The data reported in this study implicate ECRG4 as a candidate tumor suppressor gene at 2q12.2 that is frequently hypermethylated and transcriptionally down-regulated in colon carcinomas and other types of human cancers. Cancer cells are characterized by extensive epigenetic DNA alterations, including global hypomethylation as well as focal hypermethylation at CpG islands in gene promoters. DNA hypermethylation constitutes a major cause of aberrant gene silencing in cancer . However, not all aberrantly silenced genes also play functional roles in tumor development. In analogy to somatic mutations in tumor cells, which in part are neutral passenger mutations, some methylation events might be epigenetic passengers . Therefore, each candidate tumor suppressor gene identified by methylation screening methods has to be carefully evaluated for its role in tumor development and progression.
In the course of a methylation screening approach, we identified ECRG4 as a gene differentially methylated in sarcoma versus normal cells. Previously, ECRG4 had been found to be hypermethylated in esophageal cancer . To investigate its possible role in human cancer, we (1) analyzed different tumor entities for ECRG4 promoter methylation, (2) revealed a correlation between the methylation and gene silencing, (3) measured the effect of ECRG4 re-expression on the proliferation of colon cancer cells, and (4) determined the cellular localization of the ECRG4 protein.
Initially, we found the ECRG4 promoter to be frequently hypermethylated in a broad spectrum of human tumor cell lines. To exclude that hypermethylation is limited to tumor cells in culture we carried out qualitative and semiquantitative methylation analysis of primary cancer samples. In colorectal carcinomas, we found a high frequency of methylation, but, as the analyzed samples were all from advanced staged tumors, correlations to clinical features were not possible. We also found frequent methylation in astrocytic gliomas, in which the methylation frequency increased with advancing tumor grade, being particularly common in glioblastomas. This result is consistent with recent reports that ECRG4 methylation is associated with advanced disease and poor prognosis in esophageal and prostate cancer patients [18, 31]. Our findings of frequent ECRG4 methylation in various cancer cell lines may suggest that ECRG4 might be methylated in even other tumor types. Hence, ECRG4 might belong to the set of genes that are frequently methylated in multiple types of cancer. Another example is the pro-apoptotic gene RASSF1A, which is rarely mutated but often hypermethylated in various kinds of epithelial, mesenchymal and neuroectodermal tumors . Such genes often regulate processes that are crucial for cancer development, such as apoptosis or cell cycle progression.
Our data also indicate that ECRG4 promoter methylation strongly correlates with transcriptional silencing. None of the methylated cancer cell lines showed expression of ECRG4 transcripts. Furthermore, the expression level of ECRG4 was significantly lower in colon cancer samples than in normal mucosa, which agrees with the recent result that ECRG4 expression is lower in esophageal cancer than in surrounding normal epithelium . It should be noted that two normal colon tissues also showed weak ECRG4 expression. This could be due to a methylation field effect in normal tissue surrounding tumor tissue. Alternatively, it cannot be ruled out that ECRG4 silencing might be regulated by other mechanisms than DNA methylation. Then again, ECRG4 expression could be reactivated by demethylating treatment, which is a known feature of epigenetically silenced tumor suppressor genes [33, 34].
By ECRG4 overexpression or ECRG4 treatment, we demonstrate an inhibitory effect of this protein on colorectal carcinoma cell growth. Using two independent assays of cell viability and cell proliferation, we found reduced growth rates after ECRG4 transfection or culturing of cells in ECRG4 containing medium. An additional hint supporting a growth suppressive effect of ECRG4 in tumor cells comes from stabile transfection experiments, in which we observed that the expression of the transgene rapidly declined, presumably by de novo methylation (data not shown). Recent findings show that ECRG4 acts as a tumor suppressor in esophageal cancer cells in vitro and in vivo . Our data provide first hints that ECRG4 may serve a tumor suppressor function also in colorectal carcinoma cells.
Finally, we demonstrate that ECRG4 is a secreted protein. The ECRG4 gene codes for a small protein of 148 aa (17 kDa) of yet unknown function and without significant homology to other known proteins, except for a 31% homology to the mouse IgG V region . A putative N-terminal signal peptide suggests that ECRG4 might be a secreted protein. We found that an ECRG4-eGFP fusion protein localized to the Golgi apparatus within the cell, which would be consistent with the secretory pathway of proteins. In addition, we detected the ECRG4-eGFP fusion protein in the cell culture medium of transfected cells. Recently, the ECRG4 protein was renamed in the Uniprot database http://www.uniprot.org as "augurin", a protein which had been identified in a bioinformatics approach as a putative new peptide hormone . Mirabeau et al. showed that augurin is a secreted protein which is processed from its prepro-form. However, only limited augurin expression was found in murine endocrine tissues. In addition to its potential expression in endocrine glands, ECRG4 is expressed in normal epithelium of the colon and esophagus and in fat tissue as shown here and in other studies [18, 19]. The findings of Mirabeau et al. (2007) support the hypothesis that ECRG4 functions as an extracellular signaling molecule. Interestingly, unlike other peptide hormones, the signaling of ECRG4 is not growth promoting but rather growth limiting.
In conclusion, we show that the ECRG4 gene is frequently hypermethylated in a variety of human cancer cells. Hypermethylation associates strongly with transcriptional silencing in colorectal carcinomas. Furthermore, ECRG4 displays growth-inhibitory effects on colon cancer cells in vitro and may function as a secreted extracellular signaling molecule. Thus, restoring ECRG4 expression in the tumor, either by epigenetic therapy or application of recombinant protein, may represent a promising novel therapeutic approach in various types of cancers.