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.

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.

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.

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.

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.