Aberrant activation of Wnt/β-catenin signaling plays a central role in the pathogenesis of a wide variety of malignancies and is typically caused by mutations in core Wnt pathway components driving constitutive, ligand-independent signaling. In multiple myelomas (MMs), however, these pathway intrinsic mutations are rare despite the fact that most tumors display aberrant Wnt pathway activity. Recent studies indicate that this activation is caused by genetic and epigenetic lesions of Wnt regulatory components, sensitizing MM cells to autocrine Wnt ligands and paracrine Wnts emanating from the bone marrow niche. These include deletion of the tumor suppressor CYLD, promotor methylation of the Wnt antagonists WIF1, DKK1, DKK3, and sFRP1, sFRP2, sFRP4, sFRP5, as well as overexpression of the co-transcriptional activator BCL9 and the R-spondin receptor LGR4. Furthermore, Wnt activity in MM is strongly promoted by interaction of both Wnts and R-spondins with syndecan-1 (CD138) on the MM cell-surface. Functionally, aberrant canonical Wnt signaling plays a dual role in the pathogenesis of MM: (I) it mediates proliferation, migration, and drug resistance of MM cells; (II) MM cells secrete Wnt antagonists that contribute to the development of osteolytic lesions by impairing osteoblast differentiation. As discussed in this review, these insights into the causes and consequences of aberrant Wnt signaling in MM will help to guide the development of targeting strategies. Importantly, since Wnt signaling in MM cells is largely ligand dependent, it can be targeted by drugs/antibodies that act upstream in the pathway, interfering with Wnt secretion, sequestering Wnts, or blocking Wnt (co)receptors.

AIM: To investigate the CDH1, DLEC1 and SFRP5 gene methylation panel for advanced epithelial ovarian carcinoma (EOC).
MATERIALS & METHODS: One hundred and seventy-seven advanced EOC specimens were evaluated by methylation-specific PCR. We also used The Cancer Genome Atlas dataset to evaluate the panel.
RESULTS: The presence of two or more methylated genes was significant in recurrence (hazard ratio [HR]: 1.91 [1.33-2.76]; p = 0.002) and death (HR: 1.96 [1.26-3.06]; p = 0.006) in our cohort. In The Cancer Genome Atlas dataset, the presence of two or three methylated genes was significant in death (HR: 1.59 [1.15-2.18]; p = 0.0047) and close to the significance level in recurrence (HR: 1.37 [0.99-1.88]; p = 0.058).
CONCLUSION: The CDH1, DLEC1 and SFRP5 methylation panel is a potential prognostic biomarker for advanced EOC.

Aim: The aim of this study is to explore the prognostic significance and clinicopathological correlations of the Wnt pathway genes in a cohort of surgically treated patients with oral squamous cell carcinoma (OSCC) patients.
Settings and Design: A prospective genetic study on patients with OSCC was carried out during the period from July 2014 to January 2016. Informed consent from patients and institutional ethical approval for the study was obtained and the guidelines were strictly followed for collection of samples.
Subjects and Methods: Clinical data and mRNA expression analysis of ten genes in the canonical Wnt pathway were evaluated and their relationships with clinical and demographic variables were studied in 58 tissue samples. Wnt-3a, β-catenin, secreted frizzled-related proteins sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor 1, dickkopf-1, c-MYC, and cyclin-D1 from cancer (n = 29) and normal (n = 29) tissue samples were investigated using quantitative reverse transcription-polymerase chain reaction.
Statistical Analysis: Descriptive statistics were used to summarize the sample characteristics and clinical variables. If the data were normal, then parametric tests were used; otherwise, nonparametric alternatives were used. All the analyses were carried out using SPSS version 23.0 (IBM SPSS Inc., USA).
Results: Expression of sFRP-1, sFRP-2, and sFRP-5 in control samples and expression of c-MYC and cyclin D1 in cancer samples showed statistical significance. Significant expression of Wnt3A was observed among patients who had recurrence and were deceased.
Conclusion: Wnt3A, β-catenin, and cyclin D1 are recognized as key components of Wnt/β-catenin signaling. However, in this study, there was no significant expression of all the three genes in OSCC. The proto-oncogene c-MYC showed statistically significant upregulation in cancer tissue samples suggesting that the OSCC among South Indian population is primarily not mediated by the canonical Wnt signaling pathway.

