Comprehensive Profiling of ac4C RNA Modification Identifies CERCAM in Cancer-Associated Fibroblasts as a Key...

N4-acetylcytidine (ac4C) RNA modification has emerged as a critical epigenetic regulator in tumorigenesis and progression. However, the comprehensive landscape of ac4C modification in colorectal cancer (CRC), particularly its orchestration of the tumor microenvironment (TME) and stromal crosstalk, remains largely unexplored.

This study aimed to comprehensively delineate the landscape of N4-acetylcytidine (ac4C) RNA modification in colorectal cancer (CRC) to uncover its role in shaping the tumor microenvironment and clinical outcomes. Integrated bioinformatic and in vitro analyses revealed that ac4C patterns dictate aggressive stromal crosstalk and identified CERCAM as a cancer-associated fibroblast (CAF)-specific driver of tumor progression, providing a novel framework for prognostic prediction and personalized targeted therapy.

Transcriptomic and clinical data of CRC patients were curated from TCGA and GEO databases (GSE14333, GSE17536, GSE38832, GSE39582). The ac4C modification patterns were quantified using single-sample Gene Set Enrichment Analysis (ssGSEA). Single-cell RNA sequencing (scRNA-seq) analyses were performed via the scCancer Explorer database. A prognostic ac4C score was constructed to evaluate clinical outcomes and therapeutic vulnerabilities. The functional role of the identified target gene, CERCAM, was validated using in vitro co-culture experiments.

Patients with high ac4C scores exhibited worse prognosis, higher clinical stages, and metastasis (N2, M1). High ac4C levels positively correlated with epithelial-mesenchymal transition (EMT), TGF-β signaling, and extracellular matrix (ECM) receptor interactions, whereas low ac4C levels were associated with cell cycle, fatty acid metabolism, and microsatellite stability (MSS). The TME of high-ac4C tumors was characterized by higher Stromal, Immune, and ESTIMATE scores, elevated immune checkpoints (LAG3, CD14, LILRB2, SIRPA, CD8A), and strong correlations with macrophages and NK cells. scRNA-seq analysis revealed that CERCAM, a key gene within the ac4C network, was predominantly expressed in cancer-associated fibroblasts (CAFs) rather than tumor cells. DNA methylation of CERCAM was significantly elevated in metastatic CRC compared to primary tumors. In vitro experiments confirmed that CERCAM was overexpressed in CAFs and CRC tissues. Co-culturing CRC cells (RKO, HCT116) with CERCAM-knockdown CAFs significantly suppressed tumor cell proliferation, migration, and invasion. Furthermore, the ac4C score effectively predicted tumor mutational landscape differences and sensitivities to agents like pazopanib, gefitinib, and conventional chemotherapies.

This study delineates the crucial role of ac4C modification in shaping the CRC microenvironment, identifies CERCAM as a CAF-specific driver of tumor progression, and provides a translational framework for ac4C-based prognostic prediction and personalized targeted therapy.