SFRP1 Promoter Methylation Research Articles

GRIK1-AS1 deficiency accelerates endometriosis progression by boosting DNMT1-dependent SFRP1 promoter methylation in endometrial stromal cells.

Endometriosis, a gynecological disease that affects up to 10% of women, is a major cause of pain and infertility. Deregulation of the epigenome is accountable for the onset and progression of endometriosis, although its exact mechanism is unknown. The purpose of the current study is to examine the role of the long non-coding RNA (lncRNA) GRIK1-AS1 in the epigenetic regulation of endometrial stromal cell proliferation and the development of endometriosis. Endometriosis datasets were screened to identify GRIKI-AS1 as dramatically declining in endometriosis. Gain or loss of function endometrial stromal cell (ESC) models were established. The anti-proliferation phenotype was investigated using in vitro and in vivo experiments. Epigenetic regulatory network analyses were conducted to suggest the intrinsic molecular mechanism. With bioinformatic and clinical data, we observed that GRIK1-AS1 and SFRP1 were expressed at low levels in endometriosis. Overexpressed GRIK1-AS1 inhibited ESC proliferation, while SFRP1 knockdown rescued the antiproliferative ability of GRIK1-AS1. Specifically, methylation-dependent expression inhibition of SFRP1 was revealed in ESCs. Mechanistically, GRIK1-AS1 hampers the occupancy of DNMT1 in SRFP1 promoter, leading to hypomethylation of SFRP1 and upregulated SFRP1 expression, thereby potentially suppressing Wnt signaling and its adverse proliferative effect. Therapeutically, lentivirus-mediated upregulation of GRIK1-AS1 inhibited endometriosis disease progression in vivo. Our study is a proof-of-concept demonstration for GRIKI-AS1-associated endometriosis pathogenesis and highlights a potential intervention target.

Abstract B047: Promoter hypermethylation of SFRP1 as a prognostic and predictive blood-based biomarker in patients with stage IV pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a disease with an abysmal prognosis. Unfortunately, no reliable prognostic or predictive blood-based biomarkers are currently available. An increasingly promising prognostic and predictive tool in cancer is the analysis of genetic and epigenetic alterations in cell-free DNA. One such alteration, promoter hypermethylation (ph) of SFRP1, has recently been linked to poor prognosis in gemcitabine-treated patients with stage IV PDAC. This study aimed to further examine phSFRP1 as a clinically relevant prognostic and predictive biomarker in FOLFIRINOX-treated patients with stage IV PDAC. Methods: Serum samples from patients with stage IV PDAC, treated with first-line FOLFIRINOX, were obtained from two danish biobanks. All blood samples were drawn before the initiation of palliative treatment. Based on a bisulfite treatment, we conducted a methylation-specific polymerase chain reaction analysis of the promoter region of the gene SFRP1. Methylation status was dichotomized based on the measured Cycle threshold. Survival according to SFRP1 promoter methylation status was assessed with Kaplan-Meier curves, log-rank tests, and Cox proportional hazard regression models. The adjusted Cox regression model included SFRP1 promoter methylation status, WHO performance status, age > 65 years, and sex. Results: Fifty-two FOLFIRINOX-treated patients with stage IV PDAC were included in the study. Patients with phSFRP1 (n = 23) had a median overall survival (mOS) of 6.8 months, significantly shorter than patients with unmethylated (um) SFRP1 (n = 29) who had a mOS of 15.7 months (log-rank test, p < 0.01). Likewise, in both crude and adjusted Cox-regression analysis, patients with phSFRP1 had an hazard ratio for death of 2.78 (95% CI 1.55-4.99) and 2.82 (95% CI 1.55-5.14) respectively, compared to patients with umSFRP1. Conclusions: Results indicate that FOLFIRINOX-treated stage IV PDAC patients with phSFRP1 have significantly shorter survival than patients with umSFRP1. Combined with previous literature, this indicates the value of phSFRP1 as a blood-based prognostic biomarker in patients with stage IV PDAC, regardless of the given first-line palliative chemotherapy. In addition, it indicates that phSFRP1 may also have predictive value for chemotherapy sensitivity in patients with stage IV PDAC. This knowledge may facilitate individualized treatment of patients with stage IV PDAC. Additionally, SFRP1 could be a valuable target for treatment with hypomethylating drugs to improve treatment response. Citation Format: Benjamin E. Stubbe, Stine D. Henriksen, Poul H. Madsen, Anders C. Larsen, Henrik B. Krarup, Inge S. Pedersen, Julia S. Johansen, Carsten P. Hansen, Ole Thorlacius-Ussing. Promoter hypermethylation of SFRP1 as a prognostic and predictive blood-based biomarker in patients with stage IV pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B047.

