Aberrant CpG Island Hypermethylation Research Articles

TRIM29 promotes antitumor immunity through enhancing IGF2BP1 ubiquitination and subsequent PD-L1 downregulation in gastric cancer

Tripartite motif-containing protein 29 (TRIM29) is a member of TRIM family protein which has been reported to play a role in the progress of inflammatory and cancer diseases. However, its specific role in gastric cancer (GC) has yet to be fully understood. Here, we investigated the expression of TRIM29 in gastric cancer and its functions in the antitumor immunity. TRIM29 expression was lower in tumor tissues than that in paired normal tissues. Lower expression of TRIM29 was related to aberrant hypermethylation of CpG islands in TRIM29 gene. Comprehensive proteomics and immunoprecipitation analyses identified IGF2BP1 as TRIM29 interactors. TRIM29 interacted with IGF2BP1 and induced its ubiquitination at Lys440 and Lys450 site by K48-mediated linkage for protein degradation. IGF2BP1 promoted PD-L1 mRNA stability and expression in a 3′UTR and m6A-dependent manner. Functionally, TRIM29 enhanced antitumor T-cell immunity in gastric cancer dependent on the IGF2BP1/PD-L1 axis in vivo and in vitro. Clinical correlation analysis revealed that TRIM29 expression in patient samples was associated with CD8+ immune cell infiltration in the GC microenvironment and the overall survival rates of GC patients. Our findings revealed a crucial role of TRIM29 in regulating the antitumor T-cell immunity in GC. We also suggested that the TRIM29/IGF2BP1/PD-L1 axis could be used as a diagnostic and prognostic marker of gastric cancer and a promising target for GC immunotherapy.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer.

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIMTo use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODSThe primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.RESULTSThe best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.CONCLUSIONIn summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.

Global Methylome Scores Correlate with Histological Subtypes of Colorectal Carcinoma and Show Different Associations with Common Clinical and Molecular Features.

Simple SummaryAberrant patterns of methylation at specific genome sequences (CpG) are driving forces for the carcinogenesis process of different cancers, including colorectal cancer (CRC) and, typically, include gene promoter hypermethylation and global genome hypomethylation. Despite that CRC is diagnosed based on histological evaluation, its association with global methylation patterns has not been established so far. By studying the methylation status of 450,000 CpG sites in 117 colorectal specimens, the authors obtained global scores reflecting the methylation level at different sequence distances from the genes. The authors identified that histological CRC subtypes show different global methylation scores and that the shift from promoter hypermethylation to genomic hypomethylation occurs at a small sequence between 250 bp and 1 Kb from the gene TSS.Background. The typical methylation patterns associated with cancer are hypermethylation at gene promoters and global genome hypomethylation. Aberrant CpG island hypermethylation at promoter regions and global genome hypomethylation have not been associated with histological colorectal carcinomas (CRC) subsets. Using Illumina’s 450 k Infinium Human Methylation beadchip, the methylome of 82 CRCs were analyzed, comprising different histological subtypes: 40 serrated adenocarcinomas (SAC), 32 conventional carcinomas (CC) and 10 CRCs showing histological and molecular features of microsatellite instability (hmMSI-H), and, additionally, 35 normal adjacent mucosae. Scores reflecting the overall methylation at 250 bp, 1 kb and 2 kb from the transcription starting site (TSS) were studied. Results. SAC has an intermediate methylation pattern between CC and hmMSI-H for the three genome locations. In addition, the shift from promoter hypermethylation to genomic hypomethylation occurs at a small sequence between 250 bp and 1 Kb from the gene TSS, and an asymmetric distribution of methylation was observed between both sides of the CpG islands (N vs. S shores). Conclusion. These findings show that different histological subtypes of CRC have a particular global methylation pattern depending on sequence distance to TSS and highlight the so far underestimated importance of CpGs aberrantly hypomethylated in the clinical phenotype of CRCs.

