De Novo Gene Transcription of Connexin Mediates Cytoplasmic Fluid Exchange and Flocking Transitions in Physiological and Cancerous Epithelial Systems

Abstract The initial invasion of tumors requires a transition from a solid, jammed state to a fluid‐like, flocking, unjammed state that enables collective migration. Here, we show that de novo gene transcription is essential for the emergence of flocking in epithelial tissues and identify connexins (Cx) as key mediators of this transition. Using quiescent HaCaT keratinocytes, tumorigenic A431 epidermoid carcinoma cells, primary bronchial epithelial explants, and vocal fold carcinoma (VFC) cells, we find that flocking induction depends on transcriptional programs activated downstream of epidermal growth factor (EGF). EGF stimulation upregulates Cx26 and Cx31 and enhances gap‐junctional intercellular communication (GJIC), which is necessary—though not sufficient—to generate the large‐scale cell‐volume fluctuations and density heterogeneity that accompany unjamming. Sustained signaling through extracellular signal‐regulated kinase 1/2 (ERK1/2) and AKT serine/threonine kinase (AKT) downstream of the EGF receptor (EGFR) is required for connexin induction, linking mechanical state transitions to extracellular cues. Pharmacological inhibition and CRISPR‐Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9) knockout of connexins block unjamming and collective motility. VFC cells display constitutively elevated connexins and persistent flocking that is highly sensitive to connexin inhibition. Consistently, high Cx26 expression correlates with reduced survival across carcinomas. These findings reveal a transcriptionally controlled, connexin‐dependent mechanism that enables tissue fluidization and collective invasion.