BACKGROUND: Obesity is closely associated with colorectal cancer (CRC), but the underlying mechanism is unclear. We thus evaluated the expression of the adipokine gene family in CRC tissues and its clinicopathological implications.
METHODS: Correlations between the mRNA expression levels of the adipokine gene family (ADIPOQ, ADIPOR1/2, LEP, LEPR, RETN, RETNLB, RBP4, SFRP5, NAMPT, and SPP1) in CRC tissue and clinicopathologic factors were analyzed using data from The Cancer Genome Atlas database.
RESULTS: Tissue samples from 369 patients were analyzed, and 82 deaths occurred during follow-up (median, 670 days). Overall, mortality was associated with positive venous invasion, higher TNM stage, and increased ADIPOR1 (adiponectin receptor 1 gene) and SPP1 (secreted phosphoprotein gene 1) mRNA expression. Higher ADIPOR1 (odds ratio [OR]: 3.29, 95% confidence interval [CI]: 1.33-8.13) and SPP1 (OR: 2.31, 95%CI: 1.49-3.59) levels were independently associated with increased mortality. A Kaplan-Meier survival analysis showed shorter overall survival times in patients with higher ADIPOR1 (P = 0.006) and SPP1 (P < 0.001) expression.
CONCLUSIONS: Upregulation of ADIPOR1 and SPP1, among the adipokine gene family, in cancer tissue is associated with poor survival in CRC, suggesting a potential mechanism linking obesity and CRC. ADIPOR1 and SPP1 expression could become useful prognostic indicators after further validation.

Malignant pleural mesothelioma (MPM) is associated with asbestos exposure. Asbestos can induce chronic inflammation which in turn can lead to silencing of tumour suppressor genes. Wnt signaling pathway can be affected by chronic inflammation and is aberrantly activated in many cancers including colon and MPM.

BACKGROUND: Two molecular pathways promote the development of colorectal cancer (CRC). One is termed "microsatellite stable" (MSS) whereas the other is characterized by "microsatellite instability" (MSI or MIN). In addition, the CpG island methylation phenotype is known to be an important alteration as a third molecular type. Thus, DNA methylation is thought to provide potential biomarkers for assessment of cancer risk in normal mucosa. In addition, it is also known that colonic location is an important parameter in the development of CRC.
METHODS: We examined the surrounding normal mucosa in three parts of the colon. Next, we quantified DNA methylation levels of
RESULTS: DNA methylation levels of
CONCLUSIONS: Our results showed that DNA methylation of

Aberrant hypermethylation of Wnt antagonists has been observed in gastric cancer. A number of studies have focused on the hypermethylation of a single Wnt antagonist and its role in regulating the activation of signaling. However, how the Wnt antagonists interacted to regulate the signaling pathway has not been reported. In the present study, we systematically investigated the methylation of some Wnt antagonist genes (

Worldwide, prostate cancer (PCa) is the second cause of death from malignant tumors among men. Establishment of aberrant epigenetic modifications, such as histone post-translational modifications (PTMs) and DNA methylation (DNAme) produce alterations of gene expression that are common in PCa. Genes of the SFRP family are tumor suppressor genes that are frequently silenced by DNA hypermethylation in many cancer types. The SFRP family is composed of 5 members (SFRP1-5) that modulate the WNT pathway, which is aberrantly activated in PCa. The expression of SFRP genes in PCa and their regulation by DNAme has been controversial. Our objective was to determine the gene expression pattern of the SFRP family in prostatic cell lines and fresh frozen tissues from normal prostates (NP), benign prostatic hyperplasia (BPH) and prostate cancer (PCa), by qRT-PCR, and their DNAme status by MSP and bisulfite sequencing. In prostatic cancer cell lines, the 5 SFRPs showed significantly decreased expression levels compared to a control normal prostatic cell line (p<0.0001). In agreement, SFRP1 and SFRP5 genes showed decreased expression levels in CaP fresh frozen tissues compared to NP (p<0.01), while a similar trend was observed for SFRP2. Conversely, increased levels of SFRP4 expression were found in PCa compared to BPH (p<0.01). Moreover, SFRP2, SFRP3, and SFRP5 showed DNA hypermethylation in PCa cell lines. Interestingly, we observed DNA hypermethylation at the promoter of SFRP1 in the PC3 cell line, but not in LNCaP. However, in the LNCaP cell line we found an aberrant gain of the repressive histone posttranslational modification Histone H3 lysine 27 trimethylation (H3K27me3). In conclusion, decreased expression by DNA hypermethylation of SFRP5 is a common feature of PCa, while decreased expression of SFRP1 can be due to DNA hypermethylation, but sometimes an aberrant gain of the histone mark H3K27me3 is observed instead.

