Aberrant Promoter DNA Methylation Research Articles

Mitochondria-Associated Gene SLC25A32 as a Novel Prognostic and Immunotherapy Biomarker: From Pan-Cancer Multiomics Analysis to Breast Cancer Validation

Background. Mutations in SLC25A32 in humans cause late-onset exercise intolerance, which is associated with various neurological and metabolic diseases. However, its specific mechanism of action in tumour development is poorly understood owing to the lack of multiomics integrated analysis of SLC25A32 in pan-cancer. Methods. We used various analytical tools to comprehensively investigate the transcription, protein level, and promoter methylation of SLC25A32. Furthermore, the GSCA and cBioPortal databases were used to evaluate the inheritance impact and epigenetic alterations of SLC25A32 in pan-cancer. SLC25A32 expression and the prognostic significance of copy number alterations in multiple cancers were compared using the UCSCXenaShiny and GEPIA2.0 platforms, and its specific function in breast cancer was experimentally verified. Results. SLC25A32 is abnormally expressed at the transcriptional and protein levels in most cancer types, with aberrant DNA promoter methylation and significant gene amplification in most tumours. SLC25A32 is significantly associated with the survival prognosis of some cancers, immune infiltrating cells, tumour stemness, and immune-related markers. SLC25A32 knockdown decreased breast tumour cell proliferation, invasion, and metastasis. Conclusions. This study aimed to reveal SLC25A32 as a novel prognostic biomarker for pan-cancer prediction and immunotherapy efficacy and specifically describes its underlying mechanism of action in breast cancer. SLC25A32 is widely differentially expressed in pan-cancer with prognostic significance and is correlated with immune infiltration. Additionally, it can affect breast cancer occurrence and development.

Abstract A027: The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA

Abstract Background and purpose: The cystic fibrosis transmembrane conductance regulator (CFTR) gene is a tumor suppressor in colorectal cancer (CRC). People with cystic fibrosis, caused by biallelic germline mutations in CFTR, have a higher risk developing CRC. We also found that loss of CFTR is implicated in sporadic CRC. In a study of 90 persons diagnosed with stage II CRC, the 23% of patients with lowest CFTR expression had 30% lower disease-free survival at 3 years. The cause of decreased CFTR expression in this group is not known. We analyzed the TCGA COADREAD study to identify associations between low expression of CFTR and cancer-causing genetic alterations. Of these alterations, only BRAF-V600E mutation correlated with low expression of CFTR. The BRAF-V600E mutation is found in ~10% of CRC and is associated with global promoter hypermethylation leading to down regulation of tumor suppressor genes. Accordingly, our hypothesis was that BRAF-V600E mutation contributes to low expression of CFTR via CFTR promoter DNA methylation. Methods: In the TCGA COADREAD database 283 primary colorectal cancers diagnosed at stages II, III and IV had full information needed for this study: CFTR mRNA expression, somatic mutation, overall survival (OS), and DNA methylation status. OS was visualized using Kaplan-Meier analysis. Association between CFTR expression and methylation was determined by Pearson correlation coefficient (PCC) analysis. Associations between mutations and CFTR expression or methylation were determined by Point Biserial correlation coefficient (PBS) analysis. Results: Kaplan-Meier analysis of the 283 cases showed that overall survival was worse in the 25% of cases with lowest CFTR expression (p=0.035). The BRAF-V600E mutation was significantly associated with lower CFTR expression (PBS r=-0.38, p<0.0001) and 81% of BRAF V600E mutations were found in the 25% of cases with lowest CFTR expression. In contrast mutations in APC and KRAS showed a positive association with CFTR expression (PBS r=0.25, p<0.0001; PBS r=0.13, p=0.028 respectively). The CFTR promoter is hypermethylated in several cancers and BRAF-V600E mutation is associated with aberrant promoter DNA methylation. We examined DNA methylation at 9 CpG sites in the CFTR promoter and found that low CFTR expression significantly associated with increased DNA methylation at these 9 sites (PCC for average of 9 sites r= -0.56, p=<0.001). The association between BRAF-V600E mutation and CFTR methylation was modest but significant (PBS r=0.16, p=0.0063). Conclusions: BRAF-V600E mutation was significantly associated with CRC cases expressing low levels of CFTR, a subset with poor overall survival, as was CFTR promoter methylation. BRAF-V600E showed a modest association with CFTR promoter methylation and so may play a role in down-regulation of CFTR expression. However, association of BRAF-V600E with CFTR promoter methylation was modest compared to its association with CFTR expression suggesting that BRAF-V600E mutation may play additional roles in CRC tumors expressing low levels of CFTR. Citation Format: Patricia Scott, Anna Prizment, Rahul Bhattacharya, Zachary Blankenheim, Nathan Pankratz, Timothy Starr, Robert Cormier. The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A027.

