ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma.
BACKGROUND
Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes.


METHODS
In a methylation screening approach, we identified ECRG4 as a differentially methylated gene. We analyzed different cancer cells for ECRG4 promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The ECRG4 coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an ECRG4-eGFP fusion gene.


RESULTS
We found a high frequency of ECRG4 promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of ECRG4 was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. ECRG4 hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated in vitro by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of ECRG4 in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium.


CONCLUSIONS
ECRG4 is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.
Götze
Silke
S
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany. silke.goetze@mpi-dortmund.mpg.de
Feldhaus
Valeska
V
Traska
Thilo
T
Wolter
Marietta
M
Reifenberger
Guido
G
Tannapfel
Andrea
A
Kuhnen
Cornelius
C
Martin
Dirk
D
Müller
Oliver
O
Sievers
Sonja
S
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D005910:Glioma / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins
C476999:ECRG4 protein, human; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins