Phase Separation of PHLDB2 Drives EMT and Tumor Progression in Triple‐Negative Breast Cancer

Background Liquid–liquid phase separation (LLPS) has emerged as a critical mechanism underlying oncogenic signaling and transcriptional regulation; however, its involvement in triple‐negative breast cancer (TNBC) remains largely unknown. The pleckstrin homology‐like domain family B member 2 (PHLDB2) has been implicated in cytoskeletal organization and cell migration, but its role in phase separation–mediated tumor progression has not been explored. Methods We conducted an integrative multi‐omics analysis combining transcriptomic profiling, bioinformatic modeling, and molecular biology experiments to elucidate the function of PHLDB2 in TNBC. Expression, mutation, and copy number variation of phase separation–related genes were analyzed using TCGA, METABRIC, and GTEx datasets. PHLDB2 knockdown and overexpression models were established in MDA‐MB‐231 and HCC38 cells to assess cell proliferation, epithelial–mesenchymal transition (EMT), and extracellular matrix (ECM)–related pathways. Immunofluorescence and fluorescence recovery after photobleaching (FRAP) assays were performed to evaluate the phase separation properties of PHLDB2. In vivo, xenograft and pulmonary metastasis models were used to validate the effects of Phldb2 knockdown on TNBC growth and metastasis. Results PHLDB2 was significantly upregulated in TNBC and correlated with poor clinical prognosis. Gene ontology and KEGG analyses revealed its enrichment in ECM–receptor interaction and focal adhesion pathways. PHLDB2 knockdown inhibited cell proliferation and migration, suppressed EMT by upregulating E‐cadherin and downregulating N‐cadherin, vimentin, Snail, and MMP‐2. PONDR analysis identified extensive intrinsically disordered regions (IDRs) within PHLDB2, and FRAP assays confirmed its ability to form dynamic, reversible condensates consistent with LLPS. In vivo, Phldb2 depletion markedly reduced tumor growth and pulmonary metastases and prolonged survival in TNBC‐bearing mice. Conclusion Our findings demonstrate that PHLDB2 promotes TNBC progression by mediating ECM remodeling and EMT activation through phase separation–driven signal organization. These results establish PHLDB2 as a novel phase separation–dependent oncogenic regulator and highlight its potential as a prognostic biomarker and therapeutic target in TNBC.