Abstract 480: Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer

Abstract Background: Aberrant CpG island hypermethylation is an early event during carcinogenesis and a significant mechanism of tumor suppressor gene silencing. Assessment of tumor specific methylation changes in tissue or body fluids might provide insights into disease mechanisms and discovery of biomarkers. Ovarian cancer remains the most lethal gynaecological malignancy. Non-specific symptoms at early stage, presentation of disease at advanced stage and lack of sufficiently sensitive screening modalities have rendered significant challenge to its prompt diagnosis. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant promoter hypermethylation in cell free tumor-specific DNA can be indicative of highly specific cancer signals. Methods: Herein, we concurrently evaluated the promoter methylation of HIC1 and SOX1 gene in 120 ovarian tissue samples and validated in 70 matched serum cell free DNA of cancerous and non cancerous samples by multiplex MethyLight assay (a high throughput quantitative, TaqMan based real time PCR technique). Additionally, promoter methylation pattern of these genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panel. Results: In singleplex assay, high methylation sensitivity and specificity was evident for SOX1 (78.8% and 80.0%, respectively; AUC= 0.88) and HIC1 (80.0% and 77.14%, respectively; AUC=0.85) in tissue samples. In the multiplex assay, when either or both gene promoter showed methylation, the highly sensitivity and specific discrimination of EOC patients from normal controls was observed for SOX1 + HIC1 (83.53% and 88.57%, respectively; AUC=0.89). We identified DNA methylation of HIC1 and SOX1 as best discriminator between cancer and non-neoplastic tissue. Methylated SOX1 and HIC1 occurred in 53.3% and 71.1% of OC serum samples when their promoter methylation was analyzed in singleplex assay (AUC=0.81 and 0.88, respectively). In cell free DNA, this novel panel achieved a sensitivity of 80.0% and a specificity of 96.0% with an area under the ROC curve of 0.93 for discriminating OC samples from normal control. The results of MethyLight were highly concordant with those of clonal bisulfite sequencing. Methylated Individual gene as well as in combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell free DNA. Conclusion: We report a QPCR based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation based detection of circulating cell free tumor DNA in clinical management of ovarian cancer. Citation Format: Alka Singh, Manisha Sachan. Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 480.

The Role of H3K4 Trimethylation in CpG Islands Hypermethylation in Cancer.

CpG methylation in transposons, exons, introns and intergenic regions is important for long-term silencing, silencing of parasitic sequences and alternative promoters, regulating imprinted gene expression and determining X chromosome inactivation. Promoter CpG islands, although rich in CpG dinucleotides, are unmethylated and remain so during all phases of mammalian embryogenesis and development, except in specific cases. The biological mechanisms that contribute to the maintenance of the unmethylated state of CpG islands remain elusive, but the modification of established DNA methylation patterns is a common feature in all types of tumors and is considered as an event that intrinsically, or in association with genetic lesions, feeds carcinogenesis. In this review, we focus on the latest results describing the role that the levels of H3K4 trimethylation may have in determining the aberrant hypermethylation of CpG islands in tumors.

Aging of Preleukemic Thymocytes Drives CpG Island Hypermethylation in T-Cell Acute Lymphoblastic Leukemia