The H2A.Z-KDM1A complex promotes tumorigenesis by localizing in the nucleus to promote SFRP1 promoter methylation in cholangiocarcinoma cells

BackgroundIntrahepatic cholangiocarcinoma (ICC), originating from the bile ducts, is the second most common primary liver malignancy, and its incidence has recently increased. H2A.Z, a highly conserved H2A variant, is emerging as a key regulatory molecule in cancer. However, its underlying mechanism of action in ICC cells remains unclear. MethodsHere, we examined the expression of H2A.Z and SFRP1 in normal intrahepatic cholangiocytes, ICC cell lines, ICC tissue microarrays, and fresh specimens. The correlations between H2A.Z or SFRP1 expression and clinical features were analysed. The overall survival rate was analysed based on H2A.Z and SFRP1 expression. Immunoprecipitation was used to analyse the recruitment of KDM1A, and ChIP sequencing and BSP were used to analyse the enrichment of methylation-related molecules such as H3K4me1 and H3K4me2 in the SFRP1 promoter and reveal the underlying mechanisms. Knockdown and rescue experiments were used to determine the potential mechanism by which H2A.Z and SFRP1 promote tumorigenesis in vitro.ResultsWe showed that upregulation of H2A.Z expression is linked to downregulation of SFRP1 expression in ICC tissues and poor overall survival in patients with ICC. H2A.Z interacted with KDM1A in the nucleus to bind to the -151 ~ -136 bp region upstream of the SFRP1 promoter to increase its demethylation in ICC cells. Functionally, H2A.Z silencing inhibited the proliferation and invasion of ICC cells, and these effects were mitigated by SFRP1 silencing in ICC cells.ConclusionsOur findings reveal that H2A.Z inhibits SFRP1 expression through chromatin modification in the context of ICC by forming a complex with KDM1A in the nucleus.

Clinical Significance of ADAMTS19, BMP7, SIM1, and SFRP1 Promoter Methylation in Renal Clear Cell Carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney tumors, accounting for the majority of deaths from genitourinary cancers. The currently used nomograms for predicting patient outcomes are based on clinical-pathological characteristics only; however, a significant number of ccRCC survivors with similar radiological and histological features still demonstrate a different clinical course of the disease. This study aimed at the identification of novel DNA methylation biomarkers for the monitoring of patients with ccRCC.MethodsGene expression profiling by SurePrint G3 Human GE 8×60K Microarrays was performed in 4 ccRCC tissues and adjacent non-cancerous renal tissue (NRT) samples. Four down-regulated genes were selected for further DNA methylation status analysis in 123 ccRCC and 45 NRT samples using methylation-specific PCR (MSP).ResultsDNA methylation changes of ADAMTS19, BMP7, SIM1, and SFRP1 were cancer-specific with significantly (P<0.050) higher methylation frequency (37%, 20%, 18%, and 42%, respectively) in tumor tissues. The methylated status of at least one gene was significantly related to various clinical-pathological parameters, including tumor size, Fuhrman and WHO/ISUP grades, intravascular invasion, and necrosis. Moreover, the methylated status of multimarker panel ADAMTS19, BMP7 & SFRP1 was predictive for poorer overall survival (HR, 4.11; 95% CI, 1.22–13.86).ConclusionIn conclusion, DNA methylation of the three-gene panel consisting of ADAMTS19, BMP7 & SFRP1 supposedly predicts the outcome of patients diagnosed with ccRCC and possibly might be used to enrich the current prognostic tools.

