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.

Pancreatic cancer is the fourth leading cause of death in both males and females, with a 5-year relative survival rate of 8%. The Wnt signaling pathway has a significant role in the pathogenesis of many tumors, including those of pancreatic cancer. Hypermethylation of the Wnt inhibitory Factor-1 (WIF1) gene promoter have been detected in different types of cancer. In contrast, the anticancer effects of long-chain omega-3 PUFA (ALA) have been reported. Regarding its anticancer effects, in this study, we investigated the effects of various concentrations of omega-3 PUFA on expression level and promoter methylation of the WIF1 gene in MIA PaCa-2 cells in 24, 48, and 72 h after treatment. MIA PaCa-2 cells were treated with different concentrations of omega-3 PUFA (25, 50, 100, 250, 500, and 1000 μM). Cell viability assay was carried out followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and methylation-specific PCR (MSP). This investigation suggested that dietary consumption of omega-3 PUFAs (250-1000 μM) has a significant effect on the proliferation and WIF1 gene expression of the MIA PaCa-2 cancer cell line but no effect on the promoter methylation of this gene. Changes in promoter methylation were not observed in any of the treatments.

Wnt antagonist genes hypermethylation has been found in several tumors. Accordingly, the events that occur during the progression of adenoma to carcinoma have been characterized and include activation of the Wnt-pathway. Further, gastric adenoma (GA) is a premalignant lesion of gastric adenocarcinoma (GAC). In this paper, we focused our interesting on Wnt signaling path function in the pathogenesis of GAC.We compared the differences between low grade adenoma (LGA), high grade adenoma (HGA), GACs and corresponding normal gastric tissue (NGT). Specific indexes include the pathological characteristics of gastric neoplasia, Helicobacter pylori infection, β-catenin mutation status, and methylation status of Wnt antagonist genes.There was significant difference of β-catenin expression in patient with NGT, LGA, HGA, and GAC, the results respectively were 4.2%, 41.7%, 83.3%, and 91.7%. Only 1 GACs was detected exon 3 of β-catenin mutation. Wnt antagonist genes mRNA expression levels, such as APC, sFRP-1, Wif-1, and Dkk-1, were significantly reduced in GAC. Promoter methylation levels of the 4 genes were significantly elevated in GAC and HGA compared to NGT and LGA. However, there was no significant difference between HGAs and GACs. The β-catenin abnormal expression was correlated with hypermethylation of these 4 genes. Multiple gene concurrent methylation phenomenon was increased from NGTs to GACs; the amount of methylation genes in GACs and HGAs was more than NGTs and LGAs. The more methylation of the above-mentioned genes, the more severity of local inflammation. The infection rate of H pylori was significantly higher in patient with HGA (66.7%, 16/24) and GAC (58.5%, 14/24) than in LGAs (16.7%,4/24) (PHGA-LGA = .024, PGAC-LGA = .032). In addition, the present of H pylori also correlated with the β-catenin abnormal expression and the hypermethylation status of Wnt antagonist genes (P < .001). But other parameters in adenoma cases had no significantly related with infection of H pylori.Hypermethylation of Wnt antagonist genes may have a tight relationship with gastric tumorigenesis. And these genes may increase the incidence of GAC. Additionally, H pylori may have promotion function in GA formation.

BACKGROUND: Homeobox C6 (HOXC6) is one of several HOXC genes and is frequently overexpressed in multiple cancers. However, the function and mechanism of HOXC6 in glioma remain unclear.
METHODS: The expression level of HOXC6 and its relationship with prognosis in glioma were determined through the TCGA database. The expressions of HOXC6 mRNA in glioblastoma tissues and normal brain tissues were detected by qRT-PCR and Western blot. To explore the role of HOXC6 in glioma, a lentiviral vector that expressed HOXC6-shRNA was constructed and transfected into glioma U87 cells. The expression levels of HOXC6 and WNT inhibitory factor 1 (WIF-1) in the glioma U87 cells after transfection with HOXC6-shRNA were measured by real-time PCR and Western blot． CCK-8, colony formation and EdU assays were used to measure the effects of HOXC6 on U87 cell proliferation, and flow cytometry was used to monitor the changes in the cell cycle and cell apoptosis after transfection with HOXC6-shRNA. Xenograft tumors were examined in vivo for the carcinogenic effects and prognostic value of HOXC6 in glioma tissues.
RESULTS: In this study, HOXC6 was highly expressed in human glioma tissues, and a high expression of HOXC6 was associated with poor prognosis in GBM patients. We demonstrated that HOXC6 was highly expressed in human GBM tissues and three glioma cell lines. The knockdown of HOXC6 expression significantly inhibited the proliferation and colony formation ability of U87 cells by blocking cell cycle progression in the G0/G1 phase and induced apoptosis. In addition, we found that the mRNA and protein levels of WIF-1 were substantially increased after transfection with HOXC6-shRNA compared with Ctrl-shRNA in vitro. Consistent with the results of the in vitro assays, the xenograft assay and immunohistochemistry also demonstrated that in response to HOXC6 inhibition, the tumor growth and Ki-67 expression level were inhibited and the WIF-1 expression was increased in vivo.
CONCLUSIONS: In conclusion, the results of the current study indicate that HOXC6 promotes glioma U87 cell growth through the WIF-1/Wnt signaling pathway and HOXC6 might be a novel target in clinical treatment for gliomas.

MicroRNAs (miRNAs/miRs) are involved in the metastasis of hepatocellular carcinoma (HCC). In the present study, it was demonstrated that miR‑552 was upregulated in HCC tissues. High miR‑552 expression was associated with malignant clinicopathological features and decreased survival rates. The in vitro results indicated that miR‑552 overexpression promoted migration, invasion and epithelial‑mesenchymal transition in Hep3B cells. However, the knockdown of miR‑552 inhibited its oncogenic roles in Huh‑7 cells. Additionally, Wnt inhibitory factor 1 (WIF1) was demonstrated to be a direct target of miR‑552 in Hep3B and Huh‑7 cells. Additional experiments identified that miR‑552 promotes β‑catenin expression by increasing the phosphorylation of GSK3β at Ser9. In conclusion, the results suggested that miR‑552 may promote HCC progression by blocking WIF1‑mediated GSK3β dephosphorylation. miR‑552 may be a biomarker for predicting the outcomes of patients with HCC.

AIM: The prevalence and mortality rates of lung cancer in Xuanwei, Yunnan, China, are the highest in the world. The severe indoor air pollution caused by smoky coals with high benzo (a)pyrene (BaP) and quartz levels is the main environmental factor. The aim of this study was to investigate methylation profiles of promoters in eight genes in primary non-small cell lung cancers (NSCLC) exposed to smoky coals.
MATERIALS AND METHODS: Candidate genes including CDKN2A, DLEC1, CDH1, DAPK, RUNX3, APC, WIF1 and MGMT were determined for the promoter methylation status using Nested methylation-specific PCR (nMSP) in primary 23NSCLC tissues and in circulating tumor DNA (ctDNA) isolated from 42plasma samples (9matched to tissues) as well as 10healthy plasma samples, using Sanger sequencing to verify the results.
RESULTS: Seven of the 8genes, except MGMT, had relatively high methylation frequencies ranging from 39%-74% in tissues. Moreover, methylation frequencies in five genes identified in lung cancer plasma were 45% for CDKN2A, 48% for DLEC1, 76% for CDH1, 14% for DAPK, 29% for RUNX3, with a relatively good concordance of methylation among 9 tissues and paired plasma. However, the genes from all healthy plasma showed no methylation.
CONCLUSIONS: A panel of genes including CDKN2A, DLEC1, CDH1, DAPK and RUNX3 may be used as potential epigenetic biomarkers for early lung cancer detection. CDH1 promoter methylation was associated with lung cancer metastasis in areas of air pollution from buring of smoky coals. DLEC1 and CDH1 exhibited specific high methylation frequencies, different from previous reports.

Triptolide (TP) exhibits numerous biological activities, including immunosuppressive, anti‑inflammatory and antitumor effects. The aim of the present study was to investigate the role of TP as a potent therapeutic drug for the treatment of lung cancer and to investigate the underlying therapeutic mechanisms. Western blot analyses and reverse transcription‑quantitative polymerase chain reaction (PCR) were performed to investigate the expression of genes at transcriptional and translational levels, respectively. Methylation‑specific PCR assays were conducted to investigate whether TP affects the Wnt inhibitory factor‑1 (WIF‑1) methylation status and subsequently affects apoptosis, migration or the invasion of lung cancer cells. The results of the present study revealed that the methylation status of WIF‑1 in lung cancer cell lines A549 and H460 was significantly enhanced compared with the human normal bronchial epithelial cell line HBE, whereas treatment with TP was revealed to induce the demethylation of WIF‑1. The present study aimed to investigate whether the biological activities of TP are regulated by inhibiting the Wnt signaling pathway via an increase in WIF‑1 expression levels. The results of the present study revealed that Wnt signaling was suppressed in cells following treatment with TP, which was concluded by the downregulation of Axin 2 and β‑catenin expression. Further investigation demonstrated that the silencing of WIF‑1 expression with small interfering RNA reversed the TP‑induced upregulation of WIF‑1 expression, upregulated Axin 2 and β‑catenin expression and enhanced the activation of Wnt signaling. Notably, an upregulation of cellular tumor antigen p53 expression, and downregulation of matrix metalloproteinase‑9 (MMP‑9) and phosphorylated‑nuclear factor‑κB (NF‑κB) P65 (p‑P65) levels was observed following TP treatment. These results suggest that the Wnt, p53 and NF‑κB signaling pathways mediate the potent antitumor effects of TP. Notably, the silencing of WIF‑1 did not completely recover the levels of p53, MMP‑9 and p‑P65 in cells treated with TP compared with the control cells, thus suggesting that TP exhibits further functions in addition to the targeting of WIF‑1.

