Secreted Frizzled-related Protein 1 Gene Research Articles

Reduced Expression of SFRP1 is Associated with Poor Prognosis and Promotes Cell Proliferation in Breast Cancer: An Integrated Bioinformatics Approach

PurposeBreast carcinoma is the most frequent form of malignancy in women globally. Exploration of the breast cancer genome tiled the way for the validation of novel cancer biomarkers and to explore various mechanisms involved in the progression of carcinogenesis. The purpose of the research is to find an identification of potential gene linked to breast cancer (BC) progression and prognosis. MethodsThree datasets (GSE71053, GSE61724, and GSE36295) were downloaded from the Gene Expression Omnibus (GEO) database. An integrated analysis of several gene expression profile datasets was used to find differentially expressed genes (DEGs) in BC and normal breast tissue samples. Protein–protein interaction (PPI) network was used to verify hub genes associated with the pathogenesis and prognosis of BC. The functional enrichment and pathway analysis was performed by FunRich and cBioPortal. The expression pattern was assessed using COSMIC, GEPIA2, and BC-GenExMiner. ResultsThe results revealed that among the hub genes, Secreted Frizzled-related protein 1 (SFRP1) was a negative regulator of the Wnt pathway in breast cancer. Loss of SFRP1 may result in abnormal cellular proliferation, migration, and invasion, which may trigger cancer cells, leading to progression of the disease, poor prognosis, and therapy resistance. Lastly, the Kaplan–Meier plotter online database demonstrated that expression levels of the SFRP1 gene were related to lower survival. ConclusionThe findings of this research would provide some directive significance for further investigating the diagnostic and prognostic biomarker to facilitate the molecular targeting therapy of breast cancer; SFRP1 expression may be effective as a novel prognostic biomarker in early breast cancer.

Validation of SFRP1 Promoter Hypermethylation in Plasma as a Prognostic Marker for Survival and Gemcitabine Effectiveness in Patients with Stage IV Pancreatic Adenocarcinoma.

Simple SummaryPancreatic adenocarcinoma (PDAC) is a disease with an incredibly grim prognosis. Most patients die within one year of receiving the diagnosis. There are currently very few tools to help the clinician decide between treatment options and evaluate prognosis at an individual level. The aim of the current study was to assess the effect of promoter hypermethylation of secreted frizzled-related protein 1 (phSFRP1) as an independent prognostic blood-based biomarker in gemcitabine-treated patients with advanced PDAC. The study was conducted as a combined discovery and validation study. Analysis in both cohorts confirmed that patients with phSFRP1 had overall poorer survival compared to those without hypermethylation. Thus, phSFRP1 shows promise as an independent prognostic biomarker in this patient group and can hopefully aid the clinician and patient find the correct balance between quantity and quality of life.No reliable predictive blood-based biomarkers are available for determining survival from pancreatic adenocarcinoma (PDAC). This combined discovery and validation study examines promoter hypermethylation (ph) of secreted frizzled-related protein 1 (SFRP1) in plasma-derived cell-free DNA as an independent prognostic marker for survival and Gemcitabine effectiveness in patients with stage IV PDAC. We conducted methylation-specific polymerase chain reaction analysis of the promoter region of the SFRP1 gene, based on bisulfite treatment. Survival was analyzed with Kaplan–Meier curves, log-rank test, and Cox regression. The discovery cohort included 40 patients, 25 receiving Gem. Gem-treated patients with phSFRP1 had a shorter median overall survival (mOS) (4.4 months) than unmethylated patients (11.6 months). Adjusted Cox-regression yielded a hazard rate (HR) of 3.48 (1.39–8.70). The validation cohort included 58 Gem-treated patients. Patients with phSFRP1 had a shorter mOS (3.2 months) than unmethylated patients (6.3 months). Adjusted Cox regression yielded an HR of 3.53 (1.85–6.74). In both cohorts, phSFRP1 was associated with poorer survival in Gem-treated patients. This may indicate that tumors with phSFRP1 are more aggressive and less sensitive to Gem treatment. This knowledge may facilitate tailored treatment of patients with stage IV PDAC. Further studies are planned to examine phSFRP1 in more intensive chemotherapy regimens.

Transcription factor FOXC1 positively regulates SFRP1 expression in androgenetic alopecia

Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from −100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.

MBD2 and EZH2 regulate the expression of SFRP1 without affecting its methylation status in a colorectal cancer cell line.

Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.

Prognostic Relevance of SFRP1 Gene Promoter Methylation in Colorectal Carcinoma

Background:The development of colorectal carcinoma (CRC) involves many genetic and epigenetic alterations and methylation being an important epigenetic event has been described as a diagnostic and prognostic biomarker. Secreted Frizzled- Related Protein 1 (SFRP1) gene regulates diverse physiological processes via the Wnt signaling. Promoter hypermethylation of SFRP1 gene is an epigenetic regulation mechanism that downregulates SFRP1 protein level in the tumor, and happens to be one of the significant events in colorectal carcinogenesis. We studied the clinicopathological relationship of CRC including survival outcomes with SFRP1 gene promoter methylation.Methods:We evaluated promoter methylation status of SFRP1 gene by methylation-specific PCR (MS-PCR) in the tumor tissue in 54 cases of stage II-III CRC patients in north India. The MS-PCR result was further validated by bisulfite sequencing.Results:SFRP1 gene was methylated in 72.2% cases and un-methylated in 27.8%. We found, that SFRP1 gene methylation in tumor was associated with lymph node invasion (p=0.05). The mean overall survival was 22.318 months and 45.173 months respectively for patients with methylated and unmethylated SFRP1 gene (p= 0.010, log rank test), (HR = 17.313, 95% CI: 2.021-148.290 P=0.009).Conclusion:Study indicates that promoter methylation of SFRP1 gene is associated with lymph-node metastasis and poor mean overall survival and it can be a prognostic marker in CRC.

Hypermethylation of Secreted Frizzled Related Protein 1 gene promoter in different astrocytoma grades

AimTo identify the involvement of Secreted Frizzled Related Protein 1 (SFRP1) promoter hypermethylation in different malignancy grades of astrocytoma and assess its association with beta-catenin, lymphoid-enhancer factor 1, and T-cell factor 1.MethodsTwenty-six astrocytoma samples were collected from 2008-2015. Promoter hypermethylation was evaluated by methylation-specific polymerase-chain-reaction and protein expression by immunohistochemistry and stereological analysis. The staining intensity was scored by comparing immunoreactivity with normal tissue and by using 10% and 50% cut-offs.ResultsSFRP1 promoter methylation was found in 32% of astrocytomas. The number of hypermethylated samples increased in higher astrocytoma grades and was the highest in glioblastoma (P = 0.042 compared to other astrocytoma grades). There was 45.8% of samples with the lack of or weak expression of SFRP1 protein and 29.2% with strong expression. Samples with methylated promoter expressed significantly less SFRP1 than samples with unmethylated promoter (P = 0.031). Beta-catenin expression levels were elevated. Yet, glioblastomas with unmethylated SFRP1 promoter had significantly less beta-catenin (P = 0.033). Strong expression of lymphoid-enhancer factor 1 was associated to higher astrocytoma grades (P = 0.006).ConclusionSFRP1 gene was epigenetically silenced in glioblastomas when compared to low astrocytoma grades, which may suggest that the lack of its protein is involved in astrocytoma progression.

A specific haplotype in potential miRNAs binding sites of secreted frizzled-related protein 1 (SFRP1) is associated with BMD variation in osteoporosis

PurposeOsteoporosis is an important multifactorial disease which is largely influenced by Wnt signaling pathway. Considering regulatory single nucleotide polymorphisms in Wnt signaling pathway may pave the road of understanding the genetic basis of predisposition to osteoporosis. The aim of this study was to determine the possible association between variants of SFRP1 and WNT5b, and osteoporosis incidence risk. MethodsThe study population comprised 186 osteoporotic patients and 118 normal subjects from Amirkola Health and Ageing Project. rs1127379 (c.1406A>G) and rs3242 (c.3132C>T) variants in 3′UTR of SFRP1 gene, and rs3803164 (c.236C>T) in 3′UTR and rs735890 (c.622-536A>G) in intron 4 of WNT5b gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Regression analyses were used to calculate the association of genotype frequencies with bone mineral density (BMD) and bone mineral content (BMC) values of participants. Bioinformatics algorithms were used to detect the effect of each SNP on the secondary structure of mRNA, and predict putative 3′UTR microRNA target sites and splicing sites changes by related SNPs. ResultsWNT5b rs735890 was associated with lumbar spine BMD, BMC, and femoral neck BMC (P = 0.035, P = 0.007, and P = 0.038, respectively). WNT5b rs3803164, and SFRP1 rs3242 were significantly associated with lumbar spine BMD (P = 0.028 and P = 0.030, respectively). SFRP1 rs1127379 was associated with lumbar spine BMD in the male gender. Haplotype analysis showed a significant association of SFRP1 c.[1406A; 3132C] haplotype with lumbar spine BMD, and BMC (P = 0.019 and P = 0.030, respectively), and SFRP1 c.[1406G; 3132C] haplotype with lumbar spine BMC (P = 0.045). In silico analyses revealed that the G allele of SFRP1 rs1127379, and WNT5b rs3803164 appear as more possible target sites for many miRNAs. ConclusionsThis study is the first evidence of the association of WNT5b rs735890, and c.[1406A; 3132C] and c.[1406G; 3132C] haplotypes of SFRP1 with BMD variation in osteoporosis, probably by altering microRNA target sites, in elderly persons.

