A DIAPH1-SOWAHC-CFL1 Axis Regulates Macropinocytosis and Chemoresistance in Cervical Cancer

Formin mDia1 (DIAPH1) is a central regulator of actin dynamics underlying cell migration and plasma membrane remodeling, processes frequently dysregulated in cancer. Here, we uncover a previously unrecognized DIAPH1-dependent signaling axis controlling macropinocytosis and chemotherapeutic sensitivity in cervical cancer. Using label-free quantitative mass spectrometry in CRISPR/Cas9-engineered DIAPH1 knockout (KO) HeLa cells, we mapped the DIAPH1 interactome and identified SOWAHC as a novel binding partner. The interaction was validated in reciprocal co-immunoprecipitation experiments, and SOWAHC protein levels were found to depend on DIAPH1. Loss of either DIAPH1 or SOWAHC increased phosphorylation of the actin-severing protein cofilin (CFL1) under both basal and EGF-stimulated conditions, indicating reduced CFL1 activity. Functionally, DIAPH1, but not SOWAHC, was required for EGF-induced ruffling and optimal random cell migration. In contrast, macropinocytosis was impaired in both DIAPH1 and SOWAHC KO cells and was nearly abolished upon DIAPH1 loss. The persistence of ruffling in SOWAHC-deficient cells despite defective macropinocytosis uncouples ruffle formation from macropinosome closure and identifies SOWAHC as a selective regulator of macropinocytic uptake. CFL1-deficient cells exhibited constitutive membrane ruffling and preserved EGF-induced macropinocytosis, likely due to upregulation of the cofilin homolog Destrin. Importantly, DIAPH1 loss markedly sensitized HeLa cells to cisplatin, the standard-of-care therapy for advanced cervical cancer, consistent with the macropinocytosis-dependent uptake of the cisplatin receptor. Given the association of CFL1 with poor prognosis in cervical cancer, we examined the status of this axis in human tumors. Analysis of a tissue microarray comprising 322 cervical cancer cases revealed elevated DIAPH1 and SOWAHC expression in tumors relative to normal epithelium, stage-dependent expression patterns, and an inverse correlation with phospho-CFL1 levels. Together, these findings identify a DIAPH1-SOWAHC-CFL1 signaling axis that coordinates actin remodeling, macropinocytosis, and chemotherapeutic responsiveness, uncovering an unappreciated vulnerability in cervical cancer.