Canonical and Non-Canonical Mirna Degradation Shapes State Transitions and Stemness in Breast Cancer

Target-directed microRNA degradation (TDMD) is an emerging post-transcriptional mechanism that controls miRNA turnover, yet its role in human cancers remains largely unexplored. Here, we combine CRISPRi-mediated ZSWIM8 depletion, miRNA-seq, and AGO2-eCLIP to define the TDMD landscape across breast cancer subtypes. We identify 19 high-confidence TDMD substrates, including miR-29b-3p and miR-33a/b-5p, and show that TDMD shapes miRNA target occupancy and target repression. Integration with single-cell transcriptomics reveals that TDMD of miR-29b-3p triggered by NREP transcript is associated with transcriptional plasticity along the epithelial–mesenchymal axis and marks a stem-like subpopulation of malignat cells with tumor-initiating potential in triple-negative breast cancer. Unexpectedly, we also uncover a non-canonical TDMD mechanism, independent of ubiquitin ligase ZSWIM8 and the proteasome, which includes SERPINE1-triggered miR-30c-5p degradation, conferring paclitaxel resistance and enhancing sphere-forming potential. These findings establish target-directed microRNA degradation as a functional layer of miRNA regulation in cancer, linking miRNA decay to cellular state transitions and therapeutic response. Our results broaden the mechanistic spectrum of TDMD and provide a framework to investigate how regulated miRNA decay contributes to aggressive breast cancer phenotypes. Target-directed microRNA degradation (TDMD) is an emerging post-transcriptional mechanism that controls miRNA turnover, yet its role in cancer remains largely unexplored. This study maps endogenous TDMD substrates across human breast cancer cell models and highlights the impact of TDMD on miRNA function in malignant cell plasticity via canonical and non-canonical mechanisms. A novel catalogue of decay-regulated microRNAs in breast cancer highlights direct roles for target directed miRNA degradation malignant cell plasticity.