Secreted frizzled-related protein 1 (SFRP1) gene methylation changes in the human lung adenocarcinoma cells treated with L-securinine

Lung cancer remains the leading cause of cancer-related death worldwide. It is important to explore the biomarkers of diagnosis and prognosis in lung cancer. To evaluate the cytotoxicity of L-securinine and the expression and methylation of secreted frizzled-related proteins (SFRPs) genes in the human lung adenocarcinoma cells, cell counting kit-8 (CCK-8) assay was used to assess the proliferation of lung adenocarcinoma cells treated with L-securinine. Quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR were used to detect the expression and the DNA methylation of SFRPs genes, respectively. L-securinine inhibited the proliferation of lung adenocarcinoma cells and induced the upregulation of SFRP1 gene expression and the methylation changes at CpG sites in the SFRP1 promoter region. L-securinine was a potential agent in the treatment of lung cancer by upregulation of SFRP1 gene expression and changing the SFRP1 gene methylation.

Arsenic trioxide increases expression of secreted frizzled-related protein 1 gene and inhibits the WNT/β-catenin signaling pathway in Jurkat cells.

The aim of the present study was to investigate the demethylation effect of arsenic trioxide (As2O3) on the secreted frizzled-related protein 1 (SFRP1) gene and its ability to inhibit the Wingless-type MMTV integration site family (WNT) pathway in Jurkat cells. Methylation-specific polymerase chain reaction was used to examine the CpG island methylation status of the SFRP1 gene in leukemia cell lines. In addition, the effects on Jurkat cells of treatment with different concentrations of As2O3 for 48 h were investigated. Reverse transcription-quantitative polymerase chain reaction was employed to measure the expression of mRNAs, while western blot analysis was used to examine protein expression in cells. The SFRP1 gene was methylated in Jurkat cells. However, both methylated and unmethylated SFRP1 genes were detected in HL60 and K562 cells. In normal bone marrow mononuclear cells, the SFRP1 gene was unmethylated. Following treatment with As2O3 for 48 h, the SFRP1 gene was demethylated, and the mRNA and protein expression levels of the SFRP1 gene were increased. By contrast, the mRNA and protein expression levels of β-catenin and cyclin Dl were downregulated. The protein expression of c-myc was also downregulated, but As2O3 exhibited no significant effect on the mRNA expression of c-myc. Abnormal methylation of the SFRP1 gene was detected in Jurkat cells. These results suggest that As2O3 activates SFRP1 gene expression at the mRNA and protein levels in Jurkat cells by demethylation of the SFRP1 gene. Furthermore, they indicate that As2O3 regulates WNT target genes and controls the growth of Jurkat cells through the WNT/β-catenin signaling pathway.

Hydrogen Peroxide-Induced Secreted Frizzled-Related Protein 1 Gene Demethylation Contributes to Hydrogen Peroxide-Induced Apoptosis in Human U251 Glioma Cells.