During the last decade, aberrant DNA methylation has been identified as a hallmark of human cancer and several studies have highlighted the potential of DNA methylation as a clinically or diagnostically relevant biomarker. In comparison to their normal healthy counterparts, cancer cells generally display DNA hypermethylation at specific CpG islands, but the actual mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. To profile the DNA methylation landscape of human T-cell acute lymphoblastic leukemia (T-ALL), we analyzed 109 T-ALLs together with 10 stages of normal T cell development, which are considered the normal human counterparts of this disease, by 850 EPIC arrays. Here, we show that CpG islands are hypermethylated in all human T- ALLs compared to their normal counterparts. We designed a DNA methylation signature that can distinguish two types of T-ALL, with low or high levels of CpG island hypermethylation. This profile is dominated by CpGs in promoters of PRC2 target genes. T-ALLs with high levels of CpG island hypermethylation show low levels of H3K27me3 and vice versa, resulting in gene repression in both subtypes of T-ALL by different mechanisms. Furthermore, we found that aberrant CpG island hypermethylation shows a strong correlation with the epigenetic age of the leukemic T cells. By investigating the DNA methylation profile of two distinct mouse T-ALL models, the Lck-Cre Ptenfl/fland the CD2-Lmo2 transgenic mouse model, by Reduced Representation Bisulfite Sequencing, we could indeed recapitulate the DNA methylation features of the two human T-ALL subtypes in mice (Fig.1). The aggressive, fast-transforming Ptenfl/flmouse model displays low levels of CpG island hypermethylation, which correlated with human T-ALLs that have a shorter proliferative history and a worse prognosis. In contrast, murine CD2-Lmo2 T-ALLs have a longer disease latency and display a CpG island hypermethylation phenotype that is similar to human T-ALLs with a longer proliferative history. In CD2-Lmo2 mice, a pre-leukemic phase is present with self-renewing thymocytes. We elucidate that the CpG island methylation signature is gradually established in aging pre-leukemic thymocytes of 8, 16 and 24 weeks old CD2-Lmo2 mice (Fig.1). Of note, this hypermethylation phenotype is completely absent in age matched Lck-Cre Ptenfl/flmice that did not yet develop leukemia, suggesting that the proliferative history is responsible for aberrant CpG island DNA methylation observed in human T-ALL. Notably, this provides the first evidence that a pre-leukemic phase might be present in a large subset of human T-ALLs, and that epigenetic aberrations, either in the DNA methylation or histone methylation machinery are one of the first detectable alterations during T-ALL development. Finally, using patient derived xenografts (PDX), we show that DNA hypomethylation by the FDA-approved hypomethylating agent Decitabine is very effective in treating T-ALL. Gene expression profiling revealed that the anti-leukemic effect is exerted by down-regulation of the oncogenic MYC pathway. However, by profiling these PDX T-ALLs by EPIC arrays, we unexpectedly uncover that the age-related CpG island hypermethylation signature is completely resistant to Decitabine treatment. Altogether, our work demonstrates that DNA methylation reflects the epigenetic history of leukemic T cells and suggests that methylation-based subtypes of human T-ALL have followed a different trajectory towards T-cell transformation, possibly mediated by differences in the self-renewing capacity of the putative T-ALL cell-of-origin. Given that the concept of preleukemic thymocytes has only been reported in T-ALL mouse models so far, we here provide, for the first time, conceptual evidence that a pre-leukemic phase might also be involved in the pathogenesis of the human disease. Disclosures No relevant conflicts of interest to declare.

Abstract A13: DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA

Abstract Background: Accumulated evidence revealed that aberrant CpG island hypermethylation plays significant roles in carcinogenesis that can serve as a promising target for molecular detection in body fluids. Ovarian cancer remains the most lethal gynecologic malignancy and is characterized by few early symptoms, late stage presentation, and resulting poor survival, which thereby renders challenge to its prompt diagnosis. Despite a myriad of attempts at early diagnosis of ovarian cancer, this clinical aim still remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant circulating DNA methylation patterns provide highly specific cancer signals. Methods: To develop a DNA methylation-based screening assay for early diagnosis of ovarian cancer, we quantitatively assessed the promoter methylation of DAPK1, HOXA9, RASSF1A, and SOX1 genes in 105 ovarian cancer and 30 non-neoplastic ovarian specimens by means of a high-throughput quantitative, real-time PCR-based technique (MethyLight) and clonal bisulfite sequencing. The best-performing gene cassette was further evaluated for their methylation status in cell-free DNA from serum of matched 35 ovarian cancer patients and 20 normal controls. Area under the ROC (Receiver-operator characteristic) curve was used to evaluate the discriminatory performance of both individual and combined gene panel. Results: RASSF1A, HOXA9, DAPK1, and SOX1 displayed a methylation frequency of 72.72, 82.67, 68.0, and 80.0%, respectively, in tissue samples. We identified DNA methylation of HOXA9 and SOX1 as the best discriminator between cancer and non-neoplastic tissue. In the multiplex assay, the combined panel of HOXA9 and SOX1 achieved a sensitivity of 88.0% with a specificity of 86.7%, when either or both of the gene promoters showed methylation. These genes appear to have great potential to be evaluated for their methylation level in cell-free DNA as a noninvasive diagnostic marker for early diagnosis of ovarian cancer. In cell-free DNA, this novel panel achieved a sensitivity of 62.86.0% and a specificity of 95% with an area under the ROC curve of 0.81 for discriminating OC samples from normal control. The results of MethyLight were concordant with those of clonal bisulfite sequencing. Conclusion: Our findings revealed the potential of epigenetic biomarker for early detection of OC in cell-free DNA as a minimally invasive tool. Here, we have established that promoter hypermethylation can be detected in serum cell-free DNA from patients with early as well as advanced-stage ovarian cancer. Based on the multiplex MethyLight assay, study will focus on analyzing the clinical utility of HOXA9 and SOX1 as methylation biomarkers. These findings further underline the potential of multiplexing individual markers into a panel that helps to achieve higher sensitivity and specificity than individual marker and provides robustness to a noninvasive early screening test. Citation Format: Alka Singh, Jaydeep Aravindbhai Badarukhiya, Sameer Gupta, Manisha Sachan. DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A13.