Value of combined detection of ITGA4 and SFRP2 gene methylation in stool DNA in diagnosis and prognostic evaluation of colorectal tumors

To investigate the value of quantitative detection of ITGA4 and SFRP2 gene methylation in stool DNA for the early diagnosis and prognostic evaluation of colorectal tumors. Real-time PCR was used for quantitative assessment of ITGA4 and SFRP2 gene methylation levels in stool samples of 85 patients with colorectal cancer, 65 patients with colorectal adenoma and 40 healthy subjects. The 3 groups were comparable for age and gender composition. Methylated ITGA4 and SFRP2 promoters were detected in 48.2% and 62.4% of patients with colorectal cancer, respectively, with a combined positivity of 81.2%. ITGA4 and SFRP2 promoter methylation was detected in 23.1% and 43.1% of patients with colorectal adenoma, respectively, with a combined positivity of 69.2%. The positivity rates of ITGA4 and SFRP2 methylation were significantly higher in patients with colorectal cancer than in those with colorectal adenoma (P < 0.001; P= 0.001) and healthy subjects (P < 0.001; P < 0.001). In colorectal cancer group, ITGA4 and SFRP2 promoter methylation levels were correlated with postoperative tumor recurrence in colorectal cancer group, and the relapse-free survival rate was significantly lower in positive patients for ITGA4 and SFRP2 promoter methylation than in the negative patients (P=0.0002; P=0.007). Multivariate analysis with the COX proportional hazard regression model showed that methylation of ITGA4 and SFRP2 gene promoters (P=0.01) and the degree of tumor differentiation (P=0.03) were associated with the recurrence of colorectal cancer, and were independent risk factors for the recurrence of colorectal cancer. Combined detection of ITGA4 and SFRP2 gene methylation levels in stool DNA can improve the early diagnosis rate of colorectal tumor. ITGA4 and SFRP2 promoter methylation and the degree of tumor differentiation are independent risk factors for colorectal cancer recurrence.

High-risk HPV infection modulates the promoter hypermethylation of APC, SFRP1, and PTEN in cervical cancer patients of North India.

Persistent infection with oncogenic HPV and downregulation of tumor suppressor genes play an essential role in the development and progression of cervical cancer. The present study aimed to identify the promoter methylation status of APC, SFRP1, and PTEN which are important regulators of Wnt pathway and their association with high-risk HPV infection and gene expression. Methylation Specific PCR (MSP) and quantitative reverse transcription PCR (RT-qPCR) were used to detect methylation status and gene expression levels of APC, SFRP1, and PTEN in cervical cancer biopsies (110) and paired non-cancerous biopsies (28). APC promoter was methylated in 38%, SFRP1 in 95%, and PTEN in 55% of the cervical cancer biopsies. Our data showed a trend of a higher rate of methylation of the gene promoters in cervical cancer biopsies while; they were majorly un-methylated in non-cancerous biopsies. Corresponding to a higher rate of methylation in cancer biopsies, the gene expression levels of APC, SFRP1, and PTEN were reduced in cervical cancer samples in comparison to normal cervix tissues. Further, we observed that 97% cancer biopsies were HPV infected and high-risk type HPV16 and 18 infections were significantly positively associated with APC (p = 0.008 and p = 0.007), SFRP1 (p = 0.003 and p = 0.0067), and PTEN (p = 0.049 and p = 0.008) promoter methylation. APC, SFRP1, and PTEN promoter hyper-methylation is positively associated with high-risk HPV infection and inversely associated with gene expression. Our findings show that high-risk HPV infection promotes methylation of these genes and further promotes their silencing.

DNA methylation analysis with methylation-sensitive high-resolution melting (MS-HRM) reveals gene panel for glioma characteristics.

