GOLPH3-mediated sialylation of CXCR4 drives malignant progression and M2 macrophage polarization in colorectal cancer.

Sialylation, a post-translational protein modification, correlates with tumor malignancy. This study investigates how GOLPH3 regulates CXCR4 sialylation in colorectal cancer (CRC) and its impact on cancer cell phenotypes. Predicted glycosylation sites on CXCR4 were identified using the NetNGlyc-1.0 tool. The GOLPH3-CXCR4 interaction and GOLPH3-sialyltransferase associations were bioinformatically predicted (BIOGRAD) and validated experimentally via co-immunoprecipitation (Co-IP). CXCR4 sialylation was assessed by immunoprecipitation (IP) and lectin blotting (LB). Immunohistochemical (IHC) assessment of protein expression levels of Ki-67, GOLPH3, and CD206 in tissues. Functional assays, including colony formation, flow cytometry, scratch, and Transwell assays, evaluated proliferation, apoptosis, migration, and invasion in CRC. A co-culture system, flow cytometry, and Enzyme-Linked Immunosorbent Assay (ELISA) were conducted to assess macrophage M2 polarization, and a xenograft tumor model was constructed for in vivo validation. GOLPH3 promotes the interaction between CXCR4 and its ligand CXCL12 by inducing sialylation at the 180th amino acid residue of CXCR4. This regulatory mechanism plays an important role in modulating macrophage function, which manifests as facilitating cell proliferation, migration and invasion, suppressing cellular apoptosis, and promoting M2-type macrophage polarization. Furthermore, N180 mutation assays offer direct experimental evidence to validate the causal relationship within this regulatory network. These findings reveal a GOLPH3-ST6GAL1 axis that enhances CXCR4 sialylation to drive CRC progression and an immunosuppressive microenvironment via CXCL12/CXCR4 signaling, suggesting a novel regulatory mechanism underlying glycosylation modification of this chemokine receptor.