Glioblastoma multiforme is a type of central nervous system tumor with extremely poor prognosis. Previously, hydrogen peroxide (H2O2), which promotes the oxidative stress response, has been reported to induce the apoptosis of glioma cells. Recently, secreted frizzled-related protein 1 (SFRP1) has been shown to be associated with various types of malignant tumors and with H2O2-induced oxidative stress in cardiomyocytes by negatively regulating the Wnt signaling pathway. This study aimed to explore SFRP1 expression and its roles in H2O2-induced apoptosis in human glioma cells. We found that the SFRP1 level was decreased in several human glioma cell lines, including U87, U251, and SW1783 cells. In U251 cells, SFRP1 could function as a cancer suppressor gene, and the growth of U251 cells could be inhibited not only by H2O2 but also by the overexpression of SFRP1. Furthermore, we demonstrated that H2O2-induced SFRP1 gene demethylation partially contributed to H2O2-induced U251 cell apoptosis, which was verified by studies using an SFRP inhibitor (WAY-316606). Our research identified that H2O2-induced SFRP1 gene demethylation contributes to H2O2-induced apoptosis in human U251 glioma cells.

Dicer promotes tumorigenesis by translocating to nucleus to promote SFRP1 promoter methylation in cholangiocarcinoma cells

Dicer, a member of the RNase III family of endoribonucleases, has an important role in regulating methylation of CpG islands in mammal cancer cells. However, the underlying mechanism of action remains unclear. In this study, we demonstrated that upregulation of Dicer in cholangiocarcinoma (CCA) cells and its translocation to nuclues to interact with heterochromatin protein 1α (HP1α). The nuclear Dicer/HP1α complex appeared to promote both H3K9 trimethylation and DNA methylation of the secreted frizzled-related protein 1 (SFRP1) promoter. The expression of Dicer negatively correlated with that of SFRP1 and it appeared to promote CCA cell proliferation and invasion through repression of SFRP1 gene. High expression of Dicer in tumor tissues was significantly associated with larger tumor size (>3 cm) and lymph node metastasis. Our findings help characterize the role of Dicer in epigenetic regulation and tumorigenesis in the context of CCA.

Abstract A16: Different epigenetic mechanisms involved in the regulation of SFRP1 gene in prostate cancer

Abstract Among men, prostate cancer (PCa)[1] is the second most common cancer and the fifth cause of death worldwide [2]. Androgens play an important role in the development of the disease. For advanced PCa the standard therapy is androgen depletion. However, after 2 or 3 years a high percentage of patients become resistant to this therapy and castration resistant prostate cancer (CRPC) is developed. There is no successful treatment for CRPC, leading into death [3, 4]. Wingless pathway (WNT) is aberrantly activated in several cancer types and in PCa it is involved in AR activity modulation, promoting cancer development [5]. Secreted Frizzled Related Protein 1 (SFRP1) is commonly known as a WNT inhibitor and generally proposed as a tumor suppressor gene. SFRP1 can exert its tumor suppressor activity by inhibiting the AR transcriptional activity [6]. Usually in cancer, SFRP1 mRNA is down regulated and this is associated to increased DNA methylation at its promoter region [7]. In contrast, in PCa there are different reports suggesting SFRP1 repression in absence of DNA methylation [8]. In this work we aimed to investigate two different epigenetic mechanisms for SFRP1 silencing in PCa. For the in vitro model, we used normal prostate cells RWPE-1 and PCa cell lines: LNCaP, 22RV1 and PC3. We confirmed the correlation between mRNA expression loss and the increased DNA methylation of SFRP1 only in PC3 cells, but not in LNCaP and 22RV1, were instead of DNA methylation we found enrichment of H3K27me3, the Polycomb Repressive Complex 2 (PRC2) mark. To correlate this findings, we analyzed the expression of SFRP1 in fresh frozen tissue samples, 11 primary PCa, 10 Benign Prostatic Hyperplasia (BPH) and 4 normal prostates (NP). We found SFRP1 downregulation in all PCa and BPH samples compared to Normal Prostate, but DNA methylation was increased only in PCa but not in BPH, this suggests a novel repressive mechanism for SFRP1 independent of DNA methylation. To see if AR has a role in regulating mRNA SFRP1 expression, we silenced AR with a siRNA, but no SFRP1 expression levels were modified, so we concluded AR was not directly involved in SFRP1 regulation. All these data together suggest different repression mechanisms for SFRP1, one depending on DNA methylation and another one depending on H3K27me3 PRC2 repressive mechanism, Androgen receptor is not directly involved in SFRP1 downregulation, so other factors might be involved in SFRP1 expression loss in PCa.

Circulating SFRP1 promoter methylation status in gastric adenocarcinoma and esophageal square cell carcinoma.

