5hmC Expression Research Articles

Abstract PO-059: SOX2-mediated 5hmC dysregulation in GBM Stem Cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely track with the differentiation state of cells with terminally differentiated cells containing high levels of 5hmC and less differentiated cells having very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients, strongly suggesting that these enzymes activate tumor suppressing mechanisms. Our preliminary data shows that exogenous SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA partially rescues the reduction in 5hmC expression observed in GSCs expressing transgenic SOX2, thus implicating miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our preliminary results support a mechanism in which SOX2 represses TET2 leading to 5hmC loss by directly activating miR-10b-5p transcription and predicts that targeting this novel mechanism of epigenetic dysregulation by inhibiting miR-10b-5p in vivo will can lead to pre-clinical GBM therapeutics. Citation Format: Hernando Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM Stem Cells [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-059.

Abstract 152: SOX2-mediated 5hmC dysregulation in GBM stem cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. TET enzymes and regulation of 5mC and 5hmC have the capacity to function on a global scale to regulate tumor suppressing mechanisms. We have found that SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhances self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA rescued the reduction in 5hmC expression observed in GSCs, thus identifying miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our results identify a novel targetable pathway by which SOX2 regulates the epigenetic landscape of GBM-propagating cells. This novel mechanism combined with our recent reports that SOX2 regulates DNMTs and the HMGA1 chromatin remodeling protein identified a multi-dimensional epigenetic mechanism by which oncogenic signals control GBM cell stemness and tumor propagation. Citation Format: Hernando M. Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 152.

H2AX Promoter Demethylation at Specific Sites Plays a Role in STAT5-Induced Tumorigenesis.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions -77 and - 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.

Abstract B02: 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma

Abstract Background: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. The level of 5-hmC expression serves as a biomarker in adults with cancer, and we recently demonstrated the prognostic significance of tumor whole-genome 5-hmC profiling in children with neuroblastoma. Here, we used Nano-hmC-Seal technology to profile 5-hmC in circulating cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma and age-matched healthy children. Methods: Plasma was collected from 32 children with neuroblastoma at various time points during therapy and 34 well children. 5-hmC sequencing was performed using 100ng of cfDNA extracted from plasma. Metastatic disease burden was classified as high, moderate, low, or none based on Curie MIBG scores, the percentage of tumor cells in bone marrow samples, and/or measurable disseminated soft tissue disease. Results: One hundred cfDNA 5-hmC profiles were generated from 27 high-risk, 3 intermediate-risk, and 2 low-risk patients and 34 well controls. Hierarchical clustering based on 5-hmC levels of 347 genes separated the samples into three main clusters corresponding to high, moderate, and low/no disease burden in the bone, bone marrow, and/or soft tissue. Using functional gene pathways, two subsets of patients within the low/no cluster were revealed that correlated with low tumor burden or no clinically detectable metastatic disease (sensitivity 70%, specificity 76.9%). 5-hmC profiles tracked closely with changes in disease status in 15 patients with serial samples. Samples from patients with a detectable tumor burden had genes with elevated 5-hmC in pathways of neuronal stem cell maintenance and the epigenetic regulatory complexes PRC2 and CTCF/cohesion. Conclusions: cfDNA 5-hmC profiles correlate with disease burden in children with neuroblastoma and may serve as sensitive biomarkers of treatment response. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of the pathways that drive resistance to treatment. Citation Format: Mark A. Applebaum, Erin K. Barr, Jason Karpus, Meritxell Oliva, Alexandre Chlenski, Helen R. Salwen, Emma Wilkinson, Marija Dobratic, Elizabeth A. Sokol, Barbara E. Stranger, Chuan He, Susan L. Cohn. 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B02.

Diminished expression of 5hmc in Reed-Sternberg cells in classical Hodgkin lymphoma is a common epigenetic marker

Loss of the epigenetic marker 5-hydroxymethylcytosine (5hmC) has been demonstrated in a variety of neoplasms. Several recent studies have shown epigenetic alteration in Classical Hodgkin lymphoma (CHL), which may impact treatment. We demonstrate near universal depletion of 5hmC in the neoplastic Hodgkin Reed-Sternberg (H/RS) cells in all cases of CHL (49/49). We hypothesized that the addition of vitamin C–a cofactor for the ten-eleven translocation (TET) enzymes which oxidize 5-methylcytosine (5mC) to 5hmC - may replenish levels of 5hmC. The CHL cell line L428 was grown in optimal conditions and then subjected to vitamin C treatment, which demonstrated reduced cell viability as well as caspase activation and increased concentration of 5hmC. A more detailed understanding of the epigenetic landscape of CHL may help guide future therapies.

