Phase separation of MARCH1 promotes rectal adenocarcinoma progression through PHLPP2 ubiquitination

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.

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.

MARCH1 was highly expressed in READ cells. MARCH1 knockdown increased PHLPP2 protein levels in HT-29 cells. Additionally, MARCH1 knockdown inhibited cell proliferation and migration. PONDR analysis predicted an IDR in MARCH1, spanning amino acid residues 128-254. MARCH1 underwent phase separation both in vitro and in cellulo, whereas its IDR mutation disrupted this process. MARCH1-MUT increased PHLPP2 protein levels while decreasing its ubiquitination levels. Notably, MG132 treatment led to an increase in PHLPP2 protein levels in HT-29 cells transfected with MARCH1-WT. Moreover, MARCH1-MUT inhibited cell proliferation and migration.

Phase separation of MARCH1 promotes the proteasomal degradation of PHLPP2 via ubiquitination, thereby facilitating READ progression.