The secreted frizzled-related protein 1 (SFRP1) gene plays an important role in carcinogenesis of digestive system cancer. Previous studies proved that circulating DNA promoter methylation may be a suitable biomarker for cancer patients. The aim of the present study was to investigate whether the promoter methylation status of serum SFRP1 is a potential biomarker for gastric adenocarcinoma (GAC) and esophageal square cell carcinoma (ESCC). The blood samples obtained from 42 GAC and 36 ESCC patients were detected for the promoter methylation status of SFRP1 by methylation-specific polymerase chain reaction. The control group included 42 benign gastrointestinal disease volunteers (24 benign gastric disease and 18 benign esophageal disease) and 20 healthy volunteers. Serum SFRP1 methylation was evident in 30.95% (13/42) GAC patients and 38.89% (14/36) ESCC patients, which is clearly higher compared to 8.33% (2/24) in benign gastric disease, 11.11% (2/18) in benign esophageal disease and 5% (1/20) in healthy volunteers (P<0.05). The association between the serum SFRP1 promoter methylation status and the clinical pathological features were further analyzed and methylation of the SFRP1 gene was significantly associated with age >60 years in GAC patients (P=0.027). However, no correlations between the SFRP1 methylation status and other clinicopathological parameters were found. In conclusion, the SFRP1 promoter was detected frequently in the serum of GAC and ESCC patients. The detection of circulating methylated SFRP1 in the serum may be a useful biomarker for upper gastrointestinal cancer patients.

Aberrant promoter methylation of the SFRP1 gene may contribute to colorectal carcinogenesis: a meta-analysis

This meta-analysis of published cohort studies was conducted to evaluate whether promoter methylation of the secreted frizzled-related protein 1 (SFRP1) gene contributes to colorectal carcinogenesis. The Web of Science (1945 ~ 2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966 ~ 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), and the Chinese Biomedical Database (CBM) (1982 ~ 2013) were searched without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. We calculated odds ratio (OR) and its 95 % confidence interval (95 % CI) to estimate the correlations between SFRP1 promoter methylation and colorectal carcinogenesis. In the present meta-analysis, 8 cohort studies with a total of 942 patients with colorectal cancer (CRC) were included. The pooled results revealed that the frequency of SFRP1 promoter methylation in cancer tissues were significantly higher than those of normal, adjacent, and benign tissues (cancer tissues vs. normal tissues: OR = 31.49, 95 % CI = 17.57 ~ 56.44, P < 0.001; cancer tissues vs. adjacent tissues: OR = 5.95, 95 % CI 3.12 ~ 10.00, P < 0.001; cancer tissues vs. benign tissues: OR = 3.01, 95 % CI 1.72 ~ 5.27, P < 0.001; respectively). Furthermore, ethnicity-stratified analysis indicated that SFRP1 promoter methylation was strongly correlated with colorectal carcinogenesis among both Asians and Caucasians (all P < 0.05). Our findings provide empirical evidence that SFRP1 promoter methylation may be correlated with the pathogenesis of CRC.

Hyperpolarized 3He diffusion MRI and histology of secreted frizzled related protein-1 (SFRP1) deficient lungs in a Murine model

Secreted frizzled related protein-1 (SFRP1) plays a key role in many diverse processes, including embryogenesis, tissue repair, bone formation, and tumor genesis. Previous studies have shown the effects of the SFRP1 gene on lung development using the SFRP1 knockout mouse model via histological and physiological studies. In this study, the feasibility of ADC (acquired via HP 3He) to detect altered lung structure in the SFRP1 knockout (SFRP1−/−) mice was investigated, and compared to analysis by histology. This study consisted of two groups, the wild-type (WT) mice and the knockout (KO) mice with n=6 mice for each group. 3He ADC MRI and histology were performed on all of the animals. The global Lm values of WT and KO mice were 35.0±0.8μm and 38.4±3.8μm, respectively, which translated to an increase of 9.58% in the Lm of KO mice. The mean global ADCs for the WT and KO mice were 0.12±0.01cm2/s and 0.13±0.01cm2/s, respectively, which equated to a relative increase of 8.0% in the KO mice compared to the WT mice. In the sub-analysis of the anterior, medial and posterior lung regions, Lm increased by 10.50%, 6.66% and 11.84% in the KO mice, respectively, whereas the differences in ADC between the two groups in the anterior, medial, and posterior regions were 7.3%, 8.3%, and 4.6%, respectively. These results suggest that HP MRI measurements can be used as a suitable substitute for histology to obtain valuable information about lung geometry non-invasively. This technique is also advantageous as regional measurements can be performed, which can identify lung destruction more precisely. Most importantly, this approach extends far beyond the specific pathology analyzed in this study, as it can be applied to many other pathological conditions in the lung tissue, as well to many other embryonic studies.

