Phase separation of MARCH1 promotes rectal adenocarcinoma progression through PHLPP2 ubiquitination.

Background: Membrane-associated RING-CH 1 (MARCH1) is an E3 ubiquitin ligase that functions as an oncogene in colorectal cancer, but its role in rectal adenocarcinoma (READ) remains unclear. This study aimed to explore the effects of MARCH1 phase separation-mediated PHLPP2 ubiquitination on READ progression. Methods: MARCH1 expression was analyzed in normal and READ cell lines (SW480 and HT-29). The intrinsically disordered region (IDR) of MARCH1 was identified using Predictor of Natural Disordered Regions (PONDR), and an IDR point-mutant recombinant protein was generated. Lentiviruses were used to knock down MARCH1 or express IDR-mutant MARCH1 (MARCH1-MUT) in HT-29 cells. MG132 was used to inhibit the proteasome. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunoprecipitation were utilized to determine gene expression, protein, and ubiquitination levels. Cell proliferation and migration were assessed using cell counting kit-8 (CCK-8) and transwell assays. MARCH1 phase separation was evaluated using droplet formation, fluorescence recovery after photobleaching (FRAP), and immunofluorescence assays. Results: MARCH1 was highly expressed in READ cells. Conclusions: Phase separation of MARCH1 promotes the proteasomal degradation of PHLPP2 via ubiquitination, thereby facilitating READ progression.