673 Semi-quantitative 5-hmC expression by immunohistochemistry is a useful adjunctive technique to help distinguish spitz nevi from spitzoid malignant melanomas

The differentiation between Spitz nevi (SN), Atypical Spitzoid neoplasms (ASN) and Spitzoid malignant melanomas (SMM) represents a challenge to dermatopathologists. ASN are lesions in the gray zone for which a definitive histopathologic diagnosis of benign or malignant cannot be made with absolute certainty. We sought to investigate whether the major epigenetic biomarker, 5-hydroxymethylcytosine (5-hmC), is differentially expressed and can aid in the diagnosis of benign and malignant Spitzoid neoplasms.

147 The expression of 5-hydroxymethylcytosine, TET2 and retinoid acid receptor-β in male genital diseases

DNA methylation is an important epigenetic modification that is frequently altered in a wide range of diseases. 5-Hydroxymethylcytosine (5-hmC) acts as a positive transcriptional regulator in normal development and carcinogenesis. The expression and function of RAR-β are altered in most cancer cells. In this study, we investigated the expression level of 5-hmC, RAR-β and TET2 in a group of male genital diseases using immunohistochemistry. These diseases have very similar appearance and are very difficult to differentiate from each other. The aim of this study was to figure out whether these epigenetic makers can be used in differentiate these conditions. We performed Immunohistochemical staining on paraffin-embedded tissues of 14 penile psoriasis, 13 penile lichen planus, 14 erythroplasia of Queyrat, 6 penile verrucous carcinoma and 7 penile SCC. We found different expression level of 5-hmC,RAR-β and TET2 in epidermis among these five group of diseases. Compared with penile psoriasis and penile lichen planus, expression of 5-hmC was significantly lower in erythroplasia of Queyrat, penile verrucous carcinoma and penile SCC. Moreover, the expression of TET2 was significantly lower in penile SCC compared to other 4 groups of diseases. The expression of RAR-β was also lower in penile verrucous carcinoma and penile scc than that in penile psoriasis, penile lichen planus and erythroplasia of Queyrat. The expression of 5-hmC, TET2 and RAR-β was lower in malignant lesions(erythroplasia of Queyrat, penile verrucous carcinoma penile scc) than that in benign lesions(penile psoriasis, penile lichen planus), suggesting that 5-hmC, RAR-β and TET2 can be used in differentiating benign and malignant diseases with similar clinical manifestations in male genitalia and can help us detect malignant diseases early and reduce the difficulty of clinical biopsy sampling and misdiagnosis.

141 All-trans Retinoic Acid Inhibits Cell Proliferation through Upregulation of TET2 in Squamous Cell Cancer

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent non-melanoma skin cancer. All-trans retinoic acid (ATRA) has been previously proposed anti-tumor potential via inhibiting cancer cell growth by binding to the intracellular retinoic acid receptors (RAR). The ten-eleven translocation 2 (TET2) proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and loss-functions of TET2 leading to hypermethylation have been established in various cancers. However, its role in cSCC remains unknown. In the present study, we detected the expression of 5hmC, TET2 and retinoic acid receptor B (RARb) in normal skin, actinic keratosis (AK), Bowen disease and cSCC clinical samples by immunohistochemical staining. It’s significant downregulation of 5hmC and TET2 were observed in the AK, Bowen disease and cSCC tissues compared with normal skin, while expression of RARb was reduced. Human primary keratinocytes and cSCC cell lines (Scl-1 and HSC-1) were cultured in vitro, receiving stimulation of retinoic acid. The results showed that treatment with ATRA inhibited the proliferation of cSCC cell lines and increased the expressions of TET2 and RARb in cSCC cell lines. Moreover, ectopic overexpression of TET2 in cSCC cell lines induce upregulation of RARB while no obvious expression changes of TET2 were observed in RARB overexpressed cSCC cell lines. Our results suggest that TET2 and 5hmC were decreased in cSCC tissue and cSCC cell lines compared to normal tissues and human primary keratinocytes. In addition, ATRA promotes the expression of TET2 in cSCC cell lines, which is closely related to RARb. This study provides evidence that 5hmC and TET2 have the potentials to serve as biomarkers that identified presence and progression of cSCC. It identified a novel function of ATRA in promoting a TET-mediated epigenetic regulation suggesting that the availability of ATRA in cancer cells will have various effects on different epigenetic targets.