Aim: We investigated the association of WNT inhibitory factor-1 (WIF-1) gene methylation with the pathogenesis of multiple human tumors, using a meta-analysis based approach.
Materials and Methods: Electronic databases and manual search was additionally employed to retrieve relevant published literature. The cohort studies relating to tumor and WIF-1 were screened based on predefined selection criteria, and all extracted data from the selected studies were analyzed through STATA software.
Results: Sixteen studies were finally enrolled in our study involved 1112 tumor samples and 612 adjacent normal samples. The study result showed that WIF-1 gene methylations in tumor tissues were significantly higher compared with adjacent/normal tissues. The result of subgroup analysis on ethnicity revealed that in the Caucasians, Asians, and Africans, the methylation status of WIF-1 gene in tumor tissues was higher than adjacent/normal tissues. Further subgroup analysis on disease types revealed that WIF-1 gene methylation status is a widespread phenomenon that is, observed in tumor tissues of patients with multiple human tumors compared with that in adjacent/normal tissues. Interestingly, there was no significant difference in WIF-1 gene methylation between tumor tissues among patients with lung cancer, gastric cancer, astrocytoma, and adjacent/normal tissues, indicating the WIF-1 gene methylation not a general nonspecific phenomenon.
Conclusion: WIF-1 gene methylation in tumor tissues was significantly more frequent as compared to that in adjacent normal tissues, indicating that WIF-1 gene methylation may be an important event in the pathogenesis of multiple human tumors.

Wnt inhibitory factor‑1 (WIF‑1) is an important antagonist of Wnt/β‑catenin signaling by binding to Wnt ligands. The downregulation of WIF‑1 leads to the development of non‑small cell lung cancer (NSCLC). The upregulation of WIF‑1 significantly inhibits proliferation and induces apoptosis by inhibiting Wnt/β‑catenin signaling in NSCLC. However, the mechanisms underlying the inhibition of Wnt/β‑catenin signaling by WIF‑1‑mediated autophagy are poorly understood. Thus, in this study, we aimed to shed some light into these mechanisms. The upregulation of WIF‑1‑induced autophagy in NSCLC cells was detected by transmission electron microscopy, acridine orange staining, punctate GFP‑LC3 and immunoblotting‑based LC3 flux assay. Subsequently, WIF‑1‑mediated autophagy was blocked in NSCLC cells and the effects of WIF‑1‑mediated autophagy blocking were examined on the proliferation and apoptosis of NSCLC cells in vitro. Western blot analysis was used to investigate the molecular mechanisms effected by WIF‑1‑mediated autophagy in NSCLC cells. Finally, combination treatment with WIF‑1 and an autophagy agonist was used to examine the tumor growth inhibitory effects of WIF‑1 in vivo. The results revealed that the upregulation of WIF‑1 induced autophagy in NSCLC cells. WIF‑1‑mediated autophagy was demonstrated to inhibit Wnt/β‑catenin signaling by downregulating dishevelled‑2 (Dvl2), which contributed to the inhibition of the proliferation and the promotion of the apoptosis of NSCLC cells. Moreover, the induction of autophagy mediated by WIF‑1 was associated with to suppression of the activation of the phosphoinositide 3‑kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Finally, we found that transfection with a WIF‑1 gene overexpression vector in combination with treatment with the autophagy agonist, everolimus (RAD001) exerted synergistic antitumor effects on A549 subcutaneous tumor xenografts and pulmonary metastasis in mice. On the whole, the findings of this study demonstrated that WIF‑1‑mediated autophagy inhibits Wnt/β‑catenin signaling by downregulating Dvl2 expression in NSCLC cells. This may a novel molecular mechanism through which WIF‑1 inhibits Wnt/β‑catenin signaling. This study may provide a theoretical basis for joint therapy of NSCLC with WIF‑1 and autophagic agonists in clinical practice.

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.

INTRODUCTION AND AIM: Epigenetic alterations play an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/β-catenin pathway in liver cancer cells.
MATERIAL AND METHODS: Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/β-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of β-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays.
RESULTS: Our result suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of β-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells.
CONCLUSION: We were able to demonstrate Wnt/ β-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer.

