Alternative Promoters Drive Transcriptomic Reprogramming and Prognostic Stratification in TNBC

Abstract

Abstract 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