The INK4B-ARF-INK4A (INK/ARF) locus is composed of three tumor suppressor genes, which are kept silenced by DNA methylation in different cancer types. In addition, a non-coding RNA (ANRIL) is transcribed in the anti-sense orientation upstream of the ARF gene. The resulting divergent promoter region is bound by the chromatin insulator protein CTCF in association with histone H3 tri-methylated on lysine 4, irrespective of transcription of ANRIL and ARF. Methylation of the overlapping CpG island abolishes CTCF binding and the associated modification, which can be restored by 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment. shRNA knock down of CTCF expression dramatically reduces the induction of ANRIL and ARF, but also that of INK4A and INK4B expression by 5-Aza-dC. We propose that CTCF is an essential factor for transcription of the INK/ARF locus and that abrogation of its binding by DNA methylation contributes to the permanent silencing of several genes of the locus in tumors. less...

E-cadherin (E-cad) is an adhesion molecule associated with tumor invasion and metastasis. Its down-regulation is associated with poor prognosis for many epithelial tumor types. We have profiled E-cad in the NCI-60 cancer cell lines at the DNA, RNA, and protein levels using six different microarray platforms plus bisulfite sequencing. Here we consider the effects on E-cad expression of eight potential regulatory factors: E-cad promoter DNA methylation, the transcript levels of six transcriptional repressors (SNAI1, SNAI2, TCF3, TCF8, TWIST1, and ZFHX1B), and E-cad DNA copy number. Combined bioinformatic and pharmacological analyses indicate the following ranking of influence on E-cad expression: (1) E-cad promoter methylation appears predominant, is strongly correlated with E-cad expression, and shows a 20% to 30% threshold above which E-cad expression is silenced; (2) TCF8 expression levels correlate with (-0.62) and predict (P < 0.00001) E-cad expression; (3) SNAI2 and ZFHX1B expression levels correlate positively with each other (+0.83) and also correlate with (-0.32 and -0.30, respectively) and predict (P = 0.03 and 0.01, respectively) E-cad expression; (4) TWIST1 correlates with (-0.34) but does not predict E-cad expression; and (5) SNAI1 expression, TCF3 expression, and E-cad DNA copy number do not correlate with or predict E-cad expression. Predictions of E-cad regulation based on the above factors were tested and verified by demethylation studies using 5-aza-2'-deoxycytidine treatment; siRNA knock-down of TCF8, SNAI2, or ZFHX1B expression; and combined treatment with 5-aza-2'-deoxycytidine and TCF8 siRNA. Finally, levels of cellular E-cad expression are associated with levels of cell-cell adhesion and response to drug treatment. Mol Cancer Ther; 9(1); 1-16. less...

BACKGROUND: We previously demonstrated that a putative anti-tumor gene, peroxisomal membrane protein 4, 24 kDa (PMP24 or PXMP4), is silenced via DNA methylation of a CpG island in its 5' flanking region (5'-CGI) in prostate cancer (PCa) cells. METHODS: To identify demethylation hypersensitive site(s) in PMP24 5'-CGI, PC-3 cells with methylated 5'-CGI were treated with a low-dose of 5-aza-2'-deoxycytidine (5-aza-dC) just sufficient to reactivate gene expression, referred as the limited demethylation approach. Gel shift assays and promoter analyzes were performed to demonstrate the role of the hypersensitive site in PMP24 gene regulation. Transfection of a methylated oligonucleotide corresponding to the hypersensitive site was conducted to determine the effect of site-specific methylation on the gene expression. Bisulfite sequencing analysis was performed to reveal the methylation status of PMP24 promoter in cultured cells and microdissected samples. In situ hybridization was applied to determine expression positivity of PMP24 mRNA. RESULTS: A 5-aza-dC hypersensitive site encompasses two CpG dinucleotides in intron 1 was identified. Methylation of the first, but not the second, CpG dinucleotide of this site disrupted DNA-protein interactions and suppressed the gene expression. Using archival specimens, we found the first CpG dinucleotide of the hypersensitive site is hypermethylated with a loss of PMP24 mRNA expression in microdissected PCa cells when compared to normal prostatic epithelial cells. CONCLUSIONS: These findings support a critical role for a single intronic CpG dinucleotide in PMP24 gene regulation through DNA methylation. The data suggest that methylation-mediated silencing of PMP24 is a molecular event associated with prostate carcinogenesis. Prostate (c) 2010 Wiley-Liss, Inc. less...