BACKGROUND/AIMS: A few Rho GTPase activating proteins (RhoGAPs) have been identified as tumor suppressors in a variety of human cancers. ARHGAP17, a member of RhoGAPs, has been reported to be involved in the maintenance of tight junction and epithelial barrier. The present study aimed to explore its expression in colon cancer and the possible function in colonic carcinogenesis.
METHODS: The mRNA and protein expression was assessed by realtime PCR and immunoblotting, respectively. Cell Counting Kit-8 (CCK-8) and Transwell assays were performed to evaluate cell proliferation and invasion, respectively.
RESULTS: We found that ARHGAP17 expression was obviously lower in colon cancer specimens than in normal colonic mucosa. ARHGAP17 expression was associated with tumor stage, size and differentiation. In vitro analysis demonstrated that ARHGAP17 overexpression inhibited cell growth and invasion of HCT-8 and HCT-116 cells. In addition, an in vivo experimental metastasis model showed that ARHGAP17 overexpression restricted cancer metastasis to the lung. Mechanically, we found that Wnt signaling contributed to the functions of ARHGAP17 in colon cancer cells. Gene set enrichment analysis (GSEA) in The Cancer Genome Atlas dataset showed that the Wnt signaling pathway was negatively associated with ARHGAP17 expression. The mRNA expression of β-catenin (an important signaling transducer of canonical Wnt signaling) gene (CTNNB1) was negatively correlated with ARHGAP17 expression. Immunoblot analysis of downstream effectors of β-catenin (c-Myc/p27 and MMP7) in ARHGAP17 overexpressing colon cancer cells and metastatic tumors within the lung also validated the GSEA result. ARHGAP17 overexpression increased the phosphorylation of glycogen synthetase kinase 3β, and decreased β-catenin nuclear localization and transcriptional activity. Furthermore, inhibition of Wnt signaling by Wnt Inhibitor Factor-1 (WIF-1) in HIEC cells with ARHGAP17 knockdown significantly attenuated the promotion effects of ARHGAP17 knockdown on cell proliferation, invasion and the activation of β-catenin.
CONCLUSION: these results suggest that ARHGAP17 might serve as a tumor suppressor in colon cancer progression and metastasis through Wnt/β-catenin signaling pathway.

WNT inhibitory factor 1 (WIF‑1) is involved in the tumorigenicity and progression of several types of tumor, which has been attributed to aberrant hypermethylation of its promoter. However, the role of WIF‑1 in the pathogenesis of gallbladder cancer (GBC) remains to be fully elucidated, and the data available are insufficient to identify the upstream molecular mechanisms involved. In the present study, the methylation status of the WIF‑1 promoter was investigated using methylation‑specific polymerase chain reaction (PCR) and bisulfate sequencing PCR in GBC cells. Immunohistochemistry, reverse transcription‑quantitative PCR and western blotting were used to analyze the expression of WIF‑1 and c‑Jun. In addition, a co‑immunoprecipitation assay was designed to determine the DNA methyltransferase that was implicated in WIF‑1 methylation. The results revealed that the expression of WIF‑1 was low in GBC, and that this was caused by aberrant DNA hypermethylation. However, there were no significant correlations between the expression of WIF‑1 and certain key clinicopathological characteristics of GCB. Subsequently, a negative correlation was found between the protein expression of c‑Jun and WIF‑1 in 50 GBC specimens using immunohistochemistry. The demethylation and re‑expression of WIF‑1 was observed when the expression of c‑Jun was silenced. Finally, it was found that the knockdown of c‑Jun downregulated the expression of DNA methyltransferase 1 (DNMT1) and that c‑Jun interacted with DNMT1. Taken together, the present study suggested that c‑Jun suppressed the expression of WIF‑1 through transcriptional regulation and interaction with DNMT1 in GBC. These findings provide an alternative pathogenesis of GBC, which may be promising as a novel reference for early diagnosis or future treatment.

Colon cancer is characterized by invasion and migration. DNA methylation of CpG islands in tumor suppressor genes is considered to be an epigenetic mechanism underlying cancer development. Epigenetic silencing of a gene may be reversed by drugs, including genistein. The present study aimed to determine the effect of genistein on Wnt inhibitory factor 1 (WIF1) and invasion, and migration of colon cancer cells. The viability of HT29 colon cancer cells was suppressed by genistein in a dose dependent manner. Following 72 h of treatment with 10, 20 and 60 µmol/l genistein, increased demethylation of WIF1 was induced in a dose‑dependent manner. Additionally, the invasive/migratory abilities of cells treated with genistein decreased in a dose‑dependent manner. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were performed to identify the mRNA and protein expression levels of invasion/migration‑associated factors. Following treatment with genistein, matrix metalloproteinase (MMP) 2 and MMP9 expression levels decreased, whereas the expression of metalloproteinase inhibitor 1 and E‑cadherin increased significantly. In addition, the expression levels of proto‑oncogene Wnt‑1 (Wnt‑1)/β‑catenin pathway‑associated factors, β‑catenin, c‑Myc proto‑oncogene protein and cyclin D1 decreased in a dose‑dependent manner following treatment with genistein. The invasive/migratory abilities of cells transfected with WIF1‑small interfering (si) RNA, and those transfected with WIF1‑siRNA and treated with genistein, increased notably compared with the control group. The present study demonstrated that genistein was able to inhibit the cell invasion and migration of colon cancer cells by inducing demethylation, and recovering the activity of WIF1 by altering the expression of invasion‑associated factors, and components of the Wnt signaling pathway.