Cross-sectional and longitudinal associations between global DNA (hydroxy) methylation and exposure biomarkers of the Hebei Spirit oil spill cohort in Taean, Korea

The Hebei Spirit oil spill (HSOS) occurred on the west coast of South Korea (Taean county) on December 7, 2007, and studies revealed that exposure to the oil spill was associated with various adverse health issues in the inhabiting population. However, no studies evaluated the association between crude-oil exposure and epigenetic changes. This study aimed to investigate the HSOS exposure-associated longitudinal and cross-sectional variations in global DNA methylation (5-mc) and/or hydroxymethylation (5-hmc) and expression profiles of related genes in Taean cohort participants from 2009 (AH-baseline) and 2014 (AH-follow-up) relative to the reference group (AL). We measured global DNA 5-mc and 5-hmc levels and related gene expression levels in whole blood. We identified significant associations between HSOS exposure and AH-baseline-5-mc, AH-baseline-5-hmc, and AH-follow-up-5-hmc. HSOS exposure was associated with lower %5-mc content and higher %5-hmc content in the same individuals from both the cross-sectional and longitudinal studies. In addition, we found a strong correlation between 5-mc and DNMT3B expression, and between 5-hmc and TET1 expression. Our findings suggested that epigenetic changes are important biomarkers for HSOS exposure and that 5-hmc is likely to be more sensitive for environmental epidemiological studies.

Prognostic value of downregulated 5-hydroxymethylcytosine expression in renal cell carcinoma: a 10 year follow-up retrospective study.

5-hydroxymethylcytosine (5hmC) is converted from DNA methylation of cytosine (5mC) by the catalysis of TET proteins, and proposed to be involved in tumorigenesis. However, the prognostic value of 5hmC in renal cell carcinoma (RCC) is still unclear. This study aimed to define the clinical significance of 5hmC in RCC. We performed dot blot assays to measure the relative expression of 5hmC in RCC. We reviewed the clinical records of 310 RCC patients and performed immunohistochemical (IHC) staining of 5hmC. The overall survival (OS) and cancer specific survival (CSS) of all patients were recorded over a 10-year follow-up period. Effective prognostic nomograms which contained 5hmC were established to provide individualized OS and CSS in RCC. 5hmC expression level was significantly decreased in RCC tissues compared with those in the normal counterparts. Kaplan-Meier curves revealed that high 5hmC expression had a good prognostic impact on RCC patients. Cox multivariate survival analyses further indicated 5hmC was an independent prognostic factor for RCC survival. Nomograms constructed based on cox regression analysis were available to calculate the survival probability directly. Calibration curves displayed good agreements. The findings were validated with an independent external cohort included 77 RCC cases. Thus, we believe we have found a significative prognostic factor for RCC.

Integrin α6 signaling induces STAT3-TET3-mediated hydroxymethylation of genes critical for maintenance of glioma stem cells.

Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5’-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3+ GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 expression and 5hmC accumulation also co-segregate with integrin α6 in patient malignant glioma. Thus, ECM- integrin α6-STAT3-TET3 axis regulates hydroxymethylation of genes important for GSCs, thereby increasing GSC tumorigenicity and resistance to therapies.

Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation.

Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

Abstract B010: SOX2-mediated 5hmC dysregulation in GBM Stem Cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely track with the differentiation state of cells with terminally differentiated cells containing high levels of 5hmC and less differentiated cells having very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients, strongly suggesting that these enzymes activate tumor suppressing mechanisms. Our preliminary data shows that exogenous SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA partially rescues the reduction in 5hmC expression observed in GSCs expressing transgenic SOX2, thus implicating miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our preliminary results support a mechanism in which SOX2 represses TET2 leading to 5hmC loss by directly activating miR-10b-5p transcription and predicts that targeting this novel mechanism of epigenetic dysregulation by inhibiting miR-10b-5p in vivo will can lead to pre-clinical GBM therapeutics. Citation Format: Hernando Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM Stem Cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B010. doi:10.1158/1535-7163.TARG-19-B010

628P - Detection of 5-hydroxymethylcytosine in circulating-free DNA for early diagnosis of colorectal cancer