The recent development of next-generation sequencing technology for extensive mutation analysis, and beadarray technology for genome-wide DNA methylation analysis has made it possible to obtain integrated pictures of genetic and epigenetic alterations, using the same cancer samples. In this study, we aimed to characterize such a picture in esophageal squamous cell carcinomas (ESCCs). Base substitutions of 55 cancer-related genes and copy number alterations (CNAs) of 28 cancer-related genes were analyzed by targeted sequencing. Forty-four of 57 ESCCs (77%) had 64 non-synonymous somatic mutations, and 24 ESCCs (42%) had 35 CNAs. A genome-wide DNA methylation analysis using an Infinium HumanMethylation450 BeadChip array showed that the CpG island methylator phenotype was unlikely to be present in ESCCs, a different situation from gastric and colon cancers. Regarding individual pathways affected in ESCCs, the WNT pathway was activated potentially by aberrant methylation of its negative regulators, such as SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 (33%). The p53 pathway was inactivated by TP53 mutations (70%), and potentially by aberrant methylation of its downstream genes. The cell cycle was deregulated by mutations of CDKN2A (9%), deletions of CDKN2A and RB1 (32%), and by aberrant methylation of CDKN2A and CHFR (9%). In conclusion, ESCCs had unique methylation profiles different from gastric and colon cancers. The genes involved in the WNT pathway were affected mainly by epigenetic alterations, and those involved in the p53 pathway and cell cycle regulation were affected mainly by genetic alterations. © 2016 Wiley Periodicals, Inc.

BACKGROUND: The Wnt signaling pathway is abnormally activated in many human cancers. Secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and play an important role in carcinogenesis. SFRP promoter hypermethylation has often been identified in human cancers; however, the precise role of SFRPs in cutaneous squamous cell carcinoma (SCC) is unclear.
METHODS: The methylation status of the SFRP family was analyzed in an age-and sex-matched case-control study, including 40 cutaneous SCC cases and 40 normal controls, using the MassARRAY EpiTYPER system.
RESULTS: The methylation rate of SFRP1, SFRP2, SFRP4, and SFRP5 promoters was significantly higher in cutaneous SCC tissues than in adjacent tissue and normal skin samples.
DISCUSSION: Our manuscript mainly discussed the average methylation rate of SFRPs (SFRP1, SFRP2, SFRP4, and SFRP5) promoters are significantly high in tumor tissue samples and the average CpG island methylation rate among different pathological levels of cutaneous SCC between these genes are different.
CONCLUSIONS: Our findings suggest that promoter hypermethylation of SFRPs is associated with the development of carcinoma, and could be a useful tumor marker for cutaneous SCC and other types of cancers.

AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma (HCC).
METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer (COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function (GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of CpG islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nested-reverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ(2) test as appropriate.
RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC (P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies (P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates that the mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC.
CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.

Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of RSK4, SPARC, and HOXA9 were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.