Non-functioning pituitary adenomas (NFPAs) are the most common pituitary tumors and mainly invade the sphenoid, cavernous sinus or dura mate. Aberrant regulation of the Wnt signaling pathway plays an important role in tumorigenesis. This study was designed to investigate the relationships between secreted frizzled-related proteins (sFRPs), WIF1 genes and the invasion of NFPAs by tissue microassays (TMAs) of samples from 163 patients. Significantly weaker staining of WIF1 and sFRP4 were detected in the invasive group compared with the non-invasive group by TMAs (p = 0.002, p < 0.001). Univariate analysis showed a significant correlation between tumor invasion and low expression of WIF1 and sFRP4 (p = 0.002, p < 0.001). A similar trend was observed when analyzing the mRNA and protein levels through RT-PCR and western blot experiments. Methylation of the WIF1 promoter was significantly increased in invasive NFPAs compared with the noninvasive group (p = 0.004). The average progression free survival time in the high WIF1 group was longer than that in the low WIF1 group (p = 0.025). Furthermore, RT-PCR measured the levels of 11 miRNAs targeting WIF1 according to the Targetscan database and PubMed. The levels of miRNA-137, miRNA-374a-5p and miRNA-374b-5p in the invasive group were 0.037-fold, 0.577-fold and 0.44-fold that of the noninvasive group (p = 0.003, p = 0.049 and p = 0.047). Overexpression of miRNA-137 could inhibit the proliferation and invasion of GH3 cells through cell viability and Transwell experiments (p < 0.05). Furthermore, the WIF1 level was upregulated after overexpression of miRNA-137 compared with miRNA-137-NC (control miRNA) in GH3 cells. Our data suggest that WIF1 may be potential biomarker for the aggressiveness of NFPAs. miRNA-137 plays an important role in the Wnt signaling pathway by affecting promoter methylation of WIF1.

Glioblastoma are notorious for their highly invasive growth, diffusely infiltrating adjacent brain structures that precludes complete resection, and is a major obstacle for cure. To characterize this "invisible" tumor part, we designed a high resolution multimodal imaging approach assessing in vivo the metabolism of invasively growing glioma xenografts in the mouse brain. Animals were subjected longitudinally to magnetic resonance imaging (MRI) and

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors (

OBJECTIVE: Colon cancer is one of the most common and deadly types of gastrointestinal tumor. Despite progressive treatments, the patient prognosis has not been improved effectively.
MATERIALS AND METHODS: Expression of miRNA and mRNA were tested by Realtime PCR. Cell cycle was detected by flow cytometry. Cell viability was evaluated by MTT assay. Cell spheroid formation was determined by colony assay. Wnt signaling pathway activity was evaluated by TOP/FOP ratio. Protein expression was tested using Western blot. β-catenin binding ability was detected by ChIP assay. miRNA target gene was confirmed by luciferase assay.
RESULTS: miR-590-3p was found to be overexpressed in both glioma tissues and cell lines. miR-590-3p is upregulated in colon cancer cells and tissues compared to non-tumorigenic colon cells and normal colon tissues. miR-590-3p positively regulated cell proliferation, spheroid formation, and cell cycle in LS174T cells. Conversely, inhibition of miR-590-3p reduced these effects. We confirmed that WIF1 and DKK1 are targets of miR-590-3p. Overexpression of miR-590-3p promoted TOP flash luciferase activity, enhanced nuclear β-catenin levels and increased target genes expression of Wnt signaling pathway. The results indicated that miR-590-3p activates the Wnt/β-catenin signaling pathway.
CONCLUSIONS: We demonstrate that miR-590-3p regulates colon cancer progression via WIF1 and DKK1, which suggests that miR-590-3p may be a promising candidate for therapeutic applications in colon cancer treatment.