Myelodysplastic syndromes (MDS) represent a collection of stem cell disorders characterized by impaired hematopoiesis resulting in low peripheral blood counts. The majority of patients with MDS present with symptoms related to anemia; however, bleeding and infection are the most common causes of death. The median age of diagnosis is 72 and the median survival is 2.5 years. Lenalidomide, azacitidine, and decitabine are all FDA-approved agents to treat MDS; however, the only potential cure for MDS remains stem cell transplantation. less...

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer. less...

Hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2) encodes Kunitz-type protease inhibitor that regulates HGF activity. Inspection of the human HAI-2/SPINT2 locus uncovered a large and dense CpG island within the 5' region of this gene. Analysis of cultured human gastric tumor lines indicated that HAI-2/SPINT2 expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing (BSP) confirmed the densely methylated HAI-2/SPINT2 promoter region. Forced expression of HAI-2/SPINT2 induced cell apoptosis, suppressed anchorage independent growth in vitro and tumor growth in vivo. We investigated HAI-2/SPINT2 aberrant methylation in patients with gastric cancer. The HAI-2/SPINT2 methylation was found preferentially in cancerous tissues (30 of 40, 75%) compared with non-tumor tissues (no methylation was detected), indicating that this aberrant characteristic is common in gastric malignancies. In conclusion, epigenetic inactivation of HAI-2/SPINT2 is a common event contributing to gastric carcinogenesis and may be a potential biomarker for gastric cancer. (c) 2010 UICC. less...

PURPOSE: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer. EXPERIMENTAL DESIGN: We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment. RESULTS: We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2'-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history. CONCLUSIONS: Collectively, these results suggest that cigarette smoke-induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer. Clin Cancer Res; 16(2); 398-409. less...

The pyrimidine analogs, 5-azacytidine (azacitidine, Vidaza((R))) and its deoxy derivative, 5-aza-2'-deoxycytidine (decitabine, Dacogen((R))), are the most widely used inhibitors of DNA methylation which trigger demethylation leading to a consecutive reactivation of epigenetically silenced tumor suppressor genes in vitro and in vivo.Although the antileukemic capacity of decitabine has been known for almost 40 years, its therapeutic potential in hematologic malignancies is still under intensive investigation. Multiple clinical trials have shown the promising activity of low-dose decitabine in AML, MDS, CML, and hemoglobinopathies, whereas its efficacy in solid tumors is rather limited.Clinical responses appear to be induced by both epigenetic alterations and the induction of cell-cycle arrest and/or apoptosis. Recent clinical trials have been investigating new dosing schedules, routes of administration, and combination of decitabine with other agents, including histone deacetylase (HDAC) inhibitors. less...

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in six prostate cancer cell lines and non-malignant prostate epithelial cells. 38 miRNAs showed increased expression in any prostate cancer cell line after 5-aza-2'-deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR-193b was methylated in 22Rv1 cell line at a CpG island approximately 1kb upstream of the miRNA locus. Expressing miR-193b in 22Rv1 cells using pre-miR-193b oligonucleotides caused a significant growth reduction (p<0.001) resulting from a decrease of cells in S-phase of the cell cycle (p<0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR-193b -expressing 22Rv1 cells (p<0.01). Altogether, our data suggest that miR-193b is an epigenetically silenced putative tumor suppressor in prostate cancer. (c) 2010 UICC. less...

Aberrant expression of microRNA (miRNA) has been reported in various cancers. To clarify the role of miRNA in gastric carcinogenesis, we performed miRNA microarray analysis and investigated expression changes of miRNAs in a 5-aza-2'-deoxycytidine (5-aza-CdR)-treated gastric cancer cell line, KATO-III. On microarray analysis, 5 miRNAs were found to be up-regulated (>3-fold) after 5-aza-CdR treatment compared with untreated cells. Among them, miR-181c and miR-432AS exhibited CpG islands in their upstream sequences on computational analysis, and their up-regulation was verified by reverse transcription-PCR analyses. In particular, miR-181c up-regulation was found not only in KATO-III but also in two other gastric and one colorectal cancer cell line with 5-aza-CdR treatment. Decreased expression of miR-181c was observed in 9 of 16 primary gastric carcinoma cases compared with in the corresponding non-cancerous stomach tissues. Hypermethylation signals in the upstream region of miR-181c were observed in some cultured and primary gastric carcinoma cells with negative or low miR-181c expression. Transfection of the precursor miR-181c molecule induced decreased growth of two gastric cancer cell lines, KATO-III and MKN45. As for targets of miR-181c, oncogenic NOTCH4 and KRAS were identified by cDNA microarray analysis after precursor miR-181c molecule transfection, computational searches of miRNA target databases, and reporter assaying using the 3'-UTR regions of the two genes. These results indicate that miR-181c may be silenced through methylation and play important roles in gastric carcinogenesis through its target genes, such as NOTCH4 and KRAS. less...