IntroductionLocal DNA hypermethylation is a potential source of cancer biomarkers. While the evaluation of single gene methylation has limited value, their selected panel may provide better information.AimsThis study aimed to analyze the promoter methylation level in a 7‐gene panel in brain tumors and verifies the usefulness of methylation‐sensitive high‐resolution melting (MS‐HRM) for this purpose.MethodsForty‐six glioma samples and one non‐neoplastic brain sample were analyzed by MS‐HRM in terms of SFRP1, SFRP2, RUNX3, CBLN4, INA, MGMT, and RASSF1A promoter methylation. The results were correlated with patients’ clinicopathological features.ResultsDNA methylation level of all analyzed genes was significantly higher in brain tumor samples as compared to non‐neoplastic brain and commercial, unmethylated DNA control. RASSF1A was the most frequently methylated gene, with statistically significant differences depending on the tumor WHO grade. Higher MGMT methylation levels were observed in females, whereas the levels of SFRP1 and INA promoter methylation significantly increased with patients’ age. A positive correlation of promoter methylation levels was observed between pairs of genes, for example, CBLN4 and INA or MGMT and RASSF1A.ConclusionsOur 7‐gene panel of promoter methylation can be helpful in brain tumor diagnosis or characterization, and MS‐HRM is a suitable method for its analysis.

Association between variation of circulating 25-OH vitamin D and methylation of secreted frizzled-related protein 2 in colorectal cancer

BackgroundsColorectal cancer (CRC) results from the accumulation of epigenetic and genetic changes in colon cells during neoplasic transformation, which the activation of Wingless (Wnt) signaling pathway is a common mechanism for CRC initiation. The Wnt pathway is mainly regulated by Wnt antagonists, as secreted frizzled-related protein (SFRP) family. Indeed, SFRP2 is proposed as a noninvasive biomarker for CRC diagnosis. Vitamin D also antagonizes Wnt signaling in colon cancers cells. Several studies showed that vitamin D was able to alter DNA methylation, although this mechanism is not yet clear. Therefore, the aim of this study was to find an association between circulating 25-OH vitamin D (30th percentile of vitamin D) and the SFRP2 methylation.MethodsA total of 67 CRC patients were included in the study. These patients were subdivided into two groups based on their 30th percentile vitamin D (20 patients were below, and 47 participants were above the 30th percentile of vitamin D). We investigated the SFRP2 methylation in peripheral blood mononuclear cells (PBMCs), visceral adipose tissue (VAT), CRC tumor tissue, and adjacent tumor-free area. We also determined the relationship between SFRP2 methylation and methylation of carcinogenic and adipogenic genes. Finally, we tested the effect of vitamin D on the SFRP2 methylation in human colorectal carcinoma cell lines 116 (HCT116) and studied the association of neoadjuvant therapy under the 30th percentile vitamin D with SFRP2 promoter methylation.ResultsSFRP2 methylation in tumor area was decreased in patients who had higher levels of vitamin D. SFRP2 promoter methylation was positively correlated in tumor area with insulin and homeostasis model assessment of insulin resistance (HOMA-IR) but negatively correlated with HDL-c. SFRP2 methylation was also correlated with T cell lymphoma invasion and metastasis 1 (TIAM1) methylation in tumor area and CCAAT/enhancer-binding protein alpha (C/EBPα) in VAT. Treatment with vitamin D did not affect SFRP2 methylation in HCT116 cell line. Finally, neoadjuvant treatment was correlated with higher circulating 25-OH vitamin D and SFRP2 methylation under linear regression model.ConclusionOur results showed that higher circulating vitamin D is associated with low SFRP2 promoter methylation. Therefore, our results could suggest that vitamin D may have an epigenetic effect on DNA methylation. Finally, higher vitamin D could contribute to an improvement response to neoadjuvant treatment.

The Prognostic Value of DNA Methylation Markers in Renal Cell Cancer: A Systematic Review

Introduction: DNA methylation is an epigenetic event that occurs in normal tissues but changes in both the early and late stages of multiple tumor entities, including renal cell cancer (RCC). Many studies have shown that DNA methylation markers are relevant to RCC prognosis, but none of these markers have entered into clinical routine. Furthermore, because of its potential reversibility, DNA methylation might provide a new target for RCC therapy strategies. Materials and methods: Following PRISMA guidelines, we performed a systematic literature search up to February 2019. After selection for eligibility, a total of 56 studies were identified for analysis. Each study was categorized and the level of evidence assessed. Only articles reporting on DNA methylation markers and their association with survival were included. Descriptive statistical analyses were conducted with R statistical software. Results: We identified promoter methylation of SFRP1, GATA5, NEFH, GREM1, and BCN1 as associated with survival in RCC. Moreover, we found evidence that methylation signatures, i.e., grouping of different potential gene markers, might be of better prognostic value than single gene marker investigations. Nevertheless, because of the heterogeneous features of the studies in terms of design, methodology, patient cohorts, and statistics, the true clinical impact of these methylation markers for prognosis in RCC patients remains uncertain. Conclusion: This systematic review elucidates the potential impact of DNA methylation on survival of patients with RCC. Several promising prognostic markers, especially methylation signatures, were identified, which is encouraging, but prospective validations are necessary to establish their true clinical value.

