Identification of 20 genes aberrantly methylated in human breast cancers

Abstract

Aberrant methylation of CpG islands (CGI) not only plays a role in gene silencing, but is also a potential cancer biomarker. To identify more CGI aberrantly methylated in human breast cancers, we carried out a genome-wide search for aberrant methylation, using methylation-sensitive-representational difference analysis. CGI in 5 0 upstream regions of 20 genes, TSPAN-2, AK5, LOC284999, HOXD11, FLJ25161, XT3, PCDH10, PCDHGB6, SIM1, LOC346978, COE2, TDH (FLJ25033), LOC346419, FLJ33790, GJB2, AMN, LOC201164, DLX4, DCC and FOXA2, were found to be methylated in at least one of 8 breast cancer cell lines. Fifteen of the 20 genes were methylated in more than one of 21 primary breast cancers in Stages I or II, and especially, those of LOC346978, HOXD11, SIM1, PCDHGB6 and FLJ25161 were methylated in more than 10 cancers. All the breast cancers had some aberrant methylation. Key words: MS-RDA; breast cancer; CpG island; methylation Aberrant methylation of CpG islands (CGI) in promoter regions is known to be involved in silencing of tumor-suppressor genes. 1,2Because the aberrant methylation of tumor-suppressor genes is specific to cancer cells, its detection in biopsy specimens and free DNA in the blood is drawing attention as a tumor marker.3 Aberrantly methylated DNA molecules embedded in the vast majority of normal, unmethylated DNA molecules can be sensitively detected by various techniques, such as methylation-specific PCR (MSP) and quantitative MSP.4,5 Detection of aberrant methylation is more advantageous than detection of mutation in terms of sensitivity. One of the current limitations to the use of aberrant methylation as a tumor marker is the lack of target CGI specifically methylated in cancer cells.Uncovering novel aberrantly methylated CGI is important not only for discovery of novel tumor-suppressor genes, but also for identification of tumor markers. To make a genome-wide screening for novel aberrant methylation, restriction landmark genomic scanning (RLGS), methylation-sensitive-representational difference analysis (MS-RDA) and cDNA microarrays combined with treatment with a demethylating agent have been used.6-8 Most studies, including our previous analysis of human breast cancers, 9,10 have attempted to identify genes silenced in cancers. These studies have paid little attention to the other CGI that are potentially useful as tumor biomarkers even if not related with gene silencing.In our study, to search for aberrantly methylated 5 0 CGI as candidate tumor markers, we analyzed a human breast cancer cell line, MDA-MB-468, by MS-RDA with recent modifications for efficient isolation of CGI. 11,12Material and methods Cell lines, tumor samples, and DNA/RNA extraction MCF-7, ZR-75-1, BT474, T-47D, MDA-MB-231, MDA-MB-468 and SK-BR-3 were purchased from the American Type Culture Collection (Rockville, MD), and YMB-1-E was obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan). Human mammary epithelial cells (HMEC) were purchased from Cambrex (East Rutherford, NJ). Breast cancers (1 carcinom...

Induction of sodium iodide symporter gene and molecular characterisation of HNF3β/FoxA2, TTF-1 and C/EBPβ in thyroid carcinoma cells

“…Similarly, co-transfection of HNF3b/FoxA2 and TTF-1 did not induce the expression of TPO, TG or TSHR mRNAs in BHP cells (data not shown), indicating that other molecule(s) might be required to induce endogenous mRNA expression of these thyroid-related differentiation genes. Interestingly, HNF3b/FoxA2 is a methylated gene in breast and lung cancer cells; and overexpression of HNF3b/FoxA2 in a lung cancer cell line leads to growth arrest and apoptosis (Halmos et al, 2004;Miyamoto et al, 2005). Here, we report for the first time that the presence of aberrant methylation of HNF3b/FoxA2 in thyroid carcinoma cell lines, and forced expression of the gene, resulted in growth inhibition.…”

“…The HNF3b/FoxA2 gene has a CpG island in its promoter; and the region is often methylated in breast and lung cancers (Halmos et al, 2004;Miyamoto et al, 2005) prompting us to examine thyroid carcinoma cells. The great majority of the 21 CpG sites in the promoter were methylated in BHP (subline 2-7) and NPA papillary thyroid carcinoma cells ( Figure 4A FRO (data not shown).…”

Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach

“…In this context it is of particular interest that DLX4 has also been suggested to regulate trophoblast invasion (Quinn et al, 1998). We should also mention that a CpG island in the 5 0 upstream region of DLX4 was recently reported to be methylated in breast cancer cell lines (Miyamoto et al, 2005).…”

Aberrant methylations in cancer cells: Where do they come from?

“…Now, owing to completion of the sequencing of the human genome, it has become evident that, even if limited to CGI in promoter regions or putative promoter regions (5′ regions) of genes, most cancers have multiple aberrant methylations. (18)(19)(20)(21) These aberrant methylations are considered to provide a good source of tumor markers,…”

“…Now, owing to completion of the sequencing of the human genome, it has become evident that, even if limited to CGI in promoter regions or putative promoter regions (5′ regions) of genes, most cancers have multiple aberrant methylations. (18)(19)(20)(21) These aberrant methylations are considered to provide a good source of tumor markers, (22) and targets for chemotherapeutics. (23,24) In contrast with these rapidly progressing areas of epigenetics, the etiology of aberrant DNA methylation needs more attention.…”