This study investigated the expression profile, immunoregulatory roles, genomic alterations, and prognostic significance of Centromere Protein N (CENPN) in invasive breast cancer (BRCA). The authors aimed to determine whether CENPN expression differs between tumor and adjacent normal tissues, how CENPN relates to tumor immune features and genomic changes, and whether modulating CENPN affects malignant phenotypes in breast cancer cells.
The analysis integrated public databases and an institutional tissue cohort. Public resources included The Cancer Genome Atlas (TCGA) for expression and clinical correlations; the Human Protein Atlas (HPA) for immune cell expression patterns; cBioPortal for CENPN genomic alterations; GEPIA 2.0 to identify CENPN-related genes; STRING to visualize interaction networks; and TIMER 2.0 to assess correlations with immune cell infiltration and immune markers. Functional enrichment employed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA). Experimental validation used 17 paired tumor and adjacent normal tissues from patients treated surgically at Yantai Mountain Hospital between January 2022 and April 2023. In vitro assays included CCK-8 proliferation assays and wound healing migration assays in MCF-7 breast cancer cells, with additional testing of chemosensitivity to 5-fluorouracil and gemcitabine following CENPN knockdown.
TCGA analysis showed significantly higher CENPN expression in BRCA tissues compared with adjacent normal tissues (P < 0.05). In the authors’ institutional cohort of 17 paired samples, CENPN positivity was detected in 82.4% of breast cancer tissues versus 5.9% of paired paracancerous tissues (P < 0.001), validating the TCGA finding at the tissue level.
Survival analyses reported that high CENPN expression correlated with poorer outcomes across multiple endpoints: overall survival, recurrence-free survival, post-progression survival, and distant metastasis-free survival, with hazard ratios and P values provided in the source abstract.
Data from the HPA indicated elevated CENPN expression in specific blood immune cell types, notably regulatory T (T-reg) cells, naïve B cells, and myeloid dendritic cells. TIMER 2.0 correlation analyses associated tumor CENPN expression with infiltration levels of multiple immune cell types in BRCA, including B cells, CD4+ and CD8+ T cells, macrophages, neutrophils, and dendritic cells. CENPN expression showed positive correlations with marker genes for CD8+ T cells, B cells, pan-T cell markers, and T-cell exhaustion markers.
The authors also reported a negative correlation between CENPN expression and tumor mutational burden (TMB) in BRCA patients (r = -0.196, P < 0.001). Correlations with 20 common immune checkpoint genes were mixed: CENPN positively correlated with five checkpoint genes and negatively correlated with the remaining fifteen, suggesting a complex relationship with immune checkpoint biology and potential relevance for predicting immunotherapy response.
cBioPortal analysis of genomic alterations in BRCA revealed that invasive lobular carcinoma exhibited the highest frequency of CENPN alterations. Across BRCA cases, gene amplification was the most common alteration type. Invasive mixed mucinous breast carcinoma had the highest mutation frequency in CENPN, and a notable missense substitution, K329N, was identified as a potential driver mutation in BRCA.
In vitro experiments evaluated the phenotypic effects of CENPN knockdown in the MCF-7 luminal breast cancer cell line. CCK-8 assays demonstrated that reduction of CENPN expression significantly suppressed cell proliferation. Wound healing assays showed impaired migration after CENPN knockdown. Additionally, CENPN knockdown enhanced the chemosensitivity of MCF-7 cells to the cytotoxic agents 5-fluorouracil and gemcitabine, indicating that CENPN influences both intrinsic malignant behavior and response to chemotherapy in this model.
Bioinformatic enrichment analyses of genes related to CENPN indicated functional associations concentrated in cell-cycle and DNA-related processes. GO terms included cell cycle, DNA metabolic processes, cell division, nuclear lumen, chromosomes, nucleoplasm, and molecular functions such as ATP, nucleotide, and small-molecule binding. KEGG pathway enrichment highlighted DNA replication, cellular senescence, mismatch repair, homologous recombination, the p53 signaling pathway, and the FOXO signaling pathway. These pathway associations align with a role for CENPN-related networks in proliferative and genomic maintenance processes.
Across multi-database analyses and institutional validation, CENPN was consistently overexpressed in BRCA tissue relative to adjacent normal tissue and associated with worse survival outcomes. The relationship between CENPN expression and multiple immune cell populations, immune checkpoint genes, and a negative correlation with TMB suggests CENPN may participate in tumor immune regulation and could have utility as a biomarker for immunotherapy stratification, though the directionality and mechanism remain to be defined.
Genomic alteration patterns (amplification predominating) and enrichment of CENPN-related genes in DNA repair and cell-cycle pathways support a potential role in tumor proliferation and genomic stability. Functional assays in MCF-7 cells provided experimental support that CENPN contributes to proliferation, migration, and chemotherapy response in vitro.
Based on the presented data, the authors conclude that CENPN is a potential prognostic biomarker and a candidate therapeutic target for immunotherapy in BRCA. Translation to clinical practice would require further mechanistic studies and validation in larger, independent clinical cohorts.
All authors declared no conflicts of interest.