Abstract
VWA2 encodes AMACO, a secreted protein up-regulated in most colorectal carcinomas (CRC), constituting a promising biomarker. The mechanism responsible for its aberrant up-regulation has not been previously described. In this work, we analyzed VWA2 DNA methylation in over 400 primary CRCs. No epigenetic alterations were found in its promoter-associated CpG island. However, the region located downstream of the transcriptional start site was hypomethylated in most CRCs. ChIP-Seq revealed increased levels of the active mark H3K4me3 and reduction of the repressive mark H3K27me3. In contrast, several CRC cell lines exhibited hypermethylation of VWA2. 5-AZA-2-deoxycitidine treatment led to transcriptional activation of VWA2, supporting a functional link between DNA methylation and transcription. VWA2 expression in primary CRCs correlated with that of Myc and Myc-target genes. Transcriptional up-regulation of VWA2 is extremely frequent (78%) and strong (average fold change >15) in CRC, but not in other types of cancer. VWA2 undergoes hypomethylation in the majority of CRCs. This alteration could partly underlie the previously reported over-expression of AMACO. Co-expression profiling suggests that VWA2 might be a constituent of a larger oncogenic transcriptional program regulated by c-Myc. Up-regulation of VWA2 is virtually exclusive of CRC, reinforcing its potential as a specific biomarker.
Highlights
The human gene VWA2, located in chromosome 10q25.3, encodes the von Willebrand factor A domain-containing protein 2 (Uniprot Q5GFL6, known as AMACO)[1,2]
The resultant sequence mapped at a NotI site located 637 bp downstream of the transcriptional start site (TSS) of the gene VWA2, outside the CpG island (CGI) that completely covers its first exon (Fig. 1a)
We extended the number of colorectal cancers (CRC) cases analyzed by methylation sensitive amplified fragment length polymorphism (MS-AFLP) to a total of 81
Summary
The human gene VWA2, located in chromosome 10q25.3, encodes the von Willebrand factor A domain-containing protein 2 (Uniprot Q5GFL6, known as AMACO)[1,2]. Double immunoelectron microscopy labeling on P0 mouse skin showed that Fras[1] and AMACO co-localized in distinct regions, named anchoring plaques, in the dermis close to the basal membrane. Based on these observations, it has been suggested that the functions of AMACO in tumors somehow mimic its role during development, inducing matrix remodeling and signaling that might promote metastases[7]. In 2005 AMACO was found to be aberrantly up-regulated in about 80% of human colorectal cancers (CRC), and in the majority of the colonic adenomas This protein was named colorectal cancer secreted protein 2 (CCSP-2)[10]. Since AMACO is an extracellular-matrix protein that can be secreted into the blood stream, detection of AMACO in plasma was proposed as a candidate serological biomarker of colon neoplasia, and specific immunodetection methods have been developed based on AMACO overexpression[11]
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