ANO1 stabilizes partial EMT to drive colorectal cancer metastasis through PI3K/AKT/mTOR and ERK signaling pathways

Colorectal cancer (CRC) metastasis remains a clinically intractable challenge, where the partial epithelial-mesenchymal transition (partial EMT) state is a key driver of metastatic plasticity and treatment resistance. The calcium-activated chloride channel ANO1 (TMEM16A) is frequently overexpressed in epithelial tumors, yet its mechanism in regulating metastasis and partial EMT remains undefined. In this study, we identify ANO1 as a key stabilizer of the partial EMT state. ANO1 was upregulated in CRC tissues and correlated with poor patient survival. Functionally, ANO1 knockdown robustly suppressed CRC cell proliferation, migration, and invasion, and potently induced early apoptosis. Proteomic profiling linked ANO1 depletion to the downregulation of cytoskeletal and adhesion networks. Given the centrality of these processes to EMT, we specifically examined key EMT-associated molecules. Western blot results showed that ANO1 silencing reduced expression of mesenchymal markers (N-cadherin, vimentin, α-SMA) and EMT transcription factors (Zeb1, Snail, Twist), while also downregulating the epithelial marker E-cadherin. Immunofluorescence revealed that ANO1 depletion disrupted a spatially organized hybrid subcellular architecture, and flow cytometry demonstrated that ANO1 knockdown selectively depleted the E-cadherin+/vimentin+ partial EMT subpopulation. Collectively, these data indicated that ANO1 depletion actively dismantles the organized architecture of the partial EMT state rather than driving a canonical phenotypic reversal. Transcriptomic and integrated multi‑omics analyses further revealed that ANO1 loss induced widespread uncoupling between transcriptional and proteomic regulation, with the mTOR and MAPK pathways enriched among “transcript‑only” changes. Consistently, ANO1 silencing reduced phosphorylation of PI3K, AKT, mTOR, and ERK without altering total protein levels, and pharmacological inhibition of either pathway phenocopied the dissolution of the partial EMT state. In summary, our work delineates a novel mechanism wherein ANO1, acting as an upstream hub, licenses metastatic progression by co‑activating PI3K/AKT/mTOR and ERK signaling to stabilize a plastic, survival‑competent partial EMT state, thereby nominating it as a promising therapeutic target for intercepting CRC dissemination.