Opposing Roles of KLHL14 and X1 Isoforms in the Regulation of E-Cadherin-mediated Epithelial-Mesenchymal Transition

KLHL14 belongs to the Kelch-like (KLHL) family of E3 ubiquitin ligases. Proteins of this family share a conserved dimerization domain (BTB), a linker domain (BACK), and a C-terminal Kelch domain responsible for substrate recognition [1]. KLHL14 has recently emerged as a tumor suppressor in several cancers [2, 3]. Although it was originally implicated in the inhibition of epithelial-mesenchymal transition (EMT) in amniotic epithelial stem cells (AECs), its precise role and molecular mechanisms remain unclear [4]. Understanding KLHL14 function is highly relevant, as EMT is involved not only in cancer progression but also in stem cell development and tissue regeneration. Moreover, KLHL14 is expressed as two isoforms: the full-length form (KLHL14) and a short splice variant known as X1, which lacks the substrate-binding domain. However, the role of X1 has remained completely unexplored to date. To elucidate the roles of KLHL14 and X1 in EMT, we employed two complementary models: AECs, a physiological EMT model, and HepG2, an epithelial hepatocarcinoma cell line with high endogenous KLHL14 expression. We investigated the contribution of both isoforms through gain- and loss-of-function approaches using c-Myc-tagged overexpression constructs and isoform-specific siRNA-mediated silencing. Our results show that both isoforms contribute to the maintenance of epithelial identity through modulation of E-Cadherin, via two distinct mechanisms. Full-length KLHL14, predominantly localized at the plasma membrane in AECs and HepG2 cells, co-localized with E-Cadherin at adherens junctions. This result was further confirmed in situ in the whole amniotic membrane (AM). In contrast, X1 exhibited an endoplasmic reticulum (ER)-like pattern. Co-immunoprecipitation confirmed the specific interaction of both isoforms with E-Cadherin. This interaction was also observed in the AM, supporting the physiological relevance of our findings. Mechanistically, KLHL14 promotes E-Cadherin degradation via the ubiquitin-proteasome and lysosomal pathways, whereas X1 enhances its de novo synthesis. Functionally, silencing of both isoforms leads to overall E-Cadherin downregulation, increased cell proliferation, and EMT induction. Remarkably, these effects are conserved in the native AM, where silencing reduces E-Cadherin expression, disrupts adherens junctions, and increases Vimentin levels. Overall, this study reveals the opposing functions of KLHL14 and X1 in balancing E-Cadherin-mediated EMT regulation. While KLHL14 promotes E-Cadherin degradation, X1 supports its expression, together modulating epithelial stability. These findings uncover a previously unrecognized regulatory axis and underscore KLHL14 as a crucial pivot for epithelial integrity and plasticity. Elucidating this mechanism opens new perspectives for targeting EMT in regenerative strategies and anti-cancer therapies, where reprogramming cell fate and preventing aberrant transitions remain major clinical challenges.