Alternative Promoters Drive Transcriptomic Reprogramming and Prognostic Stratification in TNBC

Pre-transcriptional regulation through alternative promoter usage is a critical yet underexplored mechanism influencing gene expression in Triple-Negative Breast Cancer (TNBC), a highly aggressive and heterogeneous breast cancer subtype. While short-read RNA sequencing data are widely available, they offer limited resolution in accurately capturing transcript-level diversity. To overcome this, we focused on promoter-level quantification to infer active promoter usage and investigate transcriptional regulation dynamics in TNBC. Using RNA-seq data from 360 TNBC tumors and 88 adjacent normal tissues, we identified TNBC-specific and subtype-enriched Active Alternative Promoters (AAPs). Integration with H3K4me3 and H3K27ac ChIP-seq data confirmed a key promoter switching event in the HDAC9 gene: the promoter pr1077 was downregulated while another promoter pr1079 was specifically activated in TNBCs. This switch was epigenetically supported by differential enrichment of histone marks, implicating HDAC9 promoter switching as a tumor-specific regulatory mechanism. Further, we identified subtype-specific alternative promoters in TNBC, including basal subtype-enriched activity of SEC31A and reduced promoter usage of AKAP9, which were not reflected at the gene expression level but were evident through promoter-level analysis. Next, we identified alternative promoters of HUWE1 and FTX as independent predictors of relapse-free survival (RFS) in TNBC. Their prognostic value remained significant after adjusting for copy number alterations and transcriptomic subtypes. A 4-feature model integrating these two promoter activities with two clinical variables (Tumor size, Ki67 index) achieved an AUROC of 0.73 and improved patient risk stratification, with a Net Reclassification Improvement (NRI) of 0.40-0.48 over the clinical-only model, underscoring the potential of promoter activity as a biomarker in TNBC.