BACKGROUND: There is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. Here, we investigated whether epigenetic silencing of Wnt antagonists is the biological driver for this increased Wnt activity in human tissues and how these methylation changes correlate with MSI (Microsatelite Instability) and CIMP (CpG Island Methylator Phenotype) statuses as well as known mutations in genes driving colorectal neoplasia.
METHODS: We conducted a systematic analysis by pyrosequencing, to determine the promoter methylation of CpG islands associated with 17 Wnt signaling component genes. Methylation levels were correlated with MSI and CIMP statuses and known mutations within the APC, BRAF and KRAS genes in 264 matched samples representing the progression from normal to pre-invasive adenoma to colorectal carcinoma.
RESULTS: We discovered widespread hypermethylation of the Wnt antagonists SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17 in the transition from normal to adenoma with only the Wnt antagonists SFRP1, SFRP2, DKK2 and WIF1 showing further significant increase in methylation from adenoma to carcinoma. We show this to be accompanied by loss of expression of these Wnt antagonists, and by an increase in nuclear Wnt pathway activity. Mixed effects models revealed that mutations in APC, BRAF and KRAS occur at the transition from normal to adenoma stages whilst the hypermethylation of the Wnt antagonists continued to accumulate during the transitions from adenoma to carcinoma stages.
CONCLUSION: Our study provides strong evidence for a correlation between progressive hypermethylation and silencing of several Wnt antagonists with stepping-up in Wnt pathway activity beyond the APC loss associated tumour-initiating Wnt signalling levels.

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma, and is characterised in part by activation of the Wnt signalling pathway. We previously demonstrated that constitutive activation of the Wnt signalling pathway by epigenetic silencing of secreted frizzled-related protein 4 (SFRP4) is observed in this tumour. Increasing evidence shows that the Wnt signalling pathway cross-talks with other developmental pathways, including the Hedgehog (HH) pathway. The HH pathway is stimulated by inactivating mutations of PTCH1, which have a well-described oncogenic role in basal cell carcinoma (BCC) of the skin. We employed polymerase chain reaction followed by direct sequencing to detect inactivating mutations of PTCH1 using archival tissue samples of 30 oesophageal BSCCs. The frequency of PTCH1 mutation was compared to that of Wnt component genes that we reported previously. We found PTCH1 mutations in 53.3% (16/30) of cases, revealing T1195S as a hotspot mutation. This frequency is quite high for cancers other than BCC of the skin, and PTCH1 mutations were almost mutually exclusive with mutations in APC, Axin1 and Axin2. Considering the fact that activation of Wnt signalling via down-regulation of APC and SFRP5 due to promoter methylation is observed in BCC of the skin, Wnt signalling activation in oesophageal BSCC might be a secondary effect of the PTCH1-inactivating mutations. These findings suggest that the HH and Wnt pathways coordinately contribute to tumourigenesis in oesophageal BSCC. Furthermore, this study provides a potential therapeutic application for HH pathway inhibitors in oesophageal BSCC with highly malignant potential.

BACKGROUND: Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.
RESULTS: Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.
CONCLUSIONS: The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.

DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n=14) and paraffin-embedded primary bladder tumors (n=61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p=0.026), TWIST1 (p=0.009), SFRP4 (p=0.002), and CCND2 (p=0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.

Methylation of secreted frizzle-related protein (SFRP) genes activates Wnt/ß-catenin signaling and promotes tumor development. This study investigated whether SFRP5 gene methylation causes multidrug resistance (MDR) in leukemia through the Wnt/ß-catenin signaling, leading to the upregulation of the mdr1 gene and its product, P-glycoprotein (P-gp). Methylation-specific PCR identified SFRP5 gene methylation in cultured bone mononuclear cells from 7/12 patients with acute leukemia and in four human leukemia cell lines (HL-60, Raji, U937 and KG1a). Western blotting revealed absent SFRP5 protein expression in cells from 5/7 patients with SFRP5 gene methylation and in all cell lines. Treatment with a demethylation agent (DAC) rescued SFRP5 expression. mdr1 mRNA and P-gp protein were detected in cells from 3/5 patients with absent SFRP5, and in the KG1a cell line; these cells also had the highest levels of activated ß-catenin. In cells from these three patients, DAC rescued SFRP5 expression and downregulated mdr1 and P-gp. SFRP5 protein expression was rescued in transgenic KG1a/SFRP5 cells, compared with KG1a/eGFP or untransfected KG1a cells. mdr1 and P-gp in KG1a/SFRP5 cells were downregulated. Doxorubicin IC50 values were significantly lower in KG1a/SFRP5 (0.573±0.131 μM) than in KG1a (0.963±0.115) or KG1a/eGFP (0.917±0.138) cells (P<0.05). We conclude that SFRP5 gene methylation in leukemia cells activates Wnt/ß-catenin signaling to upregulate mdr1/P-gp expression and cause MDR. Recovery of SFRP5 expression reversed MDR in the KG1a leukemia cell line. Our results suggest that modulating SFRP5 methylation could decrease MDR in leukemia patients.

