DNA Methylation In Promoter Region Research Articles

Abstract PO3-24-05: Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-05.

Single-cell transcriptomic sequencing data reveal aberrant DNA methylation in SMAD3 promoter region in tumor-associated fibroblasts affecting molecular mechanism of radiosensitivity in non-small cell lung cancer

ObjectiveNon-small cell lung cancer (NSCLC) often exhibits resistance to radiotherapy, posing significant treatment challenges. This study investigates the role of SMAD3 in NSCLC, focusing on its potential in influencing radiosensitivity via the ITGA6/PI3K/Akt pathway.MethodsThe study utilized gene expression data from the GEO database to identify differentially expressed genes related to radiotherapy resistance in NSCLC. Using the GSE37745 dataset, prognostic genes were identified through Cox regression and survival analysis. Functional roles of target genes were explored using Gene Set Enrichment Analysis (GSEA) and co-expression analyses. Gene promoter methylation levels were assessed using databases like UALCAN, DNMIVD, and UCSC Xena, while the TISCH database provided insights into the correlation between target genes and CAFs. Experiments included RT-qPCR, Western blot, and immunohistochemistry on NSCLC patient samples, in vitro studies on isolated CAFs cells, and in vivo nude mouse tumor models.ResultsFifteen key genes associated with radiotherapy resistance in NSCLC cells were identified. SMAD3 was recognized as an independent prognostic factor for NSCLC, linked to poor patient outcomes. High expression of SMAD3 was correlated with low DNA methylation in its promoter region and was enriched in CAFs. In vitro and in vivo experiments confirmed that SMAD3 promotes radiotherapy resistance by activating the ITGA6/PI3K/Akt signaling pathway.ConclusionHigh expression of SMAD3 in NSCLC tissues, cells, and CAFs is closely associated with poor prognosis and increased radiotherapy resistance. SMAD3 is likely to enhance radiotherapy resistance in NSCLC cells by activating the ITGA6/PI3K/Akt signaling pathway.

CLDN6 inhibited cellular biological function of nonsmall cell lung cancer cells through suppressing aerobic glycolysis via the RIP1/ASK1/JNK axis.

Claudin-6 (CLDN6) has been extensively studied in different tumors to date. However, in the case of nonsmall cell lung cancer (NSCLC), CLDN6 has a largely unknown role and molecular mechanism. We detected the expression of CLDN6 in NSCLC tissues and cells using reverse transcription-quantitative polymerase chain reaction (PCR) and western blot assays. A gain-of-function experiment was performed to evaluate the biological effects of CLDN6 on NSCLC cell behaviors. Methylation-specific PCR was utilized to detect the DNA methylation of CLDN6 gene promoter region. The interaction of CLDN6 and receptor interacting protein 1 (RIP1) was determined by coimmunoprecipitation assay. Furthermore, the modulation of CLDN6 on RIP1/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) axis was confirmed. The results showed that in NSCLC tissues and cells, CLDN6 expression level was declined, and was associated with a high level of DNA methylation. CLDN6 overexpression suppressed the viability, invasion, migration, and promoted cell apoptosis. Besides, the enhanced expression of CLDN6 reduced the glycolysis and the dysfunction of mitochondrial respiration of NSCLC cells. Mechanistic investigation confirmed that CLDN6 interacted with RIP1 and inhibited cellular biological function of NSCLC cells via RIP1/ASK1/JNK axis. Besides, CLDN6 overexpression inhibited tumor growth in vivo. In conclusion, CLDN6 inhibited NSCLC cell proliferation through inactivating aerobic glycolysis via the RIP1/ASK1/JNK axis.

MiR-290 Family Maintains Pluripotency and Self-Renewal by Regulating MAPK Signaling Pathway in Intermediate Pluripotent Stem Cells.

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are derived from pre- and post-implantation embryos, representing the initial "naïve" and final "primed" states of pluripotency, respectively. In this study, novel reprogrammed pluripotent stem cells (rPSCs) were induced from mouse EpiSCs using a chemically defined medium containing mouse LIF, BMP4, CHIR99021, XAV939, and SB203580. The rPSCs exhibited domed clones and expressed key pluripotency genes, with both X chromosomes active in female cells. Furthermore, rPSCs differentiated into cells of all three germ layers in vivo through teratoma formation. Regarding epigenetic modifications, the DNA methylation of Oct4, Sox2, and Nanog promoter regions and the mRNA levels of Dnmt3a, Dnmt3b, and Dnmt1 were reduced in rPSCs compared with EpiSCs. However, the miR-290 family was significantly upregulated in rPSCs. After removing SB203580, an inhibitor of the p38 MAPK pathway, the cell colonies changed from domed to flat, with a significant decrease in the expression of pluripotency genes and the miR-290 family. Conversely, overexpression of pri-miR-290 reversed these changes. In addition, Map2k6 was identified as a direct target gene of miR-291b-3p, indicating that the miR-290 family maintains pluripotency and self-renewal in rPSCs by regulating the MAPK signaling pathway.