Recent studies showed that genetic aberrations in the

Cervical cancer is primarily caused by Human papillomavirus (HPV) infection, but other factors such as smoking habits, co-infections and genetic background, can also contribute to its development. Although this cancer is avoidable, it is the fourth most frequent type of cancer in females worldwide and can only be treated with chemotherapy and radical surgery. There is a need for biomarkers that will enable early diagnosis and targeted therapy for this type of cancer. Therefore, a systems biology pipeline was applied in order to identify potential biomarkers for cervical cancer, which show significant reports in three molecular aspects: DNA sequence variants, DNA methylation pattern and alterations in mRNA/protein expression levels. CDH1, CDKN2A, RB1 and TP53 genes were selected as putative biomarkers, being involved in metastasis, cell cycle regulation and tumour suppression. Other ten genes (CDH13, FHIT, PTEN, MLH1, TP73, CDKN1A, CACNA2D2, TERT, WIF1, APC) seemed to play a role in cervical cancer, but the lack of studies prevented their inclusion as possible biomarkers. Our results highlight the importance of these genes. However, further studies should be performed to elucidate the impact of DNA sequence variants and/or epigenetic deregulation and altered expression of these genes in cervical carcinogenesis and their potential as biomarkers for cervical cancer diagnosis and prognosis.

BACKGROUND: Integrator complex subunit 6 (INTS6) was found to play a tumour suppressing role in certain types of solid tumours. In this study, we wanted to determine the expression level of INTS6 in hepatocellular carcinoma (HCC) and evaluate its clinical characteristics and mechanisms in HCC patients (Lui and Lu, European Journal of Cancer, 51:S94, 2015).
METHODS: First, we used a microarray analysis to explore the mRNA expression levels in HCC and paired normal liver tissues; second, we used qRT-PCR to measure the INTS6 mRNA levels in a cohort of 50 HCC tissues and adjacent normal liver tissues; third, we used Western blot analyses to detect the INTS6 protein levels in 20 paired HCC and normal liver tissues; fourth, we used immunohistochemistry to determine the INTS6 expression levels in 70 archived paraffin-embedded HCC samples. Finally, we investigated the suppressive function of INTS6 in the Wnt pathway.
RESULTS: Herein, according to the microarray data analysis, the expression levels of INTS6 were dramatically down-regulated in HCC tissues vs. those in normal liver tissues (p<0.05). qRT-PCR and Western blot analyses showed that the INTS6 mRNA and protein expression was significantly down-regulated in tumour tissues compared to the adjacent normal liver tissues (p<0.05). Immunohistochemical assays revealed that decreased INTS6 expression was present in 62.9% (44/70) of HCC patients. Correlation analyses showed that INTS6 expression was significantly correlated with serum alpha-fetoprotein levels (AFP, p =0.004), pathology grade (p =0.005), and tumour recurrence (p =0.04). Kaplan-Meier analysis revealed that patients with low INTS6 expression levels had shorter overall and disease-free survival rates than patients with high INTS6 expression levels (p =0.001 and p =0.001). Multivariate regression analysis indicated that INTS6 was an independent predictor of overall survival and disease-free survival rates. Mechanistically, INTS6 increased WIF-1 expression and then inhibited the Wnt/β-catenin signalling pathway.
CONCLUSION: The results of our study show that down-regulated INTS6 expression is associated with a poorer prognosis in HCC patients. This newly identified INTS6/WIF-1 axis indicates the molecular mechanism of HCC and may represent a therapeutic target in HCC patients.

BACKGROUND: Male breast cancer (MBC) represents a poorly characterised group of tumours, the management of which is largely based on practices established for female breast cancer. However, recent studies demonstrate biological and molecular differences likely to impact on tumour behaviour and therefore patient outcome. The aim of this study was to investigate methylation of a panel of commonly methylated breast cancer genes in familial MBCs.
METHODS: 60 tumours from 3 BRCA1 and 25 BRCA2 male mutation carriers and 32 males from BRCAX families were assessed for promoter methylation by methylation-sensitive high resolution melting in a panel of 10 genes (RASSF1A, TWIST1, APC, WIF1, MAL, RARβ, CDH1, RUNX3, FOXC1 and GSTP1). An average methylation index (AMI) was calculated for each case comprising the average of the methylation of the 10 genes tested as an indicator of overall tumour promoter region methylation. Promoter hypermethylation and AMI were correlated with BRCA carrier mutation status and clinicopathological parameters including tumour stage, grade, histological subtype and disease specific survival.
RESULTS: Tumours arising in BRCA2 mutation carriers showed significantly higher methylation of candidate genes, than those arising in non-BRCA2 familial MBCs (average AMI 23.6 vs 16.6, p = 0.01, 45% of genes hypermethylated vs 34%, p < 0.01). RARβ methylation and AMI-high status were significantly associated with tumour size (p = 0.01 and p = 0.02 respectively), RUNX3 methylation with invasive carcinoma of no special type (94% vs 69%, p = 0.046) and RASSF1A methylation with coexistence of high grade ductal carcinoma in situ (33% vs 6%, p = 0.02). Cluster analysis showed MBCs arising in BRCA2 mutation carriers were characterised by RASSF1A, WIF1, RARβ and GTSP1 methylation (p = 0.02) whereas methylation in BRCAX tumours showed no clear clustering to particular genes. TWIST1 methylation (p = 0.001) and AMI (p = 0.01) were prognostic for disease specific survival.
CONCLUSIONS: Increased methylation defines a subset of familial MBC and with AMI may be a useful prognostic marker. Methylation might be predictive of response to novel therapeutics that are currently under investigation in other cancer types.