Aberrant macrophage infiltration and activation has been implicated in gastric inflammation and carcinogenesis. Overexpression of Wnt5a and downregulation of SFRP5, a Wnt5a antagonist, were both observed in gastric cancers recently. This study attempted to explore whether Wnt5a/SFRP5 axis was involved in macrophage chemotaxis and activation. It was found that both Wnt5a transfection and recombinant Wnt5a (rWnt5a) treatment upregulated CCL2 expression in macrophages, involving JNK and NFκB signals. Conditioned medium from Wnt5a-treated macrophages promoted macrophage chemotaxis mainly dependent on CCL2. SFRP5 from gastric epithelial cells (GECs) inhibited Wnt5a-induced CCL2 expression and macrophage chemotaxis. In addition, Wnt5a treatment stimulated macrophages to produce inflammatory cytokines and COX-2/PGE2, which was also suppressed by SFRP5 from GECs. These results demonstrate that Wnt5a induces macrophage chemotaxis and activation, which can be blocked by GEC-derived SFRP5, suggesting that Wnt5a overproduction and SFRP5 deficiency in gastric mucosa may together play an important role in gastric inflammation and carcinogenesis.

Secreted frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway whose epigenetic downregulation have been shown to be involved in hepatocarcinogenesis. However, dysregulation of SFRPs induced by hepatitis B virus (HBV) X protein (HBx) has never been studied in HBV-related hepatocellular carcinoma (HBV-HCC). In this study, we sought to determine the clinical significance and underlying mechanism of HBx-induced SFRPs dysregulation in hepatoma cells and HBV-HCC patients. Our results showed that SFRP1 and SFRP5 expression were dramatically decreased by HBx in hepatoma cells. The repressed expression in hepatoma cells was partially rescued by a DNA methylation inhibitor and synergistically increased by a combination treatment with a histone deacetyltransferases inhibitor. In addition, we identified that SFRP1 and SFRP5 promoters were hypermethylated in both HBx-expressing hepatoma cells and HBV-HCC tissues. Downregulation of SFRP1 and SFRP5 in HBV-HCC tissues was significantly correlated with overexpression of DNA methyltransferase 1 (DNMT1) and poor tumor differentiation. HBx facilitated the binding of DNMT1 and DNMT3A to SFRP1 and SFRP5 promoters, and resulted in epigenetic silencing of SFRP1 and SFRP5. Moreover, overexpression of SFRP1, SFRP5 or RNA interference mediated silencing of DNMT1 inactivated the Wnt signaling pathway and decreased the expression levels of Wnt target genes c-Myc and CyclinD1, thus impeding HCC growth in vitro and in vivo, and regressing HBx-induced epithelial-mesenchymal transition (EMT). Our findings strongly suggest that epigenetic silencing of SFRP1 and SFRP5 by HBx allows constitutive activation of Wnt signaling pathway and hence contributes to hepatocarcinogenesis.

Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field.

