SRSF7 controls RNA splicing programs driving triple-negative breast cancer metastasis

Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype in which metastasis is the major cause of mortality. However, the contribution of RNA splicing to TNBC metastatic progression remains incompletely understood. Methods We performed a high-content, imaging-based RNA interference (RNAi) screen targeting 244 spliceosome components in two highly motile TNBC cell lines to systematically identify regulators of cell migration. Cell motility was assessed using phagokinetic track (PKT) and random cell migration (RCM) assays, while proliferation, cell death, and cytoskeletal phenotypes were evaluated by high-content imaging, Sulforhodamine B (SRB) assays, Annexin V/propidium iodide (PI) staining, immunofluorescence, and western blotting. Transcriptomic consequences of candidate splicing factor depletion were examined by next-generation RNA sequencing (NGS), followed by differential gene expression, pathway enrichment, and alternative splicing analysis. SRSF7 function was further validated using inducible CRISPR-Cas9 knockout models and in vivo tail-vein metastasis mouse models. Lung colonization and metastatic burden were assessed by longitudinal bioluminescent imaging, lung weight measurement, and ink perfusion-based quantification of macrometastases. Clinical relevance was evaluated using TNBC patient gene expression cohorts and Kaplan–Meier survival analysis. Results The systematic RNAi screen identified a subset of splicing factors that selectively regulate TNBC cell motility while exerting only limited effects on cell proliferation and survival, with SRSF7 emerging as a central regulator. Functional validation using siRNA-mediated depletion and inducible CRISPR-Cas9 perturbation demonstrated that SRSF7 promotes TNBC cell migration and invasion, whereas its loss suppresses metastatic lung colonization in vivo. Transcriptomic profiling revealed that SRSF7 orchestrates a broad gene expression program enriched for extracellular matrix (ECM) organization, cell–matrix adhesion, integrin signalling, and cytoskeletal regulation. Deep-RNA sequencing identified thousands of SRSF7-dependent splicing events, including recurrent exon-skipping alterations affecting genes implicated in migratory and adhesive phenotypes. Clinically, elevated SRSF7 expression was associated with poor survival outcomes in TNBC patients, linking its molecular function to disease progression. Conclusions Our findings identify SRSF7 as a critical spliceosome-associated regulator of TNBC metastatic progression and a potential therapeutic vulnerability in aggressive breast cancer. These results highlight RNA-splicing regulation as an important mechanism underlying TNBC metastasis and support further investigation of splicing-factor dependencies for therapeutic intervention.