Low methylation levels of the SFRP1 gene are associated with the basal-like subtype of breast cancer

Epigenetic analyses have shown that aberrant DNA methylation signatures are associated with breast cancer molecular subtypes. In this study, we analyzed the methylation status of breast cancer-related genes in relation to the molecular subtypes and investigated whether the basal-like subtype displays distinct methylation profiles. By using pyrosequencing, we analyzed the DNA methylation status of 5 candidate genes in 60 breast cancer samples. We compared the methylation frequency across the molecular subtypes and analyzed the correlation between methylation levels and clinicopathological characteristics. A total of 59 cases displayed aberrant methylation. Amplification during polymerase chain reaction analysis failed in 1 case. The median methylation levels of the secreted frizzled-related protein 1 (SFRP1) gene were significantly lower in the basal-like subtype compared to the luminal A, luminal B and human epidermal growth factor receptor 2 (HER2) subtypes. Cadherin 13 (H-cadherin; CDH13) methylation levels were significantly higher in the HER2 tumors compared to the luminal A and basal-like subtypes. A comparison of the methylation status with clinicopathological characteristics revealed that the expression of bcl-2, progesterone receptor and epidermal growth factor receptor were associated with SFRP1 gene methylation status. Our results indicate that the basal-like subtype is associated with low methylation levels of the SFRP1 gene, suggesting that the methylation levels of specific breast cancer genes may potentially serve as epigenetic biomarkers and prognostic factors.

Change in gene expression profiles of secreted frizzled-related proteins (SFRPs) by sodium butyrate in gastric cancers: Induction of promoter demethylation and histone modification causing inhibition of Wnt signaling

Activation of Wnt signaling without mutation of β-catenin or APC occurs frequently in human gastric cancers. Secreted frizzled-related protein (SFRP), a negative modulator of the Wnt signaling pathway, are frequently inactivated in human gastric cancers. Inhibition of SFRP gene expression may account for the Wnt/β-catenin activation in human gastric cancer. However, the molecular mechanisms of silencing of SFRP genes are not fully understood. Sodium butyrate, a histone deacetylase (HDAC) inhibitor, is known to exhibit anti-cancer effects partly through the differentiation of various cancer cells. In the present study, we investigated: i) the relationship between the silencing of SFRP genes and Wnt signaling; ii) the mechanism of sodium butyrate mediated epigenetic regulation of SFRPs expression in human gastric cancer. We observed that nuclear β-catenin was significantly increased in gastric cancer tissues as compared to adjacent non-cancerous tissues. Nuclear β-catenin accumulation and SFRP promoter methylation in human gastric cancer cells were noted. Treatment with the DNA methyltransferase inhibitor, 5'-Aza-2-deoxycytidine (5'-Aza-dC) rapidly restored SFRPs expression. Sodium butyrate (NaB) induced demethylation and histone modification at the promoter region of SFRP1/2 restoring the SFRP expression in human gastric cancer cells. Analysis of general expression revealed that overexpression of SFRPs repressed Wnt target gene expression and induced changes in the proliferation and apoptosis related genes in human gastric cancer cells. These data suggest that aberrant epigenetic modification of SFRP genes is one of the major mechanisms by which Wnt signaling is activated in human gastric cancer cells and sodium butyrate may modulate the SFRP1/2 expression through histone modification and promoter demethylation causing anti-tumor effects.