DNA methylation of SFRP1, SFRP2, and WIF1 and prognosis of postoperative colorectal cancer patients

BackgroundAs biomarkers, DNA methylation is used to detect colorectal cancer (CRC) and make assessment of CRC prognosis. The published findings showed the association between the methylation of SFRP1, SFRP2, and WIF1, located in the Wnt signaling pathway, and the prognosis of CRC were not consistent. Our study aimed to explore the potential possibility of SFRP1, SFRP2, and WIF1 concomitant promoter methylation as prognostic biomarkers of postoperative CRC patients.MethodsAs a total of 307 sporadic postoperative CRC patients were followed up, we detected SFRP1, SFRP2, and WIF1 methylation obtained from tumor tissues and adjacent non-tumor tissues respectively on the basis of methylation-sensitive high resolution melting analysis. Univariate and multivariate Cox regressions were carried out so as to assess the potential possibility of SFRP1, SFRP2, and WIF1 promoter methylation as predictors of prognosis. Confounders in our study were controlled by Propensity Score (PS) analysis.ResultsThe SFRP1, SFRP2, and WIF1 methylation levels in tumor tissues were significantly higher than that in adjacent non-tumor tissues (P < 0.001). SFRP2 hypermethylation was significantly associated with a favorable clinical outcome at the hazard ratio (HR) of 0.343 [95% confidence intervals (CI): 0.164–0.718, P = 0.005] and 0.410 (95% CI: 0.200–0.842, P = 0.015) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1 and SFRP2 was significantly associated with a favorable clinical outcome at the HR of 0.333 (95% CI: 0.159–0.694, P = 0.003) and 0.398 (95% CI: 0.192–0.821, P = 0.013) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1, SFRP2 and WIF1 was significantly associated with a favorable clinical outcome at the HR of 0.326 (95% CI: 0.117–0.908, P = 0.032) and 0.401 (95% CI: 0.146–1.106, P = 0.077) in multivariate Cox regression and PS analysis, respectively.ConclusionsSFRP1, SFRP2, and WIF1 were frequently hypermethylated in CRC tumor tissues. It was apparent that the promoter hypermethylation of SFRP2 and co-hypermethylation of SFRP1 and SFRP2 might be considered as independent prognostic predictors for survival advantage of postoperative CRC patients.

RETRACTED: Silencing of Long Non-coding RNA HOTTIP Reduces Inflammation in Rheumatoid Arthritis by Demethylation of SFRP1

Accumulating evidence suggests long non-coding RNAs (lncRNAs) play crucial roles in the pathogenesis of rheumatoid arthritis (RA). Here, we aimed to define the role of HOXA transcript at the distal tip (HOTTIP) in RA pathogenesis in relation to SFRP1 methylation and Wnt signaling pathway. HOTTIP was found highly expressed, and SFRP1 was hypermethylated in RA synovial fibroblasts (RASFs). Next, gain- or loss-of-function experiments were conducted in RASFs to explore the effects of HOTTIP on the biological behaviors of RASFs. Silencing of HOTTIP or overexpression of SFRP1 inhibited RASF proliferation, invasion, and migration, while enhancing apoptosis. The relationship among HOTTIP, SFRP1, and Dnmt3b was determined using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) assays. The regulatory mechanisms of HOTTIP/Dnmt3b/SFRP1 were explored by altering their expression in RASFs. It was noted that HOTTIP could induce SFRP1 promoter methylation through recruitment of Dnmt3b and activate the Wnt signaling pathway. Finally, a rat RA model was established in order to evaluate the in vivo effects of HOTTIP and SFRP1, which suggested that HOTTIP silencing or SFRP1 elevation inhibited the progression of RA in vivo. Our key findings demonstrate the anti-inflammatory ability of HOTTIP silencing in RA through SFRP1 promoter demethylation. These findings support HOTTIP as a candidate anti-arthritis target.