DNMT3a-mediated upregulation of the stress inducible protein sestrin-2 contributes to malignant transformation of human bronchial epithelial cells following nickel exposure

BackgroundNickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. MethodsWe employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. ResultExposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, β-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3β/β-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3β/β-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. ConclusionOur results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/β-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.

Decoding the DSCC1 gene as a pan-cancer biomarker in human cancers via comprehensive multi-omics analyses.

While dysregulation of DSCC1 (DNA Replication And Sister Chromatid Cohesion 1) has been established in breast cancer and colorectal cancer, its associations with other tumors remain unclear. Therefore, this study was launched to explore the role of DSCC1 in pan-cancer. In this study, we investigate the biological functions of DSCC1 across 33 solid tumors, elucidating its role in promoting oncogenesis and progression in various cancers through comprehensive analysis of multi-omics data. We conducted a comprehensive analysis of DSCC1 expression using RNA-seq data from TCGA and GTEx databases across 30 cancer types. Striking variations were observed, with significant overexpression of DSCC1 identified in numerous cancers. Elevated DSCC1 level was strongly associated with poorer prognosis, shorter survival, and advanced tumor stages in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), as indicated by Kaplan-Meier curves and GEPIA2 analysis. Further investigation into the molecular mechanisms revealed reduced DNA methylation in the DSCC1 promoter region in KIRP, LIHC, and LUAD, supporting enhanced RNA transcription. Protein expression analysis via the Human Protein Atlas (HPA) corroborated mRNA expression findings, showcasing elevated DSCC1 protein in KIRP, LIHC, and LUAD tissues. Mutational analysis using cBioPortal revealed alterations in 0.4% of KIRP, 17% of LIHC, and 5% of LUAD samples, predominantly characterized by amplification. Immune cell infiltration analysis demonstrated robust positive correlations between DSCC1 expression and CD8+ T cells, CD4+ T cells, and B cells, influencing the tumor microenvironment. STRING and gene enrichment analyses unveiled DSCC1's involvement in critical pathways, emphasizing its multifaceted impact. Notably, drug sensitivity analysis highlighted a significant correlation between DSCC1 mRNA expression and responses to 78 anticancer treatments, suggesting its potential as a predictive biomarker and therapeutic target for KIRP, LIHC, and LUAD. Finally, immunohistochemistry staining of clinical samples validated computational results, confirming elevated DSCC1 protein expression. Overall, this study provides comprehensive insights into the pivotal role of DSCC1 in KIRP, LIHC, and LUAD initiation, progression, and therapeutic responsiveness, laying the foundation for further investigations and personalized treatment strategies.

DNA methylation of promoter region inhibits galectin-1 expression in BMSCs of aged mice.

Senile osteoporosis increases fracture risks. Bone marrow stromal cells (BMSCs) are sensitive to aging. Deep insights into BMSCs aging are vital to elucidate the mechanisms underlying age-related bone loss. Recent advances showed that osteoporosis is associated with aberrant DNA methylation of many susceptible genes. Galectin-1 (Gal-1) has been proposed as a mediator of BMSCs functions. In our previous study, we showed that Gal-1 was downregulated in aged BMSCs and global deletion of Gal-1 in mice caused bone loss via impaired osteogenesis potential of BMSCs. Gal-1 promoter is featured by CpG islands. However, there are no reports concerning the DNA methylation status in Gal-1 promoter during osteoporosis. In the current study, we sought to investigate the role of DNA methylation in Gal-1 downregulation in aged BMSCs. The potential for anti-bone loss therapy based on modulating DNA methylation is explored. Our results showed that Dnmt3b-mediated Gal-1 promoter DNA hypermethylation plays an important role in Gal-1 downregulation in aged BMSCs, which inhibited β-catenin binding on Gal-1 promoter. Bone loss of aged mice was alleviated in response to in vivo deletion of Dnmt3b from BMSCs. Finally, when bone marrow of young wild-type (WT) mice or young Dnmt3bPrx1-Cre mice was transplanted into aged WT mice, Gal-1 level in serum and trabecular bone mass were elevated in recipient aged WT mice. Our study will benefit for deeper insights into the regulation mechanisms of Gal-1 expression in BMSCs during osteoporosis development, and for the discovery of new therapeutic targets for osteoporosis via modulating DNA methylation status.NEW & NOTEWORTHY There is Dnmt3b-mediated DNA methylation in Gal-1 promoter in aged bone marrow stromal cell (BMSC). DNA methylation causes Gal-1 downregulation and osteogenesis attenuation of aged BMSC. DNA methylation blocks β-catenin binding on Gal-1 promoter. Bone loss of aged mice is alleviated by in vivo deletion of Dnmt3b from BMSC.

