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.
"The loss of expression of Dab2 in ovarian cancer and growth regulatory properties in cell culture studies led to the suggestion that Dab2 is a tumor suppressor in ovarian cancer
[6,7]. Subsequently, loss or reduction of Dab2 expression was found in other cancer types including rat mammary tumors
, breast cancer
[9,10], colon cancer
, esophageal cancer
, urothelial carcinomas
, prostate cancer
, head and neck cancer
, and nasopharyngeal carcinomas
. Mechanisms were also suggested for Dab2 in epithelial organization
[10,17,18], and in the regulation of Ras/MAPK
[19-22], TGF beta
[15,23,24], and Wnt
[25-28] signaling pathways. "
[Show abstract][Hide abstract] ABSTRACT: Disabled-2 (Dab2) is an endocytic adaptor protein involved in clathrin-mediated endocytosis and cargo trafficking. Since its expression is lost in several cancer types, Dab2 has been suggested to be a tumor suppressor. In vitro studies indicate that Dab2 establishes epithelial cell polarity and organization by directing endocytic trafficking of membrane glycoproteins. Dab2 also modulates cellular signaling pathways by mediating the endocytosis and recycling of surface receptors and associated signaling components. Previously, two independent gene knockout studies have been reported, with some discrepancies in the observed embryonic phenotypes. To further clarify the in vivo roles of Dab2 in development and physiology, we designed a new floxed allele to delete dab2 gene.
The constitutive dab2 deleted embryos showed a spectrum in the degree of endoderm disorganization in E5.5 and no mutant embryos persisted at E9.5. However, the mice were grossly normal when dab2 deletion was restricted to the embryo proper and the gene was retained in extraembryonic tissues using Meox2-Cre and Sox2-Cre. Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels.
The study of the new dab2 mutant allele in embryos and embryoid bodies confirms a role for Dab2 in extraembryonic endoderm development and epithelial organization. Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss. Conditional deletion indicates that Dab2 is dispensable for organ development, when the vast majority of the embryonic cells are dab2 null. However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.
"Disabled-2 isoform expression is decreased in human breast cancer Disabled-2 is expressed as two major cellular isoforms, the fulllength p96 and the p67 isoforms, which are generated from the same transcript by alternative splicing (Xu et al, 1995). Decreased Dab2 protein expression has been shown in human breast tumours by immunohistochemistry; however, the antibody utilised does not discriminate between the two isoforms (Bagadi et al, 2006). To further extend these observations, we probed the relative mRNA abundance of the p96 and p67 isoforms of Dab2 in 12 normal human breast tissue and 12 breast cancer samples using the Human Breast Cancer Rapid-Scan Panel (Origene). "
[Show abstract][Hide abstract] ABSTRACT: Metastatic tumour cells are characterised by acquisition of migratory and invasive properties; properties shared by cells, which have undergone epithelial-to-mesenchymal transition (EMT). Disabled-2 (Dab2) is a putative tumour suppressor whose expression has been shown to be downregulated in various cancer types including breast cancer; however, its exact function in suppressing tumour initiation or progression is unclear.
Disabled-2 isoform expression was determined by RT-PCR analysis in human normal and breast tumour samples. Using shRNA-mediated technology, Dab2 was stably downregulated in two cell model systems representing nontumourigenic human mammary epithelial cells. These cells were characterised for expression of EMT markers by RT-PCR and western blot analysis.
Decreased expression of the p96 and p67 isoforms of Dab2 is observed in human breast tumour samples in comparison to normal human breast tissue. Decreased Dab2 expression in normal mammary epithelial cells leads to the appearance of a constitutive EMT phenotype. Disabled-2 downregulation leads to increased Ras/MAPK signalling, which facilitates the establishment of an autocrine transforming growth factor β (TGFβ) signalling loop, concomitant with increased expression of the TGFβ2 isoform.
Loss of Dab2 expression, commonly observed in breast cancer, may facilitate TGFβ-stimulated EMT, and therefore increase the propensity for metastasis.
British Journal of Cancer 11/2010; 103(11):1716-23. DOI:10.1038/sj.bjc.6605975 · 4.84 Impact Factor
"Previous studies suggested that DNA methylation in the promoter region of DAB2 is infrequent in esophageal and breast cancers [11,15]. In contrast to these studies, we found that promoter methylation of DAB2 is observed in 65.2% (30/46) of primary NPC. "
[Show abstract][Hide abstract] ABSTRACT: Human disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC).
We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1.
Decrease or absent of DAB2 transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant DAB2 promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways.
We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells.
BMC Cancer 06/2010; 10(1):253. DOI:10.1186/1471-2407-10-253 · 3.36 Impact Factor
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