OBJECTIVE: Epithelial ovarian cancer (EOC) remains the most lethal disease among gynecological malignancies. Prompt diagnosis is challenging because of the non-specific symptoms exhibited during the early stage of the disease. As a result, there is an urgent need for improved detection methods. In this study, we established a multiplex methylation-specific PCR (MSP) assay to improve the early detection of ovarian cancer, via identification of the methylation status of cell-free serum DNA.
METHODS: After screening, we chose seven candidate genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5 and OPCML) with a high frequency of methylation to construct the multiplex-MSP assay. When methylation of at least one of the seven genes was observed, the multiplex-MSP assay was considered positive. We performed retrospective and screening studies to verify the specificity and sensitivity of the assay in the detection of EOC.
RESULTS: The methylation status of cell-free serum DNA was examined in the preoperative serum of 202 patients, including 87 EOC patients (stage I, n=41; stage II-IV, n=46), 53 patients with benign ovarian tumors and 62 healthy controls. As expected, the multiplex MSP assay achieved a sensitivity of 85.3% and a specificity of 90.5% in stageI EOC, strikingly higher rates compared with a single CA125, which produced a sensitivity of 56.1% at 64.15% specificity [P=0.0036].
CONCLUSION: A multiplex MSP assay that analyzes the methylation status of cell-free serum DNA is a suitable and reliable approach to improve the early detection of ovarian cancer, potentially benefiting a broad range of applications in clinical oncology.

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

INTRODUCTION: For early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.
METHODS: We examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.
RESULTS: Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.
CONCLUSIONS: Tumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.

BACKGROUND: The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.
OBJECTIVE: We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.
RESULTS: Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).
CONCLUSIONS: We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.

BACKGROUND AND AIM: Except for genetic mutations, epigenetic changes are also involved in the development of human cancers. Recently, we have identified SOX1, SRY (sex determining region Y)-box 1, is hypermethylated in cervical cancer and ovarian cancer. Therefore, we investigated whether promoter hypermethylation of SOX1 is common in hepatocellular carcinoma (HCC).
METHODS: We used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to analyze the methyaltion level of the SOX1 promoter in seven HCC cell lines, 54 clinical HCCs, 42 cirrhotic livers, 21 livers with chronic hepatitis, and 15 control livers. Then, we employed quantitative MS-PCR (QMSP) to validate in an independent set of samples (60 paired HCCs and 30 control livers). Finally, we used luciferase reporter and colony formation assay to check the effect of SOX1 in HCC.
RESULTS: Promoter methylation of SOX1 was significantly frequent in HCC cell lines and clinical HCCs, cirrhotic livers, but not in control livers (P < 0.0001). There is a significant correlation between downregulation of SOX1 expression and promoter methylation. QMSP results confirmed that promoter hypermethylation of SOX1 is significantly more frequent in HCCs than control livers (P < 0.0001). The frequency of SOX1 methylation in patients with secreted frizzled-related proteins (SFRPs) methylation is significantly higher than in patients without SFRPs methylation (P < 0.0001). Furthermore, ectopic expression of SOX1 could suppress T-cell factor-dependent transcriptional activity and colony formation number in HCCs.
CONCLUSIONS: Concomitant epigenetic silencing of SOX1 and SFRPs through promoter hypermethylation is frequent in HCCs, and this might contribute to abnormal activation of canonical Wnt signal pathway.

Promoter hypermethylation of several tumour suppressor genes often occurs during breast carcinogenesis, but little is known about epigenetic silencing in the possible precursor columnar cell lesion (CCL). Promoter hypermethylation of 50 different tumour suppressor genes was assessed in normal breast tissue (N = 10), CCL (N = 15), ductal carcinoma in situ (DCIS) grade I originating in CCL (N = 5) and paired CCL (N = 15) with DCIS (N = 7) and/or invasive carcinoma (N = 14) by Methylation-specific multiplex ligation-dependent probe amplification. Increasing mean cumulative methylation levels were found from normal breast tissue to CCL to DCIS and invasive carcinoma (P < 0.001) with similar methylation levels in DCIS and invasive carcinoma. Methylation levels and frequencies (in the overall analysis and analysis of only the synchronous lesions) were the highest for RASSF1, CCND2, ID4, SCGB3A1 and CDH13. The methylation levels of ID4, CCND2, and CDH13 increased significantly from normal breast tissue to CCL and to DCIS/invasive carcinoma. RASSF1, SCGB3A1 and SFRP5 had significant higher methylation levels in CCL compared to normal breast tissue, but showed no significant differences between CCL, DCIS and invasive carcinoma. Also, no difference was found between CCLs with and without atypia, or CCLs with or without synchronous cancer. In conclusion, promoter hypermethylation for several established tumour suppressor genes is already present in CCLs, underlining that promoter hypermethylation is an early event in breast carcinogenesis. Atypia in CCL or the presence of synchronous more advanced lesions does not seem to be accompanied by higher methylation levels.