OBJECTIVE: We screened and identified the differential expression of the methylation phenotype in the whole genome of colorectal laterally spreading tumor (LSTs).
MATERIALS AND METHODS: 3 tissue samples of colorectal polypoid adenomas (PAs), 3 tissue samples of LSTs and 3 tissue samples of colon cancer were analyzed with a high-density gene chip, and about 450,000 methylation sites were detected covering approximately 95% of the CpG islands. The Delta Data screening was taken through a cluster analysis of methylation phenotype differential expression. 50 tissue samples each of PAs patients, LSTs patients, and colorectal cancer patients were selected. Methylation-specific PCR (MSP) was used to detect RASSF1A and WIF-1 methylation levels. He RT-PCR method was used to detect the relative mRNA expression levels for methylation expression identification.
RESULTS: The degree of LST methylation was higher than that of PAs, and 1234 genes were found to have a lower expression when compared to colorectal cancer samples. 764 genes had a higher expression when compared to colorectal cancer, and 559 genes lower expression when compared to PAs. The average methylation level of LSTs was higher than that of PAs, and lower than that of colorectal cancer. The chromosomal location was taken on these 1234 genes, which were higher than that of PAs, and lower than that of colorectal cancer; 518 genes were located on chromosome No. 2 (41.98%), 236 on No. 5 (19.12%), 357 on No. 8 (28.93%), and 123 on No. 10 (9.97%). According to clustering analysis, DNA differentially methylated sites were mainly on genes of cell adhesion molecules regulation, signaling pathways, energy transduction, cell cycle and apoptosis. The positive rate of RASSF1A and WIF-1 methylation in the tissues of LSTs patients were higher than that of PAs, and lower than that of colorectal cancer; differences were statistically significant (p<0.05). The relative expression levels of RASSF1A and WIF-1mRNA in the tissues of LSTs patients were lower than that of PAs, higher than that of colorectal cancer, and the difference was statistically significant (p<0.05).
CONCLUSIONS: The administration of high-density gene chip technology has a good application value to screen the differential expression of LSTs gene methylation phenotype. Results are consistent with the identification results.

BACKGROUND: This study aimed to investigate the changes in the promoter methylation and gene expression of multiple Wnt antagonists between the chronic infection and eradication of Helicobacter pylori (H. pylori) in gastric carcinogenesis.
METHODS: The levels of methylation and corresponding mRNA expression of seven Wnt antagonist genes (SFRP1, -2, -5, DKK1, -2, -3, WIF1) were compared among the patients with H. pylori-positive gastric cancers (GCs), and H. pylori-positive and H. pylori-negative controls, by quantitative MethyLight assay and real-time reverse transcription (RT)-polymerase chain reaction (PCR), respectively. The changes of the methylation and expression levels of the genes were also compared between the H. pylori eradication and H. pylori-persistent groups 1 year after endoscopic resection of GCs.
RESULTS: The methylation levels of SFRP and DKK family genes were significantly increased in the patients with H. pylori-positive GCs and followed by H. pylori-positive controls compared with H. pylori-negative controls (P < 0.001). SFRP1, -2, and DKK3 gene expression was stepwise downregulated from H. pylori-negative controls, H. pylori-positive controls, and to H. pylori-positive GCs (P < 0.05). Among the Wnt antagonists, only the degrees of methylation and downregulation of DKK3 were significantly reduced after H. pylori eradication (P < 0.05).
CONCLUSION: Epigenetic silencing of SFRP and DKK family genes may facilitate the formation of an epigenetic field during H. pylori-associated gastric carcinogenesis. The epigenetic field may not be reversed even after H. pylori eradication except by DKK3 methylation.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