The Correlation between Aberrant Promoter DNA Methylation and Breast Cancer Development and Progression – A Scoping Review

Background: DNA methylation has been associated with many malignancies, including breast cancer; however, the collective type of genes associated, as well as relevant methylation signatures, have not been reported. So, the study aimed to determine the significance of methylation in breast cancer, to identify the genes involved and their corresponding signatures by reviewing original laboratory studies from 2010 to 2019. Methods: A scoping review comprising of a comprehensive literature search in PubMed, Science Direct, Africa-Wide Information, CINAHL, Health Source, Medline and Web of Science databases. The Boolean terms were “breast cancer” AND “DNA methylation” AND “gene expression and function” AND “bisulfite treatment OR bisulfite conversion” respectively. Only English-written literature of original laboratory studies on humans, published between 2010 and 2019, was included, while miRNA studies were excluded. In all studies, the genes and their methylation signatures in breast cancer patients were of primary importance. Findings: Of the 87 genes identified, 71 were hypermethylated, 13 were hypomethylated, and three were differentially methylated. Several clinical and pathological factors were also associated with DNA methylation in many of these studies. Interpretation: DNA methylation is common in breast cancer. It is associated with the development, progression and prognosis of the disease. Because it is tumor-specific and occurs early in tumorigenesis, it may have value in disease diagnosis, assessment of response to treatment and prognosis. Funding Statement: There was no funding for this study. Declaration of Interests: There are no conflicts of interest. Ethics Approval Statement: Approval was not required for a scoping review.

Early detection of the major male cancer types in blood-based liquid biopsies using a DNA methylation panel

BackgroundLung (LC), prostate (PCa) and colorectal (CRC) cancers are the most incident in males worldwide. Despite recent advances, optimal population-based cancer screening methods remain an unmet need. Due to its early onset, cancer specificity and accessibility in body fluids, aberrant DNA promoter methylation might be a valuable minimally invasive tool for early cancer detection. Herein, we aimed to develop a minimally invasive methylation-based test for simultaneous early detection of LC, PCa and CRC in males, using liquid biopsies.ResultsCirculating cell-free DNA was extracted from 102 LC, 121 PCa and 100 CRC patients and 136 asymptomatic donors’ plasma samples. Sodium-bisulfite modification and whole-genome amplification was performed. Promoter methylation levels of APCme, FOXA1me, GSTP1me, HOXD3me, RARβ2me, RASSF1Ame, SEPT9me and SOX17me were assessed by multiplex quantitative methylation-specific PCR.SEPT9me and SOX17me were the only biomarkers shared by all three cancer types, although they detected CRC with limited sensitivity. A “PanCancer” panel (FOXA1me, RARβ2me and RASSF1Ame) detected LC and PCa with 64% sensitivity and 70% specificity, complemented with “CancerType” panel (GSTP1me and SOX17me) which discriminated between LC and PCa with 93% specificity, but with modest sensitivity. Moreover, a HOXD3me and RASSF1Ame panel discriminated small cell lung carcinoma from non-small cell lung carcinoma with 75% sensitivity, 88% specificity, 6.5 LR+ and 0.28 LR–. An APCme and RASSF1Ame panel independently predicted disease-specific mortality in LC patients.ConclusionsWe concluded that a DNA methylation-based test in liquid biopsies might enable minimally invasive screening of LC and PCa, improving patient compliance and reducing healthcare costs. Moreover, it might assist in LC subtyping and prognostication.

The prevalence and persistence of aberrant promoter DNA methylation in benzene-exposed Chinese workers

Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation.

