CAPZA1 Suppressed the Progression of Esophageal Squamous Cell Carcinoma by Binding to hnRNP K and PTPB1 to Influence Its mRNA Stability.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and prevalent cancers in China, a deeper understanding at the molecular level is the cornerstone for advancing precision oncology in ESCC. This study aims to investigate the role of CAPZA1 in ESCC progression. Based on our previous whole genome sequencing (WGS) and whole exome sequencing (WES) data indicating frequent copy number loss of CAPZA1 in ESCC, as well as the presence of a specific single nucleotide polymorphism (SNP, rs373245753 T>G) in its 3'UTR via the dbSNP database (https://www.ncbi.nlm.nih.gov/snp/), we sought to determine the functional and mechanistic impact of CAPZA1 genotypes on ESCC cell behavior. MATERIALS AND METHODS: We identified the SNP rs373245753 within the 3'UTR of the CAPZA1 gene via the dbSNP database. To investigate its functional impact, we established stable ESCC cell lines overexpressing either the CAPZA1[T] or CAPZA1[G] variant and evaluated their migration and invasion capabilities using transwell assays. Subsequently, we performed subcutaneous xenograft experiments by injecting these cells into the lower limbs of mice to monitor tumor growth in vivo. Furthermore, siRNAs were used to efficiently reduce mRNA expression of CAPZA1, then xCELLigence Real-Time Cell Analyzer (RTCA)-MP system and colony formation assay were employed to detect cell proliferation ability. To elucidate the underlying molecular mechanism, we employed biotin-RNA pulldown assays coupled with mass spectrometry, along with RNA immunoprecipitation (RIP) assays, to identify RNA-binding proteins interacting with the CAPZA1 mRNA variants. Finally, we assessed the impact of these proteins on CAPZA1 mRNA stability through mRNA decay assays. RESULTS: According to the dbSNP database, we identified the SNP rs373245753 within the 3'UTR of the CAPZA1 gene. Overexpression of CAPZA1[T] significantly inhibited ESCC cell migration and invasion, while the CAPZA1[G] variant attenuated this suppression both in vivo and in vitro. Biotin-RNA pulldown combined with mass spectrometry and RIP assays showed that CAPZA1[T] mRNA binds to hnRNP K and PTBP1, enhancing its stability and tumor-suppressive function. In contrast, CAPZA1[G] mRNA preferentially bound UPF1, leading to accelerated mRNA decay and loss of tumor suppression. CONCLUSION: CAPZA1 acts as a tumor suppressor in ESCC, with its function dependent on genotype. The CAPZA1[T] variant binds hnRNP K and PTBP1 to stabilize its mRNA and inhibit tumor aggressiveness, whereas the CAPZA1[G] allele promotes UPF1-mediated mRNA decay and diminishes this protective effect. These findings reveal a novel post-transcriptional regulatory mechanism underlying ESCC progression and highlight CAPZA1 genotype as a potential prognostic marker and therapeutic target.