Aberrant epigenetic silencing of neuronatin is a frequent event in human osteosarcoma.

The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2’-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.

SAT-LB34 Repressive Epigenetic Programs Reinforce Steroidogenic Differentiation and Wnt/β-Catenin Signaling in Aggressive Adrenocortical Carcinoma

Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and >50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.

Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer.

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation-specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell-free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer-specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer-free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.

BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer.

The aberrant hypermethylation of BRCA1 promoter CpG islands induces the decreased expression of BRCA1 (Breast Cancer 1) protein. It can be detected in sporadic breast cancer without BRCA1 pathogenic variants, particularly in triple-negative breast cancers (TNBC). We investigated BRCA1 hypermethylation status (by methylation-specific polymerase chain reaction (MS-PCR) and MassARRAY® assays), and BRCA1 protein expression using immunohistochemistry (IHC), and their clinicopathological significance in 248 chemotherapy-naïve TNBC samples. Fifty-five tumors (22%) exhibited BRCA1 promoter hypermethylation, with a high concordance rate between MS-PCR and MassARRAY® results. Promoter hypermethylation was associated with reduced IHC BRCA1 protein expression (p = 0.005), and expression of Programmed death-ligand 1 protein (PD-L1) by tumor and immune cells (p = 0.03 and 0.011, respectively). A trend was found between promoter hypermethylation and basal marker staining (p = 0.058), and between BRCA1 expression and a basal-like phenotype. In multivariate analysis, relapse-free survival was significantly associated with N stage, adjuvant chemotherapy, and histological subtype. Overall survival was significantly associated with T and N stage, histology, and adjuvant chemotherapy. In addition, patients with tumors harboring BRCA1 promoter hypermethylation derived the most benefit from adjuvant chemotherapy. In conclusion, BRCA1 promoter hypermethylation is associated with TNBC sensitivity to adjuvant chemotherapy, basal-like features and PD-L1 expression. BRCA1 IHC expression is not a good surrogate marker for promoter hypermethylation and is not independently associated with prognosis. Association between promoter hypermethylation and sensitivity to Poly(ADP-ribose) polymerase PARP inhibitors needs to be evaluated in a specific series of patients.

Promoter Hypermethylation of CHODL Contributes to Carcinogenesis and Indicates Poor Survival in Patients with Early-stage Colorectal Cancer.

Aims: Aberrant hypermethylation of CpG islands is an important hallmark of colorectal cancer (CRC). We previously utilized methyl-DNA immunoprecipitation assays to identify a novel methylated gene, chondrolectin (CHODL), preferentially methylated in human CRC. In this study, we examined the epigenetic inactivation, biological effects and prognostic significance of CHODL in CRC.Main methods: The methylation status of CHODL in CRC was evaluated by bisulfite genomic sequencing (BGS). The functions of CHODL in CRC were determined by proliferation, apoptosis, cell migration and invasion assays. The impact and underlying mechanisms of CHODL in CRC were characterized by western blot and RNA-Seq analyses. The association between CHODL and CRC clinical features was examined using The Cancer Genome Atlas (TCGA) database and immunohistochemical staining.Key findings: CHODL was downregulated in 10 CRC cell lines and CRC tissues, and promoter hypermethylation contributed to its inactivation. Ectopic expression of CHODL inhibited colony formation, suppressed cell viability, induced apoptosis, and restrained cell migration and invasion in vitro and in vivo. Furthermore, high CHODL expression in CRC was a predictor of improved survival, though CHODL hypermethylation was a poor prognostic factor for CRC patients, especially those with early-stage CRC.Significance: CHODL promoter hypermethylation silences CHODL expression in CRC, and CHODL suppresses CRC tumorigenesis. CHODL methylation and expression levels can be used as potential markers to evaluate the prognosis of CRC patients.

Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing.

Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. MCTA-Seq is a promising method for the noninvasive detection of CRC.

Abstract 3787: Reduced expression of INPP4B by promoter hypermethylation in Epstein-Barr virus-associated gastric carcinoma

Abstract Background: Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is attracting increasing attention due to distinct molecular and clinical characteristics. However, its carcinogenesis still remains unclear. Previous studies demonstrated that aberrant DNA methylation is one of the pathogenic mechanisms of EBVaGC. Inositol polyphosphate 4-phosphatase type II (INPP4B), as a component of PI3K/AKT pathway, is implicated in the regulation of crucial cellular processes and tumorigenesis. This study aims to reveal the correlation between INPP4B expression and EBV infection, as well as investigate the inactivation mechanism of INPP4B in EBVaGC. Methods: EBV status and INPP4B expression were examined in gastric carcinoma (GC) tissue microarray (301 cases) using EBV RNA probe and immunohistochemistry, respectively. The correlation of EBV status and INPP4B expression was analyzed in GC by Chi-Squared Test. The INPP4B promoter methylation was investigated in GC cell lines: SNU-719 (EBV positive), AGS (EBV negative) and SGC-7901 (EBV negative) with methylation-specific polymerase chain reaction (MSPCR). After treatment with 5-Aza-2′-deoxycytidine, the INPP4B promoter methylation and expression were detected using MSPCR, western blot, and qPCR. Results: INPP4B expression is decreased in 146 of 301 GC cases (48.5%). In 272 evaluable GC cases, EBV was present in 40 cases (14.71%). In addition, Lower expression of INPP4B is significantly correlated with EBV infection (p<0.05). There was a significantly decreased expression of INPP4B in EBV positive cell line (SNU-719) compared to EBV negative cell line (AGS and SGC-7901). Moreover, aberrant CpG island hypermethylation of INPP4B was found in SNU-719 rather than AGS and SGC-7901. After treatment with 5-Aza-2′-deoxycytidine, INPP4B protein and mRNA are dramatically up-regulated, while unmethylated DNA became detectable in SNU-719. Conclusions: Our results suggest that the reduction of INPP4B expression is commonly found in GC, particularly in EBVaGC. Promoter hypermethylation may be one of the mechanisms of INPP4B inactivation. Citation Format: Yiwen Ma, Jichao Tan, Weihong Dai, Yu Zhang, Yu Zhang, Rongjin Lin, Yan Xu. Reduced expression of INPP4B by promoter hypermethylation in Epstein-Barr virus-associated gastric carcinoma [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 3787.

Targeting Mybbp1a suppresses HCC progression via inhibiting IGF1/AKT pathway by CpG islands hypo-methylation dependent promotion of IGFBP5

BackgroundMyb-binding protein 1A (Mybbp1a) is a nucleolar protein that can regulate rRNA metabolism, the stress response and carcinogenesis. However, the function of Mybbp1a in the progression of hepatocellular carcinoma (HCC) is unclear.We aimed to determine the role of Mybbp1a in HCC and the underlying mechanism. MethodsWe investigated the function of Mybbp1a in HCC cell models and the xenograft mouse model. The relationship between Mybbp1a and IGFBP5 was found through expression profile chip. The molecular mechanism of Mybbp1a regulating IGFBP5 was proved through CO-IP, CHIP, Bisulfite Sequencing and Pyrosequencing. FindingsIn this study, we observed that Mybbp1a was overexpressed in HCC tissues and associated with the poor prognosis of HCC patients. Suppression of Mybbp1a led to a reduction in the proliferation and migration ability of HCC cells through inhibiting the IGF1/AKT signaling pathway. Further study found that Mybbp1a could form a complex with DNMT1 and induce aberrant hyper-methylation of CpG islands of IGFBP5, which inhibits secretion of IGFBP5 and then activates IGF1/AKT signaling pathway. InterpretationThese findings extend our understanding of the function of Mybbp1a in the progression of HCC. The newly identified Mybbp1a may provide a novel biomarker for developing potential therapeutic targets of HCC. FundScience Technology Department of Zhejiang Province (No. 2015C03034), National Health and Family Planning Commission of China (No. 2016138643), Innovative Research Groups of National Natural Science Foundation of China (No. 81721091), Major program of National Natural Science Foundation of China (No. 91542205).