Abstract LB-141: CIMP is associated with altered microbiota composition in colorectal cancer patients

Abstract DNA methylation changes are important in cancer development and the etiology of the CpG Island Methylator Phenotype (CIMP), the most extreme form of aberrant promoter DNA methylation in cancer, can only partially be explained by genetic changes. An association between CIMP and the gut microbiota has been implicated in colorectal cancer (CRC) pathogenesis but the exact mechanisms underlying the observations are poorly understood. In order to clarify the link between CIMP and the microbiome in CRC, we studied human primary tumor and adjacent mucosal tissues (normal) from CRC patients (n=46). The patient samples were categorized by tumor sites (23 proximal and 23 distal), sex (28 males and 18 females), and age (69 yrs ±11). Bisulfite pyrosequencing and Digital Restriction Enzyme Analysis of Methylation (DREAM) were performed to determine the methylation of CpG sites in the CRC tumors. The tumor samples were selected based on their CIMP status (10 high, 10 low, and 26 negative) and the tumor sites. 16S rRNA gene sequencing using Illumina-Hi-Seq was performed on those samples. Principle Coordinate analysis of the 16S rRNA sequences showed that the microbiota of CIMP-positive (CIMP-high and -low combined) tumors was significantly different from the microbiota of the CIMP-negative tumors (PERMANOVA, P = 0.013). Linear discriminant analysis effect size (LEfSe) showed an enrichment of specific bacterial taxa in the CIMP-positive microbiota. Fusobacterium (LDA score = 4.71, p=0.033) and Erysipelotrichaceae (LDA score = 3.77, p=0.004) were most enriched in the CIMP-positive tumors along with Bacteroides. The microbiota of different tumor sites also showed significant difference with Fusobacterium (LDA score = 4.32, p=0.047) and Erysipelotrichaceae (LDA score = 3.45, p=0.039) being enriched in the proximal tumors compared to the distal tumors. All these bacterial taxa have previous been found to be associated with CRC and/or metabolic disorders. We validated the 16S rRNA data by qPCR analysis using genus-specific probes. In the same samples, Pan-Fusobacterium and Bacteroides fragilis were enriched in the CIMP-positive tumors. The median number of Pan-Fusobacterium per 100 human cells was 12-fold higher in the CIMP-positive tumors (p=0.0015) and 6-fold higher in the case of B. fragilis (p=0.0228). Thus, our data show broad difference in the microbiota of CIMP-positive CRCs compared to CIMP-negative CRCs. The significantly enriched bacterial taxa in CIMP-positive CRC suggest that these specific taxa could play an important role in aberrant DNA methylation modulation in colorectal cancer, and further studies should elucidate the mechanism underlying the CIMP/microbiota link in the context of CRC. Citation Format: Pyoung Hwa Park, Raad Z. Gharaibeh, Lauren Cole, Ang Sun, Woonbok Chung, Jaroslav Jelinek, Jillian L. Pope, Christian Jobin, Jean-Pierre J. Issa. CIMP is associated with altered microbiota composition in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-141.

