Cell Surface Localization and Shedding of the Candidate Tumor Suppressor Ligand Ecrg4 after Neutrophil Activation and Polarization

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4; an epigenetically regulated gene that, unlike other tumor suppressor genes, encodes an orphan secreted ligand‐like protein. In cultured human cell lines, Ecrg4 gene expression was low, the Ecrg4‐ encoded protein undetectable and Ecrg4 promoter hypermethylation was high (45–90%). Low gene expression was also reversible by incubation with the methylation inhibitor 5‐Aza‐ Cytidine. In contrast, Ecrg4 gene expression in fresh normal human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) was 600–800 times higher than cultured cells, the protein was readily detected in cell lysates and Ecrg4 promoter hyper‐methylation in normal leukocytes was low (<3%). Immunoblotting, cell surface biotinylation, immunofluorescent staining and cell flow analyses also established that the Ecrg4‐encoded protein was not constitutively secreted but instead exists as an extracellular, membrane‐anchored protein on the surface leukocytes. Its presence on the surface however is dynamic and upon leukocyte activation by 5 min incubations with fMLP, cell surface Ecrg4 protein is shed in concordance with fMLP‐activation of neutrophils and polarization. In light of these findings, we propose that the pathological sequelae of losing Ecrg4 expression in cancer may reflect a more global physiological response that links Ecrg4 to the biology of leukocytes in cancer surveillance, innate immunity, injury and inflammation.