Calcium-modulated GJB6-GRHL3 positive feedback loop attenuates ESCC progression through AKT signaling pathway inhibition.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy characterized by dismal outcomes, but its molecular pathogenesis remains incompletely understood. To uncover critical driver genes in ESCC, we conducted a functional screen using a CRISPR/Cas9 sgRNA library targeting 406 copy-number alteration (CNA)-related genes. Among the candidates, gap junction protein beta 6 (GJB6), a major connexin family member, emerged as a tumor suppressor that inhibits malignant phenotypes. Clinically, reduced GJB6 expression significantly correlates with ESCC development and predicts unfavorable outcomes. Mechanistically, we delineated a reciprocal regulation between GJB6 and its downstream gene GRHL3: GJB6 enhances GRHL3 expression via Ca²⁺-dependent signaling, while GRHL3 transcriptionally upregulates GJB6, thereby forming a positive feedback circuit to exert their tumor-suppressive functions. Disruption of the GJB6/GRHL3 reciprocal regulatory axis activates AKT signaling, thereby driving ESCC pathogenesis. Notably, the oncogenic effects of GJB6 ablation could be pharmacologically reversed by the AKT inhibitor capivasertib, suggesting a potential therapeutic strategy for GJB6-deficient ESCC patients. Collectively, our findings establish GJB6 as both a critical suppressor and a clinically actionable prognostic biomarker, highlighting the potential of drug repurposing approaches for ESCC treatment.