EpCAM as a novel target of MARCH8: implications for esophageal squamous cell carcinoma progression

Emerging evidence suggests a crucial role of MARCH8, a membrane-associated RING-E3 ubiquitin ligase, in cancer progression by regulating the turnover of cancer-related proteins. Our previous research identified potential MARCH8 targets using an RNAi-coupled proteomics approach, revealing that MARCH8 mediates the proteasomal degradation of E-cadherin and β2m, contributing to Esophageal Squamous Cell Carcinoma (ESCC) progression. Additionally, EpCAM emerged as a key target, and this study aims to investigate how MARCH8 regulates EpCAM stability and its implications in tumor migration. Herein, Co-immunoprecipitation (Co-IP) and Western Blotting (WB) demonstrated that MARCH8 directly interacts with EpCAM and regulates its turnover by ubiquitination and proteasomal degradation. Further, MARCH8-EpCAM co-localization was observed in ESCC cells using Immunofluorescence (IF). Additionally, Immunohistochemistry (IHC) analysis in ESCC tissues revealed a significant inverse correlation between MARCH8 and EpCAM expression (r = -0.6730, p < 0.0001). Interestingly, EpCAM showed a context-dependent expression in esophageal tissues. Adjacent non-malignant esophageal squamous epithelium showed no detectable EpCAM staining, superficial tumor regions displayed membranous EpCAM expression, whereas tumor cells at the deeper invasive front exhibited markedly reduced or lost EpCAM expression. Consistently, scratch assay coupled with IF and IHC analysis further demonstrated reduced EpCAM expression in migrating ESCC cells. Overexpression studies further revealed an inverse relationship between MARCH8 and EpCAM in migration, invasion, and β-catenin signaling, as shown by invasion/migration assays and WB analysis. Collectively, these findings establish EpCAM as a novel target of MARCH8. Given that loss of EpCAM is associated with migratory phenotypes and that MARCH8 promotes ESCC progression, MARCH8-driven EpCAM degradation may contribute to enhanced tumor cell migration and disease progression.