Influence of some methylated hepatocarcinogenesis-related genes on the response to antiviral therapy and development of fibrosis in chronic hepatitis C patients.

Background and AimEpigenetics involved in multiple normal cellular processes. Previous research have revealed the role of hepatitis C virus infection in accelerating methylation process and affecting response to treatment in chronic hepatitis patients. This work aimed to elucidate the role of promoter methylation (PM) in response to antiviral therapy, and its contribution to the development of fibrosis through hepatocarcinogenesis-related genes.MethodsA total of 159 chronic hepatitis Egyptian patients versus 100 healthy control group were included. The methylation profile of a panel 9 genes (SFRP1, p14, p73, APC, DAPK, RASSF1A, LINE1, O6MGMT, and p16) was detected in patients’ plasma using methylation-specific polymerase chain reaction (MSP).ResultsClinical and laboratory findings were gathered for patients with combined pegylated interferon and ribavirin antiviral therapy. Regarding the patients’ response to antiviral therapy, the percentage of non-responders for APC, O6MGMT, RASSF1A, SFRP1, and p16 methylated genes were significantly higher versus responders (P<0.05). Of the 159 included patients, the most frequent methylated genes were SFRP1 (102/159), followed by p16 (100/159), RASSF1A (98/159), then LINE1 (81/159), P73 (81/159), APC (78/159), DAPK (66/159), O6MGMT (66/159), and p14 (54/159). A total of 67/98 (68.4%) cases of RASSF1A methylated gene (P=0.0.024), and 62/100 (62%) cases of P16 methylated gene (P=0.03) were associated with mild-degree fibrosis.ConclusionsTo recapitulate, the PM of SFRP1, APC, RASSF1A, O6MGMT, and p16 genes increases in chronic hepatitis C patients, and can affect patients’ response to antiviral therapy. The RASSF1A and P16 genes might have a role in the distinction between mild and marked fibrosis.

Hypermethylated Promoters of Secreted Frizzled-Related Protein Genes are Associated with Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of death worldwide. Aberrant DNA methylation has been recognized as one of the most common molecular alterations in CRC. The goal of this study was to investigate the diagnostic value of SFRP1 and SFRP2 methylation for CRC. A total of 80 pairs of CRC patients were recruited to test the association of SFRP1 and SFRP2 promotor methylation with CRC. Methylation assay was performed using quantitative methylation-specific polymerase chain reaction (qMSP) method. In this study, we found the methylation levels of SFRP1 and SFRP2 in CRC tumor tissues were significantly higher than those in the adjacent non-tumor tissues (SFRP1: P = 2E-5; SFRP2: P = 0.014). Further bioinformatics analysis of TCGA data confirmed the association of the two genes with CRC (SFRP1: P = 7E-21; SFRP2: P = 5E-24). Luciferase reporter gene assay showed that the recombinant plasmids with SFRP1 and SFRP2 fragments could significantly enhance promoter activity (SFRP1: P = 0.002; SFRP2: P = 0.004). In addition, SFRP1 and SFRP2 methylation were inversely correlated with the mRNA expression displayed by TCGA data mining (SFRP1: r = -0.432, P = 4E-11; SFRP2: r = -0.478, P = 1E-13). GEO data analysis indicated that SFRP1 and SFRP2 expression were increased in three CRC cell lines (COLO320, HCT116 and HT29) after 5'-AZA-deoxycytidine treatment, suggesting that DNA methylation played an important role in regulating gene expression of the two genes. Our results confirmed that promoter methylation of SFRP1 and SFRP2 contributed to the risk of CRC.

Prognostic potential of KLOTHO and SFRP1 promoter methylation in head and neck squamous cell carcinoma.

