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1.  Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients 
Clinical Epigenetics  2015;7(1):1.
The dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.
We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).
ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4307187  PMID: 25628764
Ovarian cancer; Epigenetics; ABCA1
2.  A Mathematical Model of Bimodal Epigenetic Control of miR-193a in Ovarian Cancer Stem Cells 
PLoS ONE  2014;9(12):e116050.
Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.
PMCID: PMC4278842  PMID: 25545504
3.  FT-IR Microspectrometry Reveals the Variation of Membrane Polarizability due to Epigenomic Effect on Epithelial Ovarian Cancer 
Ovarian cancer, as well as other cancers, is primarily caused by methylation at cytosines in CpG islands, but the current marker for ovarian cancer is low in sensitivity and failed in early-stage detection. Fourier transform infrared (FT-IR) spectroscopy is powerful in analysis of functional groups within molecules, and infrared microscopy illustrates the location of specific groups within single cells. In this study, we applied HPLC and FT-IR microspectrometry to study normal epithelial ovarian cell line immortalized ovarian surface epithelium (IOSE), two epithelial ovarian cell lines (A2780 and CP70) with distinct properties, and the effect of a cancer drug 5-aza-2'-deoxycytidine (5-aza) without labeling. Our results reveal that inhibition of methylation on cytosine with 5-aza initiates the protein expression. Furthermore, paraffin-adsorption kinetic study allows us to distinguish hypermethylated and hypomethyated cells, and this assay can be a potential diagnosis method for cancer screening.
PMCID: PMC4227199  PMID: 25299694
FT-IR; methylation; paraffin-adsorption; ovarian cell; 5-aza
4.  Epithelial Progeny of Estrogen-Exposed Breast Progenitor Cells Display a Cancer-like Methylome 
Cancer research  2008;68(6):1786-1796.
Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non–estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer.
PMCID: PMC4172329  PMID: 18339859
5.  Promoter hypermethylation and silencing of tissue factor pathway inhibitor-2 in oral squamous cell carcinoma 
The treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that the gene encoding tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy.
Methylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The differences in methylation levels among the groups were compared using the Mann–Whitney U test. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the discrimination ability for detecting OSCC. Cellular TFPI-2 expression was analyzed by quantitative reverse-transcription PCR before and after treatment with 5′-aza-2′-deoxycytidine and trichostatin A, to confirm whether TFPI-2 was epigenetically silenced in OSCC cells. We investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay.
TFPI-2 was hypermethylated in OSCC tissues versus normal oral tissues (P < 0.0001), with AUROC = 0.91, when using a pyrosequencing assay to quantify the methylation level. TFPI-2 silencing in OSCC was regulated by both DNA methylation and chromatin histone modification. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2, and consequently interfered with OSCC metastasis in vivo.
Our data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.
PMCID: PMC4160550  PMID: 25179542
Oral squamous cell carcinoma; DNA methylation; Biomarker; TFPI-2; Tumor suppressor gene; Metastasis; Matrix-associated serine protease inhibitor; Matrix metalloproteinase-2
6.  Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer 
Resistance to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is down-regulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-β/SMAD4 signaling. In this study, we investigated the mechanism of down-regulation of FBXO32, another SMAD4 target gene, and the clinical significance of loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines. FBXO32 methylation was seen in ovarian cancer cell lines displaying constitutive TGF-β/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced stage ovarian tumors, significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio 1.003, P<0.05). Re-expression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-β/SMAD4 signaling and FBXO32 methylation status predicts survival in patients with ovarian cancer.
PMCID: PMC2829100  PMID: 20065949
Ovarian cancer; epigenetics; TGF-β; FBXO32
7.  Low-Dose 5-Aza-2′-deoxycytidine Pretreatment Inhibits Experimental Autoimmune Encephalomyelitis by Induction of Regulatory T Cells 
Molecular Medicine  2014;20(1):248-256.
Forkhead box P3 (Foxp3) is the major transcription factor controlling the development and function of regulatory T (Treg) cells. Previous studies have indicated epigenetic regulation of Foxp3 expression. Here, we investigated whether the deoxyribonucleic acid (DNA) methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza) applied peripherally could modulate central nervous system (CNS) inflammation, by using a mouse experimental autoimmune encephalomyelitis (EAE) model. We found that disease activity was inhibited in a myelin oligodendrocyte glycoprotein (MOG) peptide–induced EAE mouse briefly pretreated with low-dose (0.15 mg/kg) 5-Aza, ameliorating significant CNS inflammatory responses, as indicated by greatly decreased proinflammatory cytokines. On the contrary, control EAE mice expressed high levels of IFN-γ and interleukin (IL)-17. In addition, 5-Aza treatment in vitro increased GFP expression in CD4+GFP− T cells isolated from GFP knock-in Foxp3 transgenic mice. Importantly, 5-Aza treatment increased Treg cell numbers, in EAE mice, at both disease onset and peak. However, Treg inhibition assays showed 5-Aza treatment did not enhance per-cell Treg inhibitory function, but did maintain a lower activation threshold for effector cells in EAE mice. In conclusion, 5-Aza treatment prevented EAE development and suppressed CNS inflammation, by increasing the number of Treg cells and inhibiting effector cells in the periphery.
