AMOTL2 inhibits lymph node metastasis in gastric cancer by suppressing TGF-β/Smad signaling and is destabilized by the E3 ubiquitin ligase SMURF1.

Angiomotin-like protein 2 (AMOTL2) regulates cell polarity and cytoskeletal regulation, but its role in gastric cancer (GC) remains undefined. Here, we show that AMOTL2 is frequently downregulated in GC tissues and cell lines, and its low expression is associated with deeper tumor invasion, lymph node metastasis, and shorter overall survival. Gain- and loss-of-function assays demonstrated that AMOTL2 suppresses GC cell proliferation, migration, and invasion. AMOTL2 overexpression also impaired endothelial and lymphatic-endothelial tube formation and reduced the expression of angiogenic and lymphangiogenic factors. In the subcutaneous, orthotopic, and popliteal lymph node metastasis models, AMOTL2 inhibited tumor growth and lymphatic dissemination. Mechanistically, AMOTL2 suppressed TGF-β/Smad signaling, as evidenced by reduced BMP2 and TGF-β1 expression, decreased Smad1/5/9 and Smad2/3 phosphorylation, and impaired Smads nuclear accumulation. Notably, pharmacological Smad1/5/9 activation by SB4 partially reversed the suppressive phenotypes of AMOTL2 in vitro and in vivo. Co-immunoprecipitation, GST pull-down, and ubiquitination assays identified SMURF1 as an AMOTL2-interacting E3 ubiquitin ligase that binds through its WW domains and promotes AMOTL2 ubiquitination and proteasomal degradation. AMOTL2 restoration counteracted SMURF1-driven malignant phenotypes. Collectively, these findings define AMOTL2 as a potential suppressor of GC progression that is destabilized by SMURF1, suggesting that the SMURF1-AMOTL2-Smad signaling axis may contribute to GC progression and lymphatic dissemination, representing a candidate therapeutic target that warrants further validation.