Hypermethylation in the CpG island promoter regions of tumor suppressors is known to play a significant role in the development of HNSCC and the detection of which can aid the classification and prognosis of HNSCC. This study aims to profile the methylation patterns in a panel of key genes including CDKN2A, CDKN2B, KLOTHO (KL), RASSF1A, RARB, SLIT2, and SFRP1, in a group of HNSCC samples from Saudi Arabia. The extent of methylation in these genes is determined using the MethyLight assay and correlated with known clinicopathological parameters in our samples of 156 formalin-fixed and paraffin-embedded HNSCC tissues. SLIT2 methylation had the highest frequency (64.6%), followed by RASSF1A (41.3%), RARB (40.7%), SFRP1 (34.9), KL (30.7%), CKDN2B (29.6%), and CKDN2A (29.1%). KL and SFRP1 methylation were more predominant in nasopharyngeal tumors (P=0.001 and P=0.031 respectively). Kaplan Meier analysis showed that patients with moderately differentiated tumors who display SFRP1 methylation have significantly worse overall survival in comparison with other samples. In contrast, better clinical outcomes were seen in patients with KL methylation. In conclusion, our findings suggest that the detection of frequent methylation in SFRP1 and KL genes' promoters could serve as prognostic biomarkers for HNSCC.

Prognostic DNA methylation markers for renal cell carcinoma: a systematic review.

Despite numerous published prognostic methylation markers for renal cell carcinoma (RCC), none of these have yet changed patient management. Our aim is to systematically review and evaluate the literature on prognostic DNA methylation markers for RCC. We conducted an exhaustive search of PubMed, EMBASE and MEDLINE up to April 2017 and identified 49 publications. Studies were reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, assessed for their reporting quality using the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria, and were graded to determine the level of evidence (LOE) for each biomarker. We identified promoter methylation of BNC1, SCUBE3, GATA5, SFRP1, GREM1, RASSF1A, PCDH8, LAD1 and NEFH as promising prognostic markers. Extensive methodological heterogeneity across the included studies was observed, which hampers comparability and reproducibility of results, providing a possible explanation why these biomarkers do not reach the clinic. Potential prognostic methylation markers for RCC have been identified, but they require further validation in prospective studies to determine their true clinical value.

Identification of Methylation Profiles of Cancer-related Genes in Circulating Tumor Cells Population.

We performed an epigenetic analysis of the first exon of the hVIM gene and the SFRP2 in circulating tumor cells (CTCs) and correlation with the corresponding primary colorectal cancer (CRC) tissue. CTCs detection in 52 colorectal cancer patients was managed by a multi-marker immunomagnetic method with the use of quantum dots (QDs). To determine methylation levels we used high-resolution melting (HRM) technology. In the case of VIM we found 76.9% methylated samples, compared to 53.8% in tissue samples. Regarding SFRP2 promoter methylation levels in tissue and CTCs samples, 67.3% and 73.1%, were found methylated respectively. Correlation analysis of methylation levels with KRAS and BRAF mutations (performed in our previous study) demonstrates that high-methylation epigenotype strongly correlates to BRAF mutation. CTCs is a promising diagnostic tool. The combination of genetic mutations and epigenetic aberrations specifically in CTCs, will ameliorate CRC diagnosis in the future.

Wnt pathway antagonists, SFRP1, SFRP2, SOX17, and PPP2R2B, are methylated in gliomas and SFRP1 methylation predicts shorter survival.

The deregulation of Wnt signaling is observed in various cancers, including gliomas, and might be related to the methylation of the genes encoding antagonists of this signaling pathway. The aim of the study was to assess the methylation status of the promoter regions of six Wnt negative regulators and to determine their prognostic value in clinical samples of gliomas of different grades. The methylation of SFRP1, SFRP2, PPP2R2B, DKK1, SOX17, and DACH1 was analyzed in 64 glioma samples using methylation-specific polymerase chain reaction (MSP). The results were analyzed in correlation with clinicopathological data. Promoter methylation in at least one of the analyzed genes was found in 81.3 % of the tumors. All benign tumors [grade I according to the World Health Organization (WHO) classification] lacked the methylation of the studied genes, whereas grade II, III, and IV tumors were, in most cases, methylation-positive. The methylation index correlated with the patient’s age. The most frequently methylated genes were SFRP1 and SFRP2 (73.4 % and 46.9 %, respectively), followed by SOX17 (20.3 %) and PPP2R2B (10.9 %); DKK1 and DACH1 were basically unmethylated (1.6 %). SFRP1 methylation negatively correlated with patients’ survival time, and was significantly more frequent in older patients and those with higher grade tumors. Overall, the results of this study indicate that aberrant promoter methylation of Wnt pathway antagonists is common in gliomas, which may be the possible cause of up-regulation of this signaling pathway often observed in these tumors. Moreover, SFRP1 promoter methylation can be regarded as a potential indicator of glioma patients’ survival.