BackgroundA convenient and accurate method to improve the effectiveness of colorectal-cancer screening is still under exploration. This may be achieved by a diagnostic model based on 5-hydroxymethylcytosine (5-hmC). MethodsWe collected peripheral venous blood from 285 colorectal cancer patients and 285 healthy people at Zhongshan Hospital, Shanghai. The 5-hmC level of each gene locus in circulating-free DNA (cfDNA) was detected using a highly sensitive sequencing method. Of 285 subjects, 178 in each group were randomly selected as training set. The first 100 gene loci with the most significant difference of 5-hmC level were selected for establishing a diagnostic model (Logistic regression model) based on machine-learning method. The rest subjects in each group were used as validating set to validate this model. Further, we analysed the influence of several clinicopathological factors on 5-hmC level. ResultsIn training set, the comprison of the 5-hmC level suggested that the expression of 5-hmC in cfDNA was significantly different between the two groups. Of the 100 gene loci, 35 were included in the model. In internal validation, the sensitivity and specificity of the model were 88.8% and 89.9%, respectively. While the sensitivity and specificity 81.3% and 89.7%, respectively, in external validation using the validating set. The area under ROC curve of external validation was 0.946. For colorectal cancer stages I-IV, the diagnostic sensitivities were 78.7%, 81.9%, 88.2% and 95.6%, respectively. We also found that age, gender, Ras status or the site of primary tumor had no significant effect on the expression of 5-hmC. However, there was a statistically difference of 5-hmC level between stage I-III and stage IV. ConclusionsOur study identified the gene loci with significantly different 5-hmC level between colorectal cancer patients and healthy people, then we established a diagnostic model of colorectal cancer with high sensitivity and specificity. However, the utility of the model still need further verification in large-scale screening experiments. Clinical trial identificationNCT03599947 09/16/2018. Editorial acknowledgementShanghai Yibien Gene Technology Co., Ltd. Provides Testing Platform. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

TET1-mediated DNA hydroxymethylation activates inhibitors of the Wnt/\u03b2-catenin signaling pathway to suppress EMT in pancreatic tumor cells

BackgroundTen-eleven translocation 1 (TET1) is a dioxygenase that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) to induce DNA demethylation. TET1 has been reported to be absent in cancers, and to influence various oncogenes and anti-oncogenes. However the function of TET1 in pancreatic tumor remains poorly understood. In this study, we investigated the role of TET1 in the progression of pancreatic tumor and its mechanism of tumor suppression.MethodsQuantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining and dot blot were performed to detect the TET1 and 5-hmC expression in pancreatic tumor tissues and its adjacent non-tumor tissues. The clinical parameters significance of pancreatic tumor tissues was determined statistically. TET1 over-expression and knock-out cell lines were built and confirmed in vitro. Cell proliferation assay, wound-healing assays, transwell migration assay and nude mice model of orthotopic pancreatic cancer implantation were performed to assess the function of TET1 in pancreatic tumor. Western blot, qRT-PCR, immunofluorescence (IF), bisulfate sequencing (BSP), Chromatin immunoprecipitation (ChIP) were used to uncover the mechanism.ResultsTET1 levels and 5-hmC content were downregulated in pancreatic tumor tissues and cell lines, and pancreatic tumor patients with low TET1 levels had a shorter overall survival than patients with high levels of TET1. TET1 suppressed pancreatic tumor proliferation and metastasis in vivo and in vitro. TET1 bound to the secreted frizzled-related protein 2 (SFRP2) promoter and catalyzed demethylation to activate transcription of SFRP2, inhibiting both the canonical and non-canonical Wnt signaling pathways, and ultimately obstructing epithelial-mesenchymal transition (EMT) in pancreatic tumors.ConclusionWe found TET1 plays as a suppressor in pancreatic tumor progression via obstructing Wnt signaling pathways.

Photoelectrochemical biosensor for 5hmC detection based on the photocurrent inhibition effect of ZnO on MoS2/C3N4 heterojunction

A novel photoelectrochemical biosensor was fabricated for 5-hydroxymethylcytosine (5hmC) detection based on the photocurrent inhibition effect of ZnO on MoS2/C3N4 heterojunction. Firstly, the ITO electrode was modified successively with MoS2 and g-C3N4 as photoelectric materials to deliver a strong photocurrent response. Next, the 5-hydroxymethyl group (-CH2OH) of 5hmC was oxidized by KRuO4 to produce an aldehyde group (-CHO), where 5hmC was converted into 5-formylcytosine (5fC). Based on the covalent reaction with between –CHO of 5fC and –NH2 groups of g-C3N4, 5fC can be captured on electrode surface. Finally, the ZnO-PAMAM composite was covalently attached to the phosphate group of the immobilized 5fC, which could decrease the electron transfer amount of g-C3N4 to MoS2, absorption of light and consumption of electron donors thereby resulting the decrease of photocurrent. Under optimal conditions, the photocurrent shows a linear relationship with the logarithm value of 5hmC concentration from 0.01-200 nM with a low detection limit of 2.6 pM. Moreover, this method was selective and allowed to discriminate between 5hmC and 5-methylcytosine (5mC) in DNA. Finally, the photoelectrochemical biosensor was successfully applied to investigate the effect of heavy metal ion and phytohormones on 5hmC expression in rice seedlings leaves.

