Frequent loss of Dab2 protein and infrequent promoter hypermethylation in breast cancer

Abstract

Disabled-2 (Dab2), a putative tumor suppressor protein, is lost in 80-90% ovarian tumors and ovarian/breast cancer cell lines. The clinical significance of Dab2 protein in breast cancer remains yet unknown. Immunohistochemical analysis of Dab2 protein showed no detectable expression in 67/91 (74%) breast tumors, while all 10 normal tissues showed presence of Dab2 protein. We hypothesized that epigenetic silencing of Dab2 may account for loss of protein in breast cancer. Methylation of Dab2 exon 1, a putative promoter, was analyzed in six breast cancer cell lines and in 54 primary breast tumors by methylation specific PCR. Methylation was observed in MDA-MB-231 and MDA-MB-157 cells and in 6 of 54 (11%) primary breast tumors that also showed loss of Dab2 protein. Expression of Dab2 transcripts was detected in all cell lines except MDA-MB-157. However, none of these six cell lines showed detectable levels of Dab2 protein by western blotting, while non-malignant mammary epithelial cell line MCF 10A showed Dab2 protein expression. To our knowledge this is the first report showing low frequency of Dab2 (putative) promoter methylation (11%) in primary breast tumors. Frequent loss of Dab2 protein (74%) suggest that hypermethylation of Dab2 promoter may only be one of the mechanisms accounting for its loss in breast cancer. Further, in silico analysis of Dab2 3'-UTR revealed existence of miRNA complimentary to this region of the gene, suggesting microRNA mediated targeting of Dab2 mRNA might account for loss of the protein in breast cancer.

Epigenetic downregulation of human disabled homolog 2 switches TGF-β from a tumor suppressor to a tumor promoter

“…These results are consistent with previous observations, demonstrating that DAB2 is downregulated in numerous other human tumor types (22)(23)(24)(25)(26)(27)(28)(29). Analysis of the promoter region of the DAB2 gene revealed the presence of 53 CpG dinucleotides within a predicted CpG island, prompting to us to investigate aberrant promoter methylation as a potential mechanism of DAB2 silencing.…”

“…Using the weighted histoscore system, we observed a wide range of DAB2 expression in this TMA (Supplemental Figure 3). Importantly, and consistent with the microarray analysis, we found that patients with low-level DAB2 protein expression (histoscore, <70) had a significantly worse overall survival (DAB2 low average survival, 25.03 months, DAB2 high average survival, 36.62 months; log-rank c 2 test = 4.54, P = 0.033; Figure 3B). Overall survival decreased further still with even lower DAB2 expression (histoscore, <40; DAB2 low average survival, 20.61 months, DAB2 high average survival, 35.22 months; log-rank c 2 test = 7.04, P = 0.008; Supplemental Figure 4).…”

“…DAB2 was originally identified as DOC2 a gene downregulated in ovarian carcinomas (22,23). Subsequent studies have shown that DAB2 downregulation occurs in prostate (24), breast (25), esophageal (26), endometrioid (27), urothelial (28), and hepatocellular carcinomas (29), suggesting that DAB2 has tumor suppressor activity.…”

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation

“…Moreover, DAB2 was shown to regulate the Wnt signaling pathway (33) and the differentiation of Tregs (34). Because Dab2 is actively downregulated in various tumor cell lines, it has been proposed to be a tumor suppressor (35)(36)(37)(38)(39). IFN-γ, which promotes M1 phenotypic polarization in macrophages, induces the expression of IFN consensus sequence-binding protein (ICSBP, also known as IRF8), which in turn transcriptionally represses Dab2 (40).…”

Establishment and characterisation of two novel breast cancer cell lines

“…Genomic DNA was isolated from the cells using the phenolechloroform method (Blin and Stafford, 1976) and subjected to bisulphite treatment, as described previously (Bagadi et al, 2007). Methylation specific PCR was carried out using 50e100 ng of bisulphite treated DNA in a PCR mixture containing: 16.6 mM ammonium sulphate; 67 mM Tris (pH 8.8); 6.7 mM MgCl 2 ; 10 mM b-mercaptoethanol; 1.25 mM each dNTPs and primers 1.6 mM each in a 25 ml reaction.…”