PMCID: PMC4107100  PMID: 24869907
8.  ChIP-seq Defined Genome-Wide Map of TGFβ/SMAD4 Targets: Implications with Clinical Outcome of Ovarian Cancer 
PLoS ONE  2011;6(7):e22606.
Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes. Comprehensive examination of SMAD4-bound loci, revealed four distinct binding patterns: 1) Basal; 2) Shift; 3) Stimulated Only; 4) Unstimulated Only. TGFβ stimulated SMAD4-bound loci were primarily classified as either Stimulated only (74%) or Shift (25%), indicating that TGFβ-stimulation alters SMAD4 binding patterns in epithelial ovarian cancer cells. Furthermore, based on gene regulatory network analysis, we determined that the TGFβ-induced, SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells. Importantly, the TGFβ/SMAD4 target genes identified in the A2780 epithelial ovarian cancer cell line were predictive of patient survival, based on in silico mining of publically available patient data bases. In conclusion, our data highlight the utility of next generation sequencing technology to identify genome-wide SMAD4 target genes in epithelial ovarian cancer and link aberrant TGFβ/SMAD signaling to ovarian tumorigenesis. Furthermore, the identified SMAD4 binding loci, combined with gene expression profiling and in silico data mining of patient cohorts, may provide a powerful approach to determine potential gene signatures with biological and future translational research in ovarian and other cancers.
PMCID: PMC3143154  PMID: 21799915
9.  High Dickkopf-1 expression is associated with poor prognosis in patients with advanced urothelial carcinoma 
Although Dickkopf-1 (DKK1) has been demonstrated to be associated with tumorigenesis in various types of human tumors, a correlation between DKK1 and urothelial carcinoma (UC) has not been reported. In the present study, the correlation between DKK1 expression and UC progression was investigated. Seventy-five UC patients were enrolled. The expression of DKK1 in serum and UC tissue was detected by ELISA, real-time PCR and Western blotting. Prognostic significance was assessed by using Kaplan-Meier survival estimates and log-rank tests. The results showed that serum levels of DKK1 were significantly higher in the UC patients with muscle-invasive (p=0.0001) and high-grade tumors (p=0.00001) as compared to the controls. A high-serum DKK1 was also associated with poor disease-free survival in the UC patients (hazard ratio=2.44; 95% CI 1.10–5.40; p=0.028). Furthermore, DKK1 was also overexpressed in 93% (41/44) of the UC tissues. Therefore, the findings indicate that the expression of DKK1 is associated with UC progression.
PMCID: PMC3445958  PMID: 22993615
urothelial carcinoma; Dickkopf-1; disease-free survival
10.  Identification of candidate epigenetic biomarkers for ovarian cancer detection 
Oncology reports  2009;22(4):853-861.
Ovarian cancer ranks the most lethal among gynecologic neoplasms in women. To develop potential bio-markers for diagnosis, we have identified five novel genes (CYP39A1, GTF2A1, FOXD4L4, EBP, and HAAO) that are hypermethylated in ovarian tumors, compared with the non-malignant normal ovarian surface epithelia, using the quantitative methylation-specific polymerase chain reactions. Interestingly enough, multivariate Cox regression analysis has identified hypermethylation of CYP39A1 correlated with an increase rate of relapsing (P=0.032, hazard ratio >1). Concordant hypermethylation in at least three loci was observed in 50 out of 55 (91%) of ovarian tumors examined. The test sensitivity and specificity were assessed to be 96 and 67% for CYP39A1; 95 and 88% for GTF2A1; 93 and 67% for FOXD4L4; 81 and 67% for EBP; 89 and 82% for HAAO, respectively. Our data have identified, for the first time, GTF2A1 alone, or GTF2A1 plus HAAO are excellent candidate biomarkers for detecting this disease. Moreover, the known functions of these gene products further implicate dys-regulated transcriptional control, cholesterol metabolism, or synthesis of quinolinic acids, may play important roles in attributing to ovarian neoplasm. Molecular therapies, by reversing the aberrant epigenomes using inhibitory agents or by abrogating the upstream signaling pathways that convey the epigenomic perturbations, may be developed into promising treatment regimens.
PMCID: PMC2829240  PMID: 19724865
ovarian cancer; epigenetics; DNA methylation; biomarkers; quantitative methylation-specific polymerase chain reaction
11.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
PMCID: PMC1974845  PMID: 17570480

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