PURPOSE: The molecular nature and the rate-limiting step of epigenetic field defects in the evolution of left-sided colorectal cancer (LCA) remain uncertain.
MATERIALS AND METHODS: The methylation status of 27 candidate field defect markers, six classic CpG island methylator phenotype (CIMP) markers, and LINE-1 were determined in LCA and adjacent normal mucosas (ADJs) from 33 LCA patients and in left normal colorectal mucosa (LNM) from 33 age- and sex-matched controls. Hotspot mutation analyses in KRAS codons 12 and 13 and BRAF V600E were performed by genomic PCR and pyrosequencing using DNA extracted from endoscopically biopsied tissues.
RESULTS: Among the 27 candidate genes tested, we confirmed 15 differentially methylated genes in cancer (15 DMGs; ER, SFRP1, MYOD1, MGMT, CD8a, SPOCK2, ABHD9, BNIP3, IGFBP3, WIF1, MAL, GDNF, ALX4, DOK5, and SLC16A12) in comparison to ADJ samples. We further compared the methylation status of 15 DMGs of ADJs to LNM and found only methylation levels of SLC16A12 in ADJs of LCA patients to be significantly higher than that in LNM (17.3% vs. 11.5%, p=0.002). Based on the CIMP, no significant differences in methylation levels of the 15 DMGs were found between ADJs in CIMP positive LCA cases and those without CIMP. In mutation analyses, no mutation was found in ADJs, while significant KRAS mutations (6/33, 18%) were noted in LCA samples.
CONCLUSION: Epigenetic field defect marked by aberrant methylation is uncommon in normal-appearing ADJs of LCA, indicating the critical rate-limiting change of methylation is likely to occur with morphological alterations in the evolution of LCA.

Chondrosarcoma (CS) is a rare cancer, but it is the second most common primary malignant bone tumor and highly resistant to conventional chemotherapy and radiotherapy. Aberrant DNA methylation in the promoter CpG island of Wnt inhibitory factor 1 (WIF1) has been observed in different cancers. However, no studies have shown the relationship between WIF1 methylation and CS. In this study, we found promoter methylated WIF1 in both CS cell lines (CS-1 and SW1353) and tumor tissues. Western blot analysis confirmed loss WIF1 expression and activation of Wnt pathway proteins (Wnt5a/b, LRP6, and Dvl2). We subsequently examined the correlation between levels of WIF1 methylation and overall survival (OS) and progression-free survival (PFS) in CS patient samples with a follow-up spanning 234 months (mean: 57.6 months). Kaplan-Meier survival curves and log-rank tests revealed that high levels of WIF1 methylation were associated with lower OS and PFS rates (p < 0.05). Multivariate Cox hazard analysis suggested that detection of high level methylation of WIF1 could be an independent prognostic factor in OS and PFS. In conclusion, we found that WIF1 is epigenetically silenced via promoter DNA methylation in CS and propose that WIF1 methylation may serve as a potential prognostic marker for patients with CS.

Gallbladder cancer (GBC) is the most common malignant tumor in the human biliary tract, but the lack of a marker for timely diagnosis leads to an extremely poor prognosis. In this study, we assessed CpG sites in the WIF-1 promoter using bisulfite sequencing PCR and methylation-specific PCR to detect methylation in gallbladder cancer and cholecystitis tissues. WIF-1 promoter methylation was present in 36 of 50 (72.0%) gallbladder cancers but only 5 of 20 (25.0%) cholecystitis tissues (P=0.000<0.05), suggesting that WIF-1 promoter methylation might participate in the malignant transformation of cholecystitis into gallbladder cancer. WIF-1 methylation was negatively correlated with WIF-1 protein expression by immunohistochemistry, demonstrating that WIF-1 expression is downregulated by promoter hypermethylation. We analyzed the prognosis of 50 GBC patients with 5years of follow-up. Univariate analysis revealed that patients with hypermethylated WIF-1 exhibited worse overall survival than those with hypomethylated WIF-1 (χ

Wnt signal pathway genes are known to be involved with cancer development. Here we tested the hypothesis whether DNA methylation of genes part of the Wnt signaling pathway could help the diagnosis of non-small cell lung cancer (NSCLC). The methylation levels of SFRP1, SFRP2, WIF1 and PRKCB in 111 NSCLC patients were evaluated by quantitative methylation-specific PCR (qMSP). Promoter methylation levels of four candidate genes were significantly higher in tumor tissues compared with the adjacent tissues. SFRP1, SFRP2 and PRKCB genes were all shown to be good predictors of NSCLC risk (SFRP1: AUC = 0.711; SFRP2: AUC = 0.631; PRKCB: AUC = 0.650). The combined analysis showed that the methylation status of the four genes had a sensitivity of 70.3% and a specificity of 73.9% in the prediction of NSCLC risk for study cohort. A higher diagnostic value with an AUC of 0.945 (95% CI: 0.923-0.967, sensitivity: 90.6%, specificity: 93.0%) was found in TCGA cohort. In addition, SFRP1 and SFRP2 hypermethylation events were specific to male patients. Further TCGA data mining analysis suggested that SFRP1_cg15839448, SFRP2_cg05774801, and WIF1_cg21383810 were inversely associated with the host gene expression. Moreover, GEO database analysis showed that 5'-Aza-deoxycytidine was able to upregulate gene expression in several lung cancer cell lines. Subsequent dual-luciferase reporter assay showed a crucial regulatory function of PRKCB promoter. In summary, our study showed that a panel of Wnt signal pathway genes (SFRP1, SFRP2, WIF1 and PRKCB) had the potential as methylation biomarkers in the diagnosis of NSCLC.