Abstract 2576: BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage

Abstract Ovarian cancer (OC) is a lethal gynecological malignancy with a 5-year survival rate of 46%. Surgical debulking followed by platinum and taxane based chemotherapy is the standard of care for OC patients. However, the major obstacle in the use of these agents for treatment of OC patients is development of chemoresistance. An epigenetic mechanism commonly associated with development of platinum resistance is promoter DNA methylation and associated gene silencing. Transcriptional silencing of tumor suppressor genes like MutL homolog 1 (MLH1) by this mechanism has been well established in development of platinum resistance. However, the mechanism of initiation of this aberrant promoter DNA methylation is not known. Platinum agents are DNA damaging agents which crosslink guanines and form intra-strand or inter-strand adducts. We hypothesize that platinum induced DNA damage or repair of the damage results in recruitment of proteins involved in transcriptional repression to sites of damage. Recruitment of repressive proteins results in transient repression of transcription in the vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repair of damage can occasionally result in retention of repressive proteins at sites of damage causing persistent transcriptional repression and gene silencing. Such persistent repression of key loci potentially contributes to the development of platinum resistance. We demonstrate that treatment of platinum sensitive OC cells with the IC50 dose of cisplatin for 8 hours resulted in ubiquitination of H2A/H2AX by western blot analysis. We hypothesize that this ubiquitination occurs on K119 of H2A/H2AX. H2A/H2AX ubiquitination at K119 mediated by Polycomb repressive complex 1 (PRC1) has been associated with transcriptional repression and gene silencing during development and differentiation and also during DNA repair. We observe a reduction in platinum induced H2AX ubiquitination on knockdown of PRC1 complex member RING1A using western blot analysis. Using immunofluorescence, we observe that BMI1, another PRC1 complex member, localizes to sites of platinum induced DNA damage. Platinum induced lesions are repaired by different repair pathways including – Nucleotide excision repair pathway (NER), Fanconi Anemia repair pathway (FA) and Homologous recombination repair (HRR). We demonstrate that knockdown of proteins in global genome NER and HRR pathways results in a decrease in platinum induced ubiquitination. Through on-going studies we seek to further elucidate the mechanism of recruitment of BMI1, RING1A to sites of platinum induced DNA damage and their role in long-term gene silencing. Understanding this mechanism will enable us to design inhibitors to prevent the occurrence of platinum resistant tumors in OC patients. Citation Format: Shruthi Sriramkumar, Heather M. O'Hagan. BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2576.

Aberrant DNA methylation of M1-macrophage genes in coronary artery disease

M1 and M2 macrophage balance in atherosclerosis has attracted much interest. Though, it remains unknown how macrophage heterogeneity is regulated. Moreover, the regulation of macrophage polarization and activation also involve DNA methylation. However, it remains ambiguous which genes are under direct regulation by DNA methylation. Our aim was to evaluate the gene-specific promoter DNA methylation status of M1/M2 polarization markers in PBMCs of CAD patients. A case-control study was performed with 25 CAD patients and 25 controls to study the promoter DNA methylation status of STAT1, STAT6, MHC2, IL12b, iNOS, JAK1, JAK2 and SOCS5 using MS-HRM analysis. Our data indicates that there was a clear-cut difference in the pattern of gene-specific promoter DNA methylation of CAD patients in comparison to controls. A significant difference was observed between the percentage methylation of STAT1, IL12b, MHC2, iNOS, JAK1 and JAK2 in CAD patients and control subjects. In conclusion, our data show that MS-HRM assay is a rapid and inexpensive method for qualitatively identifying aberrant gene-specific promoter DNA methylation changes in CAD. Furthermore, we propose that gene-specific promoter DNA methylation based on monocyte/macrophage might aid as diagnostic marker for clinical application or DNA methylation-related drug interventions may offer novel possibilities for atherosclerotic disease management.

Abstract B07: Targeting DAB2IP in ovarian cancer stem cells

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies. Failure to eliminate ovarian cancer stem cells (OCSCs) by the end of conventional chemotherapy contributes to tumor relapse and metastasis. Understanding the molecular features of OCSCs may allow for effective targeting and eradicating these cells and tumor remission. We reported tumor-initiation and expansion capacity by ALDH+ OCSCs after platinum-based treatment of ovarian cancer xenografts. In addition, loss of DAB2IP in prostate and colon cancer promoted CSC-like features, suggesting a critical role in modulating CSC maintenance. DAB2IP, a novel member of Ras GTPase-activating (GAP) protein family, was identified as a potent tumor suppressor that interfered with many aspects of cancer progression and was frequently downregulated in tumors. In the current study, we aimed to elucidate the tumor-suppressor role of DAB2IP in ovarian cancer in the context of OCSCs and explore epigenetic approaches to upregulate its expression. OCSCs and non-CSCs subpopulations were isolated from multiple high-grade serous ovarian cancer cell lines (Kuramochi, OVCAR3, and COV362). Reduced (p&amp;lt;0.05) DAB2IP expression in OCSCs compared to non-CSC counterparts was observed, suggesting that DAB2IP suppresses OCSC maintenance. Overexpression of DAB2IP in OCSCs derived from Kuramochi decreased (p&amp;lt;0.05) OCSC population, and DAB2IP overexpression inhibited stemness-related phenotypes, including reduced (P&amp;lt;0.05) spheroid formation ability after 14-day incubation under stem cell conditions and decreased (P&amp;lt;0.05) colony formation. Decreased expression of stem cell markers (Oct4, Nanog, and Twist) in DAB2IP-overexpressing cells was also observed, indicating potential key effectors downstream of DAB2IP. Furthermore, elevated DAB2IP expression in OCSCs decreased (p&amp;lt;0.05) cisplatin IC50 and cell migration capacity in transwell assay, suggesting DAB2IP plays a role in OCSC function. Aberrant DNA promoter methylation and histone modifications were previously shown to be two major mechanisms of DAB2IP inactivation in cancer. We treated OCSCs with next-generation DNMT inhibitor guadecitabine and EZH2 inhibitor GSK-126, either alone or in combination. Single-agent guadecitabine or GSK-126 decreased ALDH expression with greater (p&amp;lt;0.05) effect in combination. Single GSK-126 was sufficient to increase DAB2IP expression (p&amp;lt;0.05) with additional increase when combined with guadecitabine, correlating with decreased ALDH expression. Collectively, our data suggested a possible combination epigenetic therapy to eliminate OCSCs, potentially through targeting DAB2IP, an important OCSC regulator. Citation Format: Xingyue Zong, Ali R. Ozes, Kenneth P. Nephew. Targeting DAB2IP in ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B07.

Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.

The clinical relevance of aberrant DNA promoter methylation is being increasingly recognized in urothelial carcinoma. The present study was conducted to explore the methylation status of patients with upper-tract urothelial carcinoma (UTUC) who experienced bladder recurrence, and to evaluate the predictive value of gene methylation for second bladder recurrence and tumor progression. A total of 85 patients with primary UTUC, who experienced bladder recurrence after radical nephroureterectomy, were enrolled between January 2001 and December 2013. Using methylation-sensitive polymerase chain reaction, the promoter methylation statuses of 10 genes were analyzed in the bladder tumor specimens. Among the patient group, 32 patients experienced second bladder recurrence, and bladder progression was detected in 16. With the exception of BRCA1, the methylation rate of the majority of genes tended to gradually increase to varying extents with the number of recurrences; a smaller proportion of primary tumors exhibited gene methylation when compared with the first recurrent tumors and second recurrent tumors. Univariate and multivariate Cox regression analyses revealed that unmethylated GDF15 [hazard ratio (HR)=0.36; 95% confidence interval (CI), 0.14-0.92] and methylated VIM (HR=2.91; 95% CI, 1.11-7.61) in the first recurrent bladder tumor, as well as male gender (HR=2.28; 95% CI, 1.06-4.87), first recurrence interval <8 months (HR=2.34; 95% CI, 1.15-4.78) and primary UTUC tumor size ≥5 cm (HR=3.48; 95% CI, 1.43-8.45) were independent risk factors for a second bladder recurrence after surgery for the first bladder recurrence; the Harrell's concordance index (c-index) for the related nomogram was 0.71 (95% CI: 0.61-0.81). Furthermore, methylated CDH1 (HR=2.91; 95% CI, 1.08-7.77) and VIM (HR=4.91; 95% CI, 1.11-21.7) in the first recurrent bladder tumor, male gender (HR=3.6; 95% CI, 1.1-11.73), and primary tumor stage T2-T4 (HR=4.57; 95% CI, 1.22-17.13), multifocality (HR=3.64; 95% CI, 1.19-11.16) and size ≥5 cm (HR=3.1; 95% CI, 1.91-10.54) for the primary UTUC were considered to be predictors of tumor progression; the c-index for the nomogram was 0.88 (95% CI, 0.69-0.92). The present findings demonstrated that promoter methylation of cancer-related genes was frequently observed in patients with urothelial carcinoma, and that the gene methylation rate of certain genes tended to gradually increase with the number of bladder recurrences. This may be used as a predictive factor for a second bladder recurrence and tumor progression after the surgical treatment of the first bladder recurrence.

Recurrent transcriptional loss of the PCDH17 tumor suppressor in laryngeal squamous cell carcinoma is partially mediated by aberrant promoter DNA methylation.

