Thrombin-processed Ecrg4 recruits myeloid cells and induces antitumorigenic inflammation

Malignant glioma is an invasive and diffuse central nervous system tumor that is characterized by extensive immune cell infiltration.1–4 Abundant infiltrating microglia constitute the primary resident innate immune response, which is accompanied by a mobilization of mononuclear cells, primarily inflammatory monocytes and T cells.5 Although up to 30% of a human glioma tumor mass comprises microglia,6 the tumor microenvironment is characterized by an overall immunosuppressive phenotype.7 For example, microglia, as the resident macrophage of the brain, become tumor permissive, enhance glioma invasion,2–4,7–9 and suppress pro-inflammatory antitumor surveillance functions.1,4,10 A hallmark of their immunosuppressive phenotype is the skewing of cytokine profiles of glioma-infiltrating microglia/macrophages and lymphocytes.1,11–13 While the specific mechanisms mediating the immunosuppressive tumor microenvironment are poorly understood, the expression of paracrine factors, including transforming growth factor β, colony stimulating factor 1, and macrophage inhibitory factor, is associated with this phenotype.1,5,14 Chromosome 2 open reading frame 40 (c2orf40), alternatively named esophageal cancer-related gene 4 (Ecrg4) is a candidate immunomodulatory factor released following brain injury, where it is expressed in normal brain neuroepithelia,15 oligodendrocytes,16 and astrocytes.17 Its expression is downregulated in various tumors,18–21 including glioma, where Ecrg4 mRNA levels are downregulated compared with matched normal brain tissue through hypermethylation of its promoter, and this pattern is more frequently observed in advanced tumor.19,21 Previous studies have shown that elevated Ecrg4 expression levels correlate with improved survival in cancers of the esophagus, prostate, and breast, suggesting a role in tumor progression.20,22,23 We have recently shown that Ecrg4 is highly expressed in leukocytes and encodes a highly conserved, membrane-tethered protein that is selectively shed from the cell surface following injury.18,24 The secreted form of Ecrg4 induces neural cell senescence,16 releases CNS hormones,25 and stimulates the pro-inflammatory phosphorylation of p65 Rel A in the nuclear factor-kappaB (NF-κB) pathway in peritoneal macrophages,18 suggesting its role as an immunomodulatory factor that is widely expressed on the cell surface and processed upon activation. In this report, we establish the capacity for Ecrg4 to mediate changes in the tumor-associated innate immune response and the effect of Ecrg4 expression on tumor burden in orthotopic brain tumor models and map a thrombin-processing site in the C-terminus of Ecrg4 that mediates the mobilization and chemotaxis of inflammatory monocytes. Thus, while Ecrg4 has been proposed to have a role in inhibiting tumor growth, a paracrine mechanism of action that accounts for its cell surface expression24 and shedding18 has not been addressed in tumor models. In this report, we provide evidence that a paracrine mechanism of Ecrg4 action leads to the reversal of the immunosuppressive glioma microenvironment by enhancing survival through the activation of microglia and recruitment of monocytes to the brain.