SPINT2 is hypermethylated in both IDH1 mutated and wild-type glioblastomas, and exerts tumor suppression via reduction of c-Met activation.

Both IDH1-mutated and wild-type gliomas abundantly display aberrant CpG island hypermethylation. However, the potential role of hypermethylation in promoting gliomas, especially the most aggressive form, glioblastoma (GBM), remains poorly understood. We analyzed RRBS-generated methylation profiles for 11 IDH1WT gliomas (including 7 GBMs), 24 IDH1MUT gliomas (including 6 GBMs), and 5 normal brain samples and employed TCGA GBM methylation profiles as a validation set. Upon classification of differentially methylated CpG islands by IDH1 status, we used integrated analysis of methylation and gene expression to identify SPINT2 as a top cancer related gene. To explore functional consequences of SPINT2 methylation in GBM, we validated SPINT2 methylation status using targeted bisulfite sequencing in a large cohort of GBM samples. We assessed DNA methylation-mediated SPINT2 gene regulation using 5-aza-2'-deoxycytidine treatment, DNMT1 knockdown and luciferase reporter assays. We conducted functional analyses of SPINT2 in GBM cell lines in vitro and in vivo. We identified SPINT2 as a candidate tumor-suppressor gene within a group of CpG islands (designated GT-CMG) that are hypermethylated in both IDH1MUT and IDH1WT gliomas but not in normal brain. We established that SPINT2 downregulation results from promoter hypermethylation, and that restoration of SPINT2 expression reduces c-Met activation and tumorigenic properties of GBM cells. We defined a previously under-recognized group of coordinately methylated CpG islands common to both IDH1WT and IDH1MUT gliomas (GT-CMG). Within GT-CMG, we identified SPINT2 as a top cancer-related candidate and demonstrated that SPINT2 suppressed GBM via down-regulation of c-Met activation.

Hypermethylation of Frizzled1 is associated with Wnt/\u03b2-catenin signaling inactivation in mesenchymal stem cells of patients with steroid-associated osteonecrosis

The Wnt/β-catenin signaling pathway is associated with the pathogenesis of steroid-induced osteonecrosis. Our investigation studied whether aberrant CpG island hypermethylation of the FZD1 gene was present in patients with osteonecrosis of the femoral head (ONFH), which results in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 21) and patients with new femoral neck fractures (n = 22), and then mesenchymal stem cells (MSCs) were isolated. We investigated cell viability, the transcription and translation levels of Wnt/β-catenin signaling-related genes, the extent of methylation at CpG islands of the FZD1 promoter, and the osteogenic and adipogenic differentiation abilities of MSCs from the control group and from the ONFH group treated with or without 5′-Aza-dC. According to the results, MSCs from the ONFH group showed a reduced proliferation ability, low transcription and translation levels of FZD1, inhibition of the Wnt/β-catenin signaling pathway, weakened osteogenesis and enhanced adipogenesis ability. Aberrant CpG island hypermethylation of FZD1 was observed in the ONFH group. Treatment with 5’-Aza-dC resulted in de novo FZD1 expression, reactivation of the Wnt/β-catenin signaling pathway and promotion of osteogenesis. Taken together, our study not only provides novel insights into the regulation of the Wnt/β-catenin signaling pathway in this disease but also reveals potential for the use of demethylating agents for the treatment of GC-associated ONFH.