Protocadherins are cell-cell adhesion molecules encoded by a large family of genes. Recent reports demonstrate recurrent silencing of protocadherin genes in tumors and provide strong arguments for their tumor supresor functionality. Loss of protocadherins may contribute to cancer development not only by altering cell-cell adhesion, that is a hallmark of cancer, but also by enhancing proliferation and epithelial mesenchymal transition of cells via deregulation of the WNT signaling pathway. In this study we have further corroborated our previous findings on the involvement of PCDH17 in laryngeal squamous cell carcinoma (LSCC). We used bisulfite pyrosequencing to analyze a cohort of primary LSCC tumors for alterations in PCDH17 promoter DNA methylation as an alternative gene inactivation mechanism to the homozygous deletions reported earlier. Moreover, we analyzed primary LSCC samples by immunohistochemistry for PCDH17 protein loss. We identified recurrent elevation of PCDH17 promoter DNA methylation in 32/81 (40%) primary tumors (P < 0.001) and therein hypermethylation of 12 (15%) cases in contrast to no tumor controls (n = 24) that were all unmethylated. Importantly, DNA demethylation by decitabine has restored low level PCDH17 expression in LSCC cell lines. In conclusion, we provide a mechanistic explanation of recurrently observed PCDH17 silencing in LSCC by demonstrating the role of promoter methylation in this process. In light of these findings and recent reports showing that PCDH17 methylation is detectable in serum of cancer patients we suggest that testing PCDH17 DNA methylation might serve as a potential biomarker in LSCC.

HOXB13 expression and promoter methylation as a candidate biomarker in gastric cancer.

Homeobox b13 (HOXB13) is considered to be a tumor suppressor gene in multiple types of human cancer. The present study aimed to identify the difference in expression of HOXB13 mRNA between gastric cancer (GC) tissues and corresponding non-malignant gastric tissues. The clinical significance of HOXB13 mRNA expression was also assessed in GC and a potential association between HOXB13 mRNA expression and DNA promoter methylation was observed. The expression of HOXB13 mRNA was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and HOXB13 methylation status was assessed by methylation-specific PCR (MSP) in 5 GC cell lines and 85 paired GC and normal gastric tissues. Kaplan-Meier survival curves were used to assess the survival of patients with GC. HOXB13 mRNA expression was significantly lower in primary GC tissues than in corresponding nonmalignant gastric tissues, and decreased HOXB13 expression was associated with poorer differentiation, lymph node metastasis, invasion depth and Tumor-Node-Metastasis (TNM) stage. Kaplan-Meier survival analysis demonstrated that HOXB13 mRNA expression was a significant prognostic indicator of GC patient survival. Furthermore, MSP revealed that the proportion of GC samples with hypermethylated HOXB13 (60.0%, 51/85) was increased compared with the corresponding nonmalignant gastric tissues (11.8%, 10/85). Decreased HOXB13 mRNA expression was due to DNA hypermethylation as following treatment with the DNA methyltransferase inhibitor 5-Aza-dC, HOXB13 expression in the GC MKN-45 cell line was upregulated. The results of the present study indicate that decreased expression of HOXB13 mRNA was associated with tumor differentiation, depth of invasion, lymph node metastases and TNM stage in GC, and it was a significant poor prognostic factor for patients with GC. Aberrant DNA promoter methylation was a crucial reason for the downregulation of HOXB13 mRNA expression.

Epigenetic biomarkers in gastrointestinal cancers: The current state and clinical perspectives.

Each year, almost 4.1 million people are diagnosed with gastrointestinal (GI) cancers. Due to late detection of this disease, the mortality is high, causing approximately 3 million cancer-related deaths annually, worldwide. Although the incidence and survival differs according to organ site, earlier detection and improved prognostication have the potential to reduce overall mortality burden from these cancers. Epigenetic changes, including aberrant promoter DNA methylation, are common events in both cancer initiation and progression. Furthermore, such changes may be identified non-invasively with the use of PCR based methods, in bodily fluids of cancer patients. These features make aberrant DNA methylation a promising substrate for the development of disease biomarkers for early detection, prognosis and for predicting response to therapy. In this article, we will provide an update and current clinical perspectives for DNA methylation alterations in patients with colorectal, gastric, pancreatic, liver and esophageal cancers, and discuss their potential role as cancer biomarkers.

