KLHL14/X1 regulates E-cadherin turnover and epithelial stability across physiological and cancer EMT models

Epithelial (E)/mesenchymal (M) plasticity (EMP) describes the reversible transition of cells along the E/M spectrum, generating hybrid E/M states that simultaneously retain mixed characteristics1. In our previous study, we demonstrated that progesterone (P4) modulates EMP in ovine AECs, promoting the acquisition of hybrid phenotypes2. KLHL14 has recently emerged as a potential marker of these functional intermediates. Specifically, KLHL14 is a Kelch-like E3 ubiquitin ligase implicated in the regulation of EMT and tumor-suppressive pathways³, although its precise function remains largely unknown. The aim of this study was to elucidate the molecular mechanisms underlying EMT inhibition and stabilization of the hybrid state. Notably, KLHL14 is expressed as two isoforms, the full-length form (KLHL14) and a short splice variant (X1), whose function remains unexplored. To investigate their roles, we used two complementary models: ovine AECs as a physiological EMT model and HepG2 cells, an epithelial hepatocarcinoma cell line with high endogenous KLHL14 expression. Both isoforms were analyzed through complementary gain- and loss-of-function approaches. Our results demonstrated that KLHL14 is expressed as a surface antigen in hybrid AECs. Moreover, co-localization and co-immunoprecipitation analyses revealed that KLHL14 interacts with E-cadherin at adherens junctions at the plasma membrane in both AECs and HepG2 cells. Conversely, the X1 isoform displayed a predominantly intracellular localization while still interacting with E-cadherin. Functionally, these distinct localizations correlated with different roles: KLHL14 promoted E-cadherin turnover via ubiquitin-dependent proteasomal and lysosomal degradation, whereas X1 appeared to enhance de novo E-cadherin synthesis. Interestingly, downregulation of either isoform rapidly induced EMT, suggesting their critical role in maintaining epithelial identity. Collectively, our findings indicate that KLHL14/X1 regulates E-cadherin stability through a conserved mechanism in both physiological and pathological contexts. These results identify a novel regulatory pathway controlling epithelial maintenance and provide a framework for the development of future EMT-targeted regenerative and anti-cancer therapies.