A Pilot Study of Aberrant CpG Island Hypermethylation of SPRED1 in Acute Myeloloid Leukemia

Background: Epigenetic silencing of tumor suppressor genes plays important role in acute myeloid leukemia (AML). Recently, SPRED1, a negative regulator of the RAS MAPK pathway, is identified as a tumour suppressor downregulated in AML. However, little is known regarding its underlying dysregulation in AML. In this study, we investigated methylation status of SPRED1 promoters and their association with mRNA levels in AML.Methods: Methylation level were measured in four regions of SPRED1 (#1: 310 bp ~ 723 bp, #2: 810 bp ~ 1299 bp, #3: 1280 bp ~ 1742 bp and #4: 1715 bp ~ 2059 bp) in a total of 16 patients with de novonon-acute promyelocytic leukemia (non-APL) and three patients who got complete remission after induction treatment using the Sequenom MassARRAY platform. Quantitative real-time polymerase chain reaction (q-RT PCR) was used to analyze SPRED1 mRNA levels.Results: AML patients had a significantly higher average methylation level than controls at regions of #1_CpG_1 (p= 0.04) and #1_CpG_11 (p =0.002). The methylation values for #1_CpG_11 were negatively correlated with mRNA levels (r= -0.558, p=0.013) but there was no significant association between #1_CpG_1 methylation status and mRNA levels (r=-0.103, p=0.675) in AML patients. There was no significant difference in the methylation level when comparing with clinical biochemical parameters and treatment response (p>0.05). Mutations of epigenetic regulation genes such as DNMT3A, TET2 and IDH1/2 were most frequently observed in patients with higher methylation levels. Decreased methylation levels were revealed in three patients who got complete remission.Conclusions: Aberrant methylation statuses of the SPRED1 promoter regions are associated with the downregulation of gene transcription in AML. The methylation level is probably associated with the treatment response of AML. Mutations of epigenetic regulation genes might be involved in the epigenetic aberration of SPRED1.

Highly sensitive detection of DNA hypermethylation in melanoma cancer cells

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a promising biomarker for early cancer detection. This methylation status is reflected in the methylation pattern of ctDNA shed from the primary tumor; however, to realize the full clinical utility of ctDNA methylation detection via liquid biopsy for early cancer diagnosis, improvements in the sensitivity and multiplexability of existing technologies must be improved. Additionally, the assay must be cheap and easy to perform in a clinical setting. We report the integration of methylation specific PCR (MSP) to melt curve analysis on giant magnetoresistive (GMR) biosensors to greatly enhance the sensitivity of our DNA hybridization assay for methylation detection. Our GMR sensor is functionalized with synthetic DNA probes that target methylated or unmethylated CpG sites in the MSP amplicon, and measures the difference in melting temperature (Tm) between the two probes (ΔTm), giving an analytical limit of detection down to 0.1% methylated DNA in solution. Additionally, linear regression of ΔTm's for serial dilutions of methylated:unmethylated mixtures allows for quantification of methylation percentage, which could have diagnostic and prognostic utility. Lastly, we performed multiplexed MSP on two different genes, and show the ability of our GMR assay to resolve this mixture, despite their amplicons’ overlapping Tm's in standard EvaGreen melt analysis. The multiplexing ability of our assay and its enhanced sensitivity, without necessitating deep sequencing, represent important steps toward realizing an assay for the detection of methylated ctDNA in plasma for early cancer detection in a clinical setting.

Loss of opioid binding protein/cell adhesion molecule-like gene expression in gastric cancer.

Previous studies have reported that the expression of the opioid binding protein/cell adhesion molecule-like (OPCML) gene was frequently downregulated in various of types of cancer. However, little is known regarding the expression of the OPCML gene in gastric cancer. The present study identified that OPCML was downregulated in the gastric cancer SGC7901, KATO III, MKN45, MKN74, SNU1, AGS, N87 and a gastric mucosa cell line GES1, compared with normal gastric tissues by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To investigate whether the downregulation of OPCML was due to promoter hypermethylation, the methylation of the OPCML promoter was assessed by methylation-specific polymerase chain reaction. Hypermethylation of the OPCML promoter was observed in the gastric cancer MKN45 cell lines, but was not as evident in normal gastric tissue. The methylation inhibitor 5-aza-2'-deoxycytidine was used to remove the methylation of the OPCML gene promoter, following which the expression of OPCML was restored. In addition, the function of the OPCML gene was studied in vitro, and it was found that the restoration expression of OPCML could lead to the suppression of cell growth. In conclusion, the present study has shown that OPCML, which acts as a tumor suppressor, was silenced in gastric cancer cell lines via aberrant hypermethylation of the promoter CpG islands, which may provide a novel molecular approach for the early diagnosis of gastric cancer.