DNA methylation regulated microRNAs in human cervical cancer

Regulation of miRNA gene expression by DNA promoter methylation may represent a key mechanism to drive cervical cancer progression. In order to understand the impact of DNA promoter methylation on miRNAs at various stages of cervical carcinogenesis, we performed DNA methylation microarray on Normal Cervical Epithelium (NCE), Cervical Intraepithelial Neoplasia (CIN I-III) and Squamous Cell Carcinoma (SCC) tissues to identify differentially methylated miRNAs followed by validation by bisulfite sequencing. Further, expression of miRNAs was analyzed by qRT-PCR in clinical tissues and cervical cancer cell lines. Transcriptional activity was determined by luciferase assay. We identified a total of 69 hypermethylated and hypomethylated miRNA promoters encompassing 78 CpG islands in all except Y chromosome, among the three groups. The candidate DNA promoters of miR-424 were significantly hypermethylated and miR-200b and miR-34c were significantly hypomethylated in SCC compared to NCE (P < 0.05). Expression of miR-424, miR-200b, and miR-34c were inversely correlated with promoter DNA methylation in tissue samples. Treatment of cell lines with 5-aza-2'-deoxycytidine showed differential expression in all three miRNAs. We observed a decrease in miRNA promoter activity following in vitro SssI methylase treatment of miR-424, miR-200b, and miR-34c. Luciferase assay demonstrated that miR-200b and miR-424 functionally interacts with 3'-UTR of HIPK3 and RBBP6 respectively and decreased their activity in presence of miR-200b and miR-424 mimics transfected in SiHa cells. Taken together, we have identified deregulation of miRNAs by aberrant DNA promoter methylation, leading to its transcriptional silencing during cervical carcinogenesis, which can be potential targets for diagnosis and therapy.

NFATC1 activation by DNA hypomethylation in chronic lymphocytic leukemia correlates with clinical staging and can be inhibited by ibrutinib.

B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL.

Diagnostic Role of Aberrant DNA Promoter Methylation in Ovarian Cancer

Diagnostic Role of Aberrant DNA Promoter Methylation in Ovarian Cancer

APC2 and CYP1B1 methylation changes in the bone marrow of acute myeloid leukemia patients during chemotherapy.

Aberrant promoter DNA methylation is a major mechanism of leukemogenesis in hematologic malignancies, including acute myeloid leukemia (AML). However, the association between promoter methylation with chemotherapeutic outcomes remains unknown. In the present study, bone marrow samples were collected prior to and following chemotherapy in 30 AML patients. Methylation-specific polymerase chain reaction technology was used to examine the promoter methylation status of adenomatous polyposis col 2 (APC2) and cytochrome P450 family 1 subfamily B polypeptide 1 (CYP1B1). The results revealed no change in the methylation status of the APC2 promoter in patients following various chemotherapy regimens. However, the methylation status of the CYP1B1 promoter changed in response to 6 different chemotherapy regimens. AML patients of the M3 subtype displayed an induction of the CYP1B1 promoter methylation levels more frequently (57.1%) than patients affected by the other subtypes (M1: 33.3%; M2: 12.5%; M4: 16.7%; M5: 0% and M6: 0%). In addition, a higher frequency of male patients (4/13) exhibited modulation of the CYP1B1 promoter methylation status compared with female patients (3/17). Furthermore, of five AML patients with a poor prognosis, two exhibited changes leading to CYP1B1 hypomethylation and two leading to CYP1B1 hypermethylation. By contrast, three other patients exhibited hypermethylation changes along with remission. This may be explained by the different chemotherapy regimens used to treat these patients or by other unknown factors. The present study revealed that CYP1B1 promoter methylation was induced during chemotherapy, whereas the APC2 promoter remained hemimethylated. Furthermore, the changes in CYP1B1 methylation were dependent on the AML subtypes and the gender of the patients.

Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma.

