Epigenetic silencing of DSC3 is a common event in human breast cancer

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure

Gene expression profiles in human gastric cancer: expression of maspin correlates with lymph node metastasis

To seek for a candidate gene that would regulate tumour progression and metastasis in gastric cancer, we investigated gene expression profiles by using DNA microarray. Tumour tissue and adjacent normal tissue were obtained from 21 patients with gastric cancer and then examined for their gene expression profiles by the Gene Chip® Human U95Av2 array, which includes 12 000 human genes and EST sequences. A total of 25 genes were upregulated and two genes were downregulated by at least four-fold in the tumour tissue. In a further analysis according to lymph node metastasis, the expressed levels of maspin, as well as carcinoembryonic antigen and nonspecific crossreacting antigen were significantly higher in tumours with lymph node metastasis than in those without it. Maspin expression in 85 gastric cancer patients was further investigated by using immunohistochemistry. Maspin expression was not observed in normal gastric epithelia without intestinal metaplasia. In contrast, maspin was expressed in 74 of 85 tumour tissues. There was a significant correlation between the incidence of maspin-positive tumour staining and lymph node metastasis. These results suggest that maspin has a potential role for tumour metastasis in gastric cancer

Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

<p>Abstract</p> <p>Background</p> <p>Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer.</p> <p>Methods</p> <p>Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay.</p> <p>Results</p> <p>The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines.</p> <p>Conclusions</p> <p>CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.</p

Maspin is a tumour suppressor that inhibits breast cancer tumour metastasis in vivo

Maspin is a member of the serpin family of serine proteases and functions as a tumour suppressor. A study using a new syngeneic mouse model for breast cancer suggests that maspin can inhibit metastasis in vivo

Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease

Glycomics using mass spectrometry

Mass spectrometry plays an increasingly important role in structural glycomics. This review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans, the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids. The given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation, lysosomal storage diseases, autoimmune diseases and cancer

Adenovirus-mediated manganese superoxide dismutase gene transfer to hamster cheek pouch carcinoma cells

As a first step in evaluating the tumor suppressor activity of the manganese superoxide dismutase (MnSOD) gene on established tumors in vivo, we used adenovirus-mediated gene transfer as a means of delivering the MnSOD cDNA to hamster cheek pouch carcinoma (HCPC-1) cells in vitro. HCPC-1 cells were transduced with the adenovirus-MnSOD construct (AdMnSOD) at multiplicities of infectivity (MOI) of 0, 10, 25, 50, 100, 150, and 200 MOI or with the adenovirus-LacZ reporter gene construct (AdLacZ) at 100 MOI. Dose-dependent increases in MnSOD immunoreactivity were seen on Western blotting and indirect immunofluorescence microscopy with increasing AdMnSOD titers. Maximal immunoreactivity was observed at 100 MOI AdMnSOD with both techniques. Moreover, we observed a concomitant 6-7-fold increase in MnSOD activity compared with parental cell levels that also peaked at 100 MOI AdMnSOD. To determine the effect of transgene-expressed MnSOD on tumor cell behavior, we examined cell growth, plating efficiency, and anchorage- dependent growth in soft agar. Cell number measured on day 13 decreased approximately 50% with 100 MOI AdMnSOD (P < 0.05) compared with parental cells. Moreover, cell doubling time increased from 38 to 44 h with 100 MOI AdMnSOD. Plating efficiency and cell growth in soft agar decreased approximately two-thirds with 100 MOI AdMnSOD (P < 0.001). These assays of the transformed phenotype in vitro all appeared to show maximal effect with 100 MOI AdMnSOD. As tumor growth in vivo is most predictable by a combination of these in vitro data, our results suggest that if MnSOD can be effectively delivered to a tumor in vivo using the adenovirus paradigm, effective tumor growth suppression can be observed