Epigenetic Inactivation of the SFRP1 Gene in Esophageal Squamous Cell Carcinoma

The secreted frizzled-related protein 1 (SFRP1) gene, as a Wnt signaling modulator, is frequently inactivated by promoter methylation in many tumors including gastric cancer, breast cancer, oral squamous cell carcinoma, and esophageal adenocarcinoma. However, the role of SFRP1 in esophageal squamous cell carcinoma (ESCC) is not clear. In this study, we investigated the epigenetic inactivation of the SFRP1 gene in ESCC. Nine ESCC cell lines, two immortalized human esophageal epithelial cell lines, twenty ESCC tissues, and paired adjacent nontumor tissues were analyzed in the study. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, reverse-transcription PCR, immunohistochemistry, and chromatin immunoprecipitation assay were used to detect SFRP1 promoter methylation, expression of the SFRP1 gene, and histone modification in the SFRP1 promoter region. The SFRP1 promoter was found to be highly methylated in 95% (19/20) of the ESCC tissues and in nine ESCC cell lines, compared with 65% (13/20) of the paired nontumor tissues. Moreover, we confirmed that complete methylation of the SFRP1 gene promoter was correlated with its greatly reduced expression level. After individual treatment with 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA), the messenger RNA (mRNA) level of the SFRP1 gene was not obviously rescued in the EC9706 cell line. Combined incubation with DAC and TSA can, however, substantially increase the SFRP1 mRNA expression level in the EC9706 cell line. Chromatin immunoprecipitation assay showed that acetylated histone H3 and H4 were found in the SFRP1 promoter region. Promoter hypermethylation of SFRP1 is a frequent event in ESCC. Promoter methylation and histone acetylation may cooperatively regulate expression of the SFRP1 gene.

Loss of SFRP1 Expression is Associated with Aberrant β-Catenin Distribution and Tumor Progression in Mucoepidermoid Carcinoma of Salivary Glands

Cytoplasmic and nuclear accumulation of beta-catenin in mucoepidermoid carcinoma (MEC) is frequently noted, but the mechanism is unknown. The methylation status of adenomatous polyposis coli (APC) and secreted frizzled-related proteins (SFRPs) was examined by methylation-specific polymerase chain reaction (MSP) assay. The association of SFRP1, beta-catenin, and cyclin D1 expression in MEC was evaluated by immunohistochemical staining. A high percentage of methylation in APC and the SFRP genes was found in MEC compared with adjacent normal tissues, in which SFRP1 (58.6%) was the most frequent methylated gene. Moreover, abundant expression of SFRP1 was noted in normal tissues, whereas reduced SFRP1 expression was detected in 71.7% (33/46) of MECs. There was significant association between methylation and reduced expression of SFRP1. Cytoplasmic/nuclear (C/N) beta-catenin and high cyclin D1 expression were found in 13/55 (23.6%) and 36/55 (65.5%) of cases, respectively. There was significant correlation between C/N beta-catenin expression and reduced SFRP1 expression (P = 0.009). In addition, SFRP1 and beta-catenin expression correlated with tumor malignancy index such as tumor grade and stage. Overall patient survival was significantly worse in patients with reduced SFRP1 and C/N beta-catenin expression (P = 0.009 and P = 0.002, respectively). Methylation of the SFRP1 gene was the major cause of reduced SFRP1 expression. Reduced SFRP1 led to C/N accumulation of beta-catenin and was associated with tumor malignancy. Therefore, examination of SFRP1 expression and beta-catenin location could be useful predictors of tumor progression and prognosis in patients with MEC.

Contrasting activity of Hedgehog and Wnt pathways according to gastric cancer cell differentiation: Relevance of crosstalk mechanisms

Gastric cancer displays different biological behaviors according to histological differentiation. The different biological behavior might involve the activation of distinct signaling pathways necessary for the growth and survival of cancer cells in gastric cancer. We investigated the differentiation-related signal interaction between Hedgehog and Wnt pathways in gastric cancer cells. Differentiation of gastric cancer cells was induced by sodium butyrate. The sonic Hedgehog (SHH) signal expressions were increased during cellular differentiation. In contrast, the expression of Wnt signaling was decreased during differentiation. Ectopic expression of glioma-associated oncogene-1 (GLI1) increased the level of secreted frizzled related protein-1 (SFRP1) transcript, whereas inhibition of GLI1 reduced the level of SFRP1 transcript. ChIP assay showed that GLI1 induced the transcriptional regulation of SFRP1 gene expression. Ectopic expression of GLI1 decreased the nuclear beta-catenin staining, but the inhibition of GLI1 induced the reversal of nuclear beta-catenin overexpression. Ectopic expression of beta-catenin also decreased the expression of GLI1 in the butyrate treated cancer cells. SHH and GLI1 immunoexpression was greater in well differentiated gastric cancer tissues compared to poorly differentiated tissues, and nuclear beta-catenin immunoexpression was lower in well differentiated compared to poorly differentiated tissues. The SHH and Wnt pathways are differentially involved according to gastric cancer cell differentiation.