) and Western blot. Functional assays [Cell Counting Kit-8 (CCK-8) proliferation, colony formation, wound‑healing migration, and Transwell migration/invasion] were used to assess malignant phenotypes. Immune relevance was explored with immunedeconv [Tumor IMmune Estimation Resource (TIMER), xCell, Microenvironment Cell Populations (MCP)‑counter, Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT), Estimating the Proportions of Immune and Cancer cells (EPIC)] and correlations with immune‑checkpoint genes (e.g., HAVCR2, SIGLEC15). Single‑cell analyses profiled cell‑type-specific P3H1 expression. Statistical analyses included Kaplan-Meier survival and receiver operating characteristic (ROC)/Youden index determinations. Human tissue immunohistochemistry (IHC) provided orthogonal validation.

P3H1 was significantly overexpressed in ESCC versus normal tissues across TCGA and GEO, confirmed by IHC of tumor/peritumoral pairs and by elevated mRNA/protein in ESCC cell lines relative to HET‑1A. Higher P3H1 is associated with larger tumor size, lymph‑node metastasis, advanced tumor-node-metastasis (TNM) stage, and poorer survival. Diagnostic performance for ESCC was high. In KYSE‑180 cells, shRNA knockdown of P3H1 reduced proliferation (CCK-8, colony formation) and impaired migration/invasion (wound healing, Transwell). Immune deconvolution linked P3H1 to multiple immune‑cell compartments-particularly T‑cell signatures-and showed positive correlations with checkpoint genes including HAVCR2 and SIGLEC15. Single‑cell analyses indicated enriched P3H1 expression in plasmacytoid dendritic cells (pDCs), suggesting a role in shaping an immunoregulatory milieu that may favor tumor progression.

Our study demonstrates that P3H1 is markedly upregulated in ESCC and contributes to tumor proliferation, migration, and invasion. In addition, P3H1 shows strong associations with immune infiltration and checkpoint pathways, suggesting a role in shaping the tumor immune microenvironment. These findings support P3H1 as a potential diagnostic biomarker and therapeutic target in ESCC.