Black raspberries protectively regulate methylation of Wnt pathway genes in precancerous colon tissue.

Ulcerative colitis is frequently an intermediate step to colon cancer. The interleukin-10 knockout mouse is a genetic model of this progression. We report that knockout mice fed 5% black raspberries (BRB) had significantly less colonic ulceration as compared with knockout mice that consumed the control diet. Dysfunction of the Wnt signaling pathway is a key event in ulcerative colitis-associated colon carcinogenesis. Therefore, we investigated the effects of BRBs on the Wnt pathway and found that the BRB-fed knockout mice exhibited a significantly lower level of β-catenin nuclear translocation. We followed-up this observation by evaluating the effect of BRBs on selected Wnt pathway antagonists. The mRNA expression levels of wif1, sox17, and qki were diminished in the knockout mice, whereas they were expressed at normal levels in knockout mice that were fed BRBs. The lower mRNA expression of these genes in the colon from the knockout mice correlated with hypermethylation of their promoter regions; BRBs decreased their promoter methylation and increased mRNA expression of these genes. This hypomethylation was associated with elevated protein expression of key proteins/enzymes that augment methylation, for example, dnmt3b, hdac1, hdac2, and mbd2 in the knockout mice; in addition, BRBs decreased the protein expression of these proteins/enzymes. The knockout mouse model recapitulates what occurs in human ulcerative colitis. Promoter methylation of CDH1 and SFRP1 was significantly higher in human ulcerative colitis tissues compared with their adjacent normal tissues. In conclusion, our results suggest that BRBs inhibit colonic ulceration and, ultimately, colon cancer partly through inhibiting aberrant epigenetic events that dysregulate Wnt signaling.

Epigenetic inactivation of the canonical wnt antagonist secreted frizzled-related protein 1 in hepatocellular carcinoma cells

Secreted Frizzled-related protein 1 (sFRP1), as one of most important Wnt antagonists, is frequently silenced by promoter hypermethylation in many types of tumor, including hepatocellular carcinoma (HCC). In this study, we aimed to investigate whether restoration of sFRP1 affected HCC metastatic behavior. sFRP1 mRNA expression and promoter methylation in HCC tissues and cell lines were examined using RT-PCR and methylation-specific PCR (MS-PCR), respectively. sFRP1 protein expression was assessed by Western Blot. We generated stable HCC cell line restoration of sFRP1 in HepG2 cells, which naturally do not express detectable sFRP1 mRNA. The effects of exogenous sFRP1 on HepG2 cell invasion were investigated using trans-well assay. Also the effects of sFRP1 re-expression on the β-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay.sFRP1 promoter methylation was frequently observed in HCC tissues (60%) and cell lines (75%). All samples with sFRP1 methylation showed down-regulation of sFRP1 expression in HCC cell lines. Demethylation treatment with 5-aza-20-deoxycytidine in HCC cells restored sFRP1 expression. Restoration of sFRP1 substantially impaired the invasive potentials of HepG2 cells. Moreover, exogenous sFRP1 caused significant decrease of β-catenin/T-cell factor-dependent transcription activity.These findings demonstrate that sFRP1 silencing due to promoter hypermethylation is a major event during tumorigenesis. sFRP1 is also a negative modulator of canonical Wnt signaling, which could contribute to metastasis in HCC progression, thus providing a possible therapeutic strategy against HCC.

Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia

AbstractSecreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)–leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.