BackgroundThe prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients and to evaluate this as a prognostic marker. Furthermore, we wanted to follow possible changes in methylation levels during treatment. Seventy-four patients were enrolled in the study, of which 59 received rituximab and CHOP-like chemotherapy. Plasma samples were collected from all patients at the time of diagnosis and from 14 healthy individuals used as controls. In addition, plasma samples were collected during and after treatment for surviving patients. In total, 158 plasma samples were analyzed for DNA methylation in the promoter regions of DAPK (DAPK1), DBC1, MIR34A, and MIR34B/C using pyrosequencing.ResultsAberrant methylation levels at the time of diagnosis were detected in 19, 16, 8, and 10 % of the DLBCL plasma samples for DAPK1, DBC1, MIR34A, and MIR34B/C, respectively. DAPK1 methylation levels were significantly correlated with DBC1 and MIR34B/C methylation levels (P < 0.001). For the entire cohort, 5-year overall survival (OS) rates were significantly lower in the groups carrying aberrant DAPK1 (P = 0.004) and DBC1 (P = 0.044) methylation, respectively. DAPK1 methylation status were significantly correlated with stage (P = 0.015), as all patients with aberrant DAPK1 methylation were stages III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7–29.3, P < 0.0007). Patients with DAPK1 methylated cell-free circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter.ConclusionsAberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating response to treatment and for prognostication. In particular, aberrant DAPK1 methylation in plasma was an independent prognostic marker that may also be used to assess treatment response.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0261-y) contains supplementary material, which is available to authorized users.

Acquired alterations of hypothalamic gene expression of insulin and leptin receptors and glucose transporters in prenatally high-glucose exposed three-week old chickens do not coincide with aberrant promoter DNA methylation.

BackgroundPrenatal exposures may have a distinct impact for long-term health, one example being exposure to maternal ‘diabesity’ during pregnancy increasing offspring ‘diabesity’ risk. Malprogramming of the central nervous regulation of body weight, food intake and metabolism has been identified as a critical mechanism. While concrete disrupting factors still remain unclear, growing focus on acquired epigenomic alterations have been proposed. Due to the independent development from the mother, the chicken embryo provides a valuable model to distinctively establish causal factors and mechanisms.AimThe aim of this study was to determine the effects of prenatal hyperglycemia on postnatal hypothalamic gene expression and promoter DNA methylation in the chicken.Methods and FindingsTo temporarily induce high-glucose exposure in chicken embryos, 0.5 ml glucose solution (30 mmol/l) were administered daily via catheter into a vessel of the chorioallantoic egg membrane from days 14 to 17 of incubation. At three weeks of postnatal age, body weight, total body fat, blood glucose, mRNA expression (INSR, LEPR, GLUT1, GLUT3) as well as corresponding promoter DNA methylation were determined in mediobasal hypothalamic brain slices (Nucleus infundibuli hypothalami). Although no significant changes in morphometric and metabolic parameters were detected, strongly decreased mRNA expression occurred in all candidate genes. Surprisingly, however, no relevant alterations were observed in respective promoter methylation.ConclusionPrenatal hyperglycemia induces strong changes in later hypothalamic expression of INSR, LEPR, GLUT1, and GLUT3 mRNA. While the chicken provides an interesting approach for developmental malprogramming, the classical expression regulation via promoter methylation was not observed here. This may be due to alternative/interacting brain mechanisms or the thus far under-explored bird epigenome.

Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis

Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis. In human coronary endothelial cells, TGFβ1 causes aberrant methylation of RASAL1 promoter, increased Ras-GTP activity, and EndMT. In end-stage failing vs. non-failing human myocardium, increased fibrosis was associated with significantly increased RASAL1 promoter methylation, decreased RASAL1 expression, increased Ras-GTP activity, and increased expression of markers of EndMT. In mice with pressure overload due to ascending aortic constriction, BMP7 significantly reduced RASAL1 promoter methylation, increased RASAL1 expression, and decreased EndMT markers as well as decreased cardiac fibrosis. The ten eleven translocation (TET) family enzyme TET3, which demethylates through hydroxymethylation, was significantly decreased in fibrotic mouse hearts, restored with BMP7, and BMP7 effects were absent in coronary endothelial cells with siRNA knockdown of TET3. Our study provides proof-in-principle evidence that transcriptional suppression of RASAL1 through aberrant promoter methylation contributes to EndMT and ultimately to progression of cardiac fibrosis. Such aberrant methylation can be reversed through Tet3-mediated hydroxymethylation, which can be specifically induced by BMP7. This may reflect a new treatment strategy to stop cardiac fibrosis.