BACKGROUND: As one of the malignant tumors most often affecting children and young adults, Ewing sarcoma (ES) is characterized by early metastasis contributing to unfavorable prognosis. However, the molecular mechanisms responsible for ES metastasis remain poorly understood. In this study, we aimed to explore whether Wnt5a, a putative pro-metastatic factor, plays a role in ES metastasis.
METHODS: Expression of Wnt5a and CXCR4 was determined by real-time PCR or Western blot in 15 ES specimens and 4 ES cell lines, A-673, RD-ES, SK-N-MC and SK-ES-1. Expression of Wnt antagonists, SFRP1, SFRP2 and SFRP5, and some components in noncanonical Wnt pathway (p-JNK, p-cJUN and p-PKC) was also analyzed in this study. Methylation status of SFRP1, SFRP2 and SFRP5 was detected by Methylation-specific PCR (MSP). Wnt5a shRNA and pcDNA3.1 SFRP5 vector were used to abrogate Wnt5a expression and overexpress SFRP5 in ES cells, respectively.
RESULTS: Wnt5a expression was positively correlated with CXCR4 expression in ES specimens. Levels of both Wnt5a mRNA and CXCR4 mRNA were significantly higher in specimens from ES patients with metastasis at diagnosis compared with specimens from those without metastasis. Recombinant Wnt5a enhanced CXCR4 expression in ES cells, which was accompanied by increased ES cell migration, whereas Wnt5a shRNA has opposite effects. SFRP5 was methylated and silenced in ES cells, and both recombinant SFRP5 and pcDNA3.1 SFRP5 vector suppressed CXCR4 expression as well as ES cell migration. Wnt5a shRNA and recombinant SFRP5 inhibited phosphorylation of JNK and cJUN, and JNK inhibitor also reduced CXCR4 expression and cell migration in ES cells.
CONCLUSIONS: Wnt5a increases ES cell migration via upregulating CXCR4 expression in the absence of Wnt antagonist SFRP5, suggesting that Wnt5a overexpression and SFRP5 deficiency may jointly promote ES metastasis.

Secreted frizzled-related protein 5 (SFRP5) is frequently found downregulated in gastric cancer due to SFRP5 gene hypermethylation, and there is a great necessity to elucidate the role of its downregulation in gastric cancer. By binding Wnt molecules, SFRP5 is generally supposed to exert negative effects on Wnt signal pathways widely linked to human cancers. This study found that macrophages over-produced Wnt5a under the stimulation of Lipopolysaccharide (LPS) or Helicobacter pylori, the most common infectious agent in human stomach. Wnt5a-conditioned medium from macrophages enhanced cell migration and CXCR4 expression in either SFRP5-negative gastric epithelial cells (GEC) harboring SFRP5 methylation or SFRP5-positive cells treated with SFRP5 small interfering RNA (siRNA). However, such induced effect was remarkably eliminated by either Wnt5a siRNA in macrophages or treatment with recombinant SFRP5. We also found that Wnt5a-conditioned medium stimulated phosphorylation of c-jun N-terminal kinase (JNK) and c-Jun, and JNK inhibitor SP600125 blocked Wnt5a-induced CXCR4 expression and cell migration in SFRP5-negative cells. Taken together, these findings suggest that epithelium-derived SFRP5 may play a probable defensive role in impeding gastric cancer progression, characteristically by inhibiting GEC migration induced by macrophage-derived Wnt5a via JNK signaling activation.

BACKGROUND: It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.
METHODS: Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).
RESULTS: We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.
CONCLUSIONS: Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.