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.