Cell surface localization and release of the candidate tumor suppressor Ecrg4 from polymorphonuclear cells and monocytes activate macrophages

Abstract

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4, an epigenetically regulated gene, which unlike other tumor suppressor genes, encodes an orphan-secreted, ligand-like protein. In human cell lines, Ecrg4 gene expression was low, Ecrg4 protein undetectable, and Ecrg4 promoter hypermethylation high (45-90%) and reversible by the methylation inhibitor 5-AzaC. In contrast, Ecrg4 gene expression in fresh, normal human PBMCs and PMNs was 600-800 times higher than in cultured cell lines, methylation of the Ecrg4 promoter was low (<3%), and protein levels were readily detectable in lysates and on the cell surface. Flow cytometry, immunofluorescent staining, and cell surface biotinylation established that full-length, 14-kDa Ecrg4 was localized on PMN and monocyte cell surfaces, establishing that Ecrg4 is a membrane-anchored protein. LPS treatment induced processing and release of Ecrg4, as detected by flow and immunoblotting, whereas an effect of fMLF treatment on Ecrg4 on the PMN cell surface was detected on the polarized R2 subpopulation of cells. This loss of cell surface Ecrg4 was associated with the detection of intact and processed Ecrg4 in the conditioned media of fresh leukocytes and was shown to be associated with the inflammatory response that follows severe, cutaneous burn injury. Furthermore, incubation of macrophages with a soluble Ecrg4-derived peptide increased the P-p65, suggesting that processing of an intact sentinel Ecrg4 on quiescent circulating leukocytes leads to processing from the cell surface following injury and macrophage activation.

Ecrg4 contributes to the anti-glioma immunosurveillance through type-I interferon signaling

“…[10][11][12][13] It was demonstrated that Ecrg4 was first produced as a precursor protein of 148 amino acids, and then was proteolytically processed into several different fragments. [10][11][12][13] Therefore, the cleaved Ecrg4 fragment(s) seems likely to transmit growth retardation signals into the cells in an autocrine/paracrine manner, rather than Ecrg4 directly inhibits cell proliferation machinery in the cells.…”

Soluble purified recombinant C2ORF40 protein inhibits tumor cell growth in vivo by decreasing telomerase activity in esophageal squamous cell carcinoma

“…An additional study also demonstrated that C2ORF40 causes cell cycle G1 phase block via interaction with Transmembrane Protease, Serine 11A in ESCC (27). Previous studies have indicated that C2ORF40 may be processed and released from the cell surface to function biologically (35,36). However, a detailed molecular mechanism for the tumor suppressing function of C2ORF40 protein remains to be clarified in ESCC.…”

Soluble purified recombinant C2ORF40 protein inhibits esophageal cancer cell proliferation by inducing cell cycle G1 phase block

“…In pervious studies, TMPRSS11A gene overexpression inhibited tumor cell growth in vitro and in vivo (15), and induced cell cycle G 1 phase arrest and cell senescence (16) in ESCC. As C2ORF40 is a putative pro-hormone protein (also known as augurin) (17,18), it may be a good candidate substrate for the transmembrane serine protease TMPRSS11A in ESCC. We previously found that C2ORF40 could directly bind to TMPRSS11A in ESCC cells, as determined by binding affinity assay and co-immunoprecipitation experiments (14).…”