Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4

Abstract

The human open reading frame C2orf40 encodes esophageal cancer related gene-4 (Ecrg4), a newly recognized neuropeptide-like precursor protein whose gene expression by cells in vitro, over-expression in mice in vivo and knock-down in zebrafish affects cell proliferation, migration and senescence, progenitor cell survival and differentiation and inflammatory function. Unlike traditionally secreted neuropeptide precursors however, we find that Ecrg4 localizes to the epithelial cell surface and remains tethered after secretion. Here, we used cell surface biotinylation to establish that 14kDa Ecrg4 localizes to the cell surface of prostate (PC3), or kidney (HEK) epithelial cells after transfection. Accordingly, this Ecrg4 is resistant to washing cells with neutral, high salt (2M NaCl), acidic (50 mM glycine, pH 2.8) or basic (100 mM Na2CO3, pH 11) buffers. Mutagenesis of Ecrg4 established that cell tethering was mediated by an NH2-terminus hydrophobic leader sequence that enabled both trafficking to the surface and tethering. Immunoblotting analyses however showed that different cells process Ecrg4 differently. Whereas PC3 cells release cell surface Ecrg4 to generate soluble Ecrg4 peptides of 6 to 14 kDa, HEK cells do neither and the 14kDa precursor resembles a sentinel attached to the cell surface. Because a phorbol ester treatment of PC3 cells stimulated Ecrg4 release from, and processing at, the cell surface, these data are consistent with a multifunctional role for Ecrg4 that is dependent on its cell of origin and the molecular form produced.

C2ORF40suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes

“…20 Moreover, further processing of this 148-amino acid molecule into smaller peptides is reportedly required for its inhibitory function 24 and smaller processed peptides are found in cell culture medium. 25,26 Thus, C2ORF40 is not only a prognostic biomarker and potential therapy target in breast cancer; C2ORF40 gene product or C2ORF40-derived peptide might potentially be a suitable biotherapeutic reagent for breast and other types of tumors. Restoring C2ORF40 expression in tumors, either by epigenetic therapy or application of recombinant C2ORF40-derived peptide, may represent a promising therapeutic approach for breast cancers.…”

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.…”