5-Hydroxymethylcytosine as a potential epigenetic biomarker in papillary thyroid carcinoma.

DNA methylation at the 5 position of cytosine (5-mC) is an epigenetic hallmark that is critical in various biological and pathological processes such as DNA methylation regulation, and initiation and development of cancers. 5-mC can be oxidized to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation family of DNA hydroxylases. Accumulating evidence has reported that loss of 5-hmC is associated with cancer development. However, its level in papillary thyroid carcinoma (PTC) remains unclear. The present study reports that the loss of 5-hmC is an epigenetic mark of PTCs, associated with their malignant biological behavior, providing diagnostic and predictive advantages over DNA hypomethylation (5-mC), an acknowledged epigenetic alteration in cancer. In addition, the 5-hmC staining levels were decreased in cases of micro-carcinoma with lymph node metastasis, which suggests that 5-hmC expression levels could be used as valuable biomarkers for predicting malignant potential and assist in the selection of therapeutic strategies in PTC; therefore, 5-hmC has the potential to provide a more precise direction for PTC therapy.

Loss of 5-hydroxymethylcytosine immunohistochemical expression is a useful diagnostic aid for distinguishing hepatocellular carcinoma in cytology fine needle aspiration specimens.

Disruption of the DNA methylation pathway involving 5-hydroxymethylcytosine (5hmC) has been implicated in hepatic tumour development. The aim of this study was to investigate the expression of 5hmC in malignant and benign hepatic mass lesions and evaluate the diagnostic utility of deficient 5hmC expression in hepatocellular carcinoma (HCC). Our study consisted of 48 fine needle aspiration (FNA) cytological cases (30 cases positive for HCC, 18 cases negative for malignancy) and 39 liver resection specimens (30 HCCs and nine hepatic adenomas [HAs]). A 5hmC immunohistochemistry score (range 0-9) was calculated by multiplying the percentage (0=no staining; 1=1%-10%; 2=11%-50%; 3=51%-100%) and intensity scores (0-3+). A score of ≤2 was considered deficient for 5hmC. In resection specimens, 5hmC expression was deficient in 90% of HCC cases. Surrounding cirrhotic nodules and 78% of HA cases exhibited intact 5hmC expression. In FNA cytological specimens, expression of 5hmC was deficient in nearly all cases of HCC (96.7%, 29/30 cases). Admixed benign hepatocytes exhibited predominantly intact expression of 5hmC with moderate to strong nuclear staining in the majority of the benign hepatocytes. 5hmC was strongly expressed by endothelial cells, lymphocytes and neutrophils in the background. Many 5hmC-negative HCC clusters were highlighted by characteristic endothelial wrapping with strong 5hmC expression in the endothelial cells. 5hmC can serve as an important diagnostic tool for the diagnosis of HCC by aiding in the distinction of HCC from HA or cirrhotic nodules in both liver resection and FNA specimens.

Metformin sensitizes endometrial cancer cells to progestin by targeting TET1 to downregulate glyoxalase I expression

Progestins has been used widely for endometrial cancer (EC) patients. However, long term use of high dose progestin often lead to progestin resistance. Our previous studies have demonstrated that metformin reversed progestin resistance through the downregulation of the expression of glyoxalase I (GLOI) in type I endometrial cancer. Recent studies have demonstrated the role of Ten-eleven translocation 1 (TET1) in endometrial cancer, but the physiological role of TET1 in GLOI-mediated progestin resistance has been poorly addressed. Immunohistochemistry was used to detect the expression of TET1, GLOI and 5hmC in various endometrium. Western blot was carried out to analyses TET1 and GLOI expression with different treatment. Cell counting kit-8 was used to evaluate cell proliferation after various treatment. Dot blot assay and HMeDIP assay were performed to detect global hydroxmethylation levels and hydroxymethylation levels in GLOI gene respectively. In current study, we found that metformin effectively sensitized progestin in endometrial cancer cell lines through the down regulation of the expression of TET1 and GLOI. Interestingly, the exogenous increase of TET1 expression enhanced total 5hmC level and hydroxymethylation modification in glyoxalase I promoter region. This effect was abated by metformin treatment. Moreover, the expression profile of TET1 and glyoxalase I in various endometrial tissue parallelized with 5hmC level. Therefore, this finding suggests that metformin sensitized progestin in endometrial cancer through the TET1-5hmC-GLOI signaling pathway.