Photoperiod Induces the Epigenetic Change of the GNAQ Gene in OVX+E2 Ewes.

GNAQ, a member of the alpha subunit encoding the q-like G protein, is a critical gene in cell signaling, and multiple studies have shown that upregulation of GNAQ gene expression ultimately inhibits the proliferation of gonadotropin-releasing hormone (GnRH) neurons and GnRH secretion, and ultimately affects mammalian reproduction. Photoperiod is a key inducer which plays an important role in gene expression regulation by affecting epigenetic modification. However, fewer studies have confirmed how photoperiod induces epigenetic modifications of the GNAQ gene. In this study, we examined the expression and epigenetic changes of GNAQ in the hypothalamus in ovariectomized and estradiol-treated (OVX+E2) sheep under three photoperiod treatments (short photoperiod treatment for 42 days, SP42; long photoperiod treatment for 42 days, LP42; 42 days of short photoperiod followed by 42 days of long photoperiod, SP-LP42). The results showed that the expression of GNAQ was significantly higher in SP-LP42 than in SP42 and LP42 (p < 0.05). Whole genome methylation sequencing (WGBS) results showed that there are multiple differentially methylated regions (DMRs) and loci between different groups of GNAQ. Among them, the DNA methylation level of DMRs at the CpG1 locus in SP42 was significantly higher than that of SP-LP42 (p < 0.01). Subsequently, we confirmed that the core promoter region of the GNAQ gene was located with 1100 to 1500 bp upstream, and the DNA methylation level of all eight CpG sites in SP42 was significantly higher than those in LP42 (p < 0.01), and significantly higher than those in SP-LP42 (p < 0.01), except site 2 and site 4 in the first sequencing fragment (p < 0.05) in the core promoter region. The expression of acetylated GNAQ histone H3 was significantly higher than that of the control group under three different photoperiods (p < 0.01); the acetylation level of sheep hypothalamic GNAQ genomic protein H3 was significantly lower under SP42 than under SP-LP42 (p < 0.05). This suggests that acetylated histone H3 binds to the core promoter region of the GNAQ gene, implying that GNAQ is epigenetically regulated by photoperiod through histone acetylation. In summary, the results suggest that photoperiod can induce DNA methylation in the core promoter region and histone acetylation in the promoter region of the GNAQ gene, and hypothesize that the two may be key factors in regulating the differential expression of GNAQ under different photoperiods, thus regulating the hypothalamus-pituitary-gonadal axis (HPGA) through the seasonal estrus in sheep. The results of this study will provide some new information to understand the function of epigenetic modifications in reproduction in sheep.

Association between DNA Methylation in the Core Promoter Region of the CUT-like Homeobox 1 (CUX1) Gene and Lambskin Pattern in Hu Sheep.

CUT-like homeobox 1 (CUX1) has been proven to be a key regulator in sheep hair follicle development. In our previous study, CUX1 was identified as a differential expressed gene between Hu sheep lambskin with small wave patterns (SM) and straight wool patterns (ST); however, the exact molecular mechanism of CUX1 expression has been obscure. As DNA methylation can regulate the gene expression, the potential association between CUX1 core promotor region methylation and lambskin pattern in Hu sheep was explored in the present study. The results show that the core promoter region of CUX1 was present at (-1601-(-1) bp) upstream of the transcription start site. A repressive region (-1151-(-751) bp) was also detected, which had a strong inhibitory effect on CUX1 promoter activity. Bisulfite amplicon sequencing revealed that no significant difference was detected between the methylation levels of CUX1 core promoter region in SM tissues and ST tissues. Although the data demonstrated the differential expression of CUX1 between SM and ST probably has no association with DNA methylation, the identification of the core region and a potential repressive region of CUX1 promoter can enrich the role of CUX1 in Hu sheep hair follicle development.

DNA methylation cooperates with H3K9me2 at HCN4 promoter to regulate the differentiation of bone marrow mesenchymal stem cells into pacemaker-like cells.

Sick sinus syndrome (SSS) is a a life-threatening disease, and biological pacemakers derived from bone marrow mesenchymal stem cells (BMSCs) have practical clinical applications. Previous studies demonstrated that epigenetics plays an important role in the differentiation of BMSCs into pacemaker-like cells. However, the underlying mechanisms remain unclear. In the present study, we investigated the role of DNA methylation and histone methylation in pacemaker cells formation and found that changes in DNA and H3K9 methylation occur in the promoter region of the pacemaker cell-specific gene HCN4. In addition, the combined addition of methylation inhibitors was able to improve the efficiency of transduction of Tbx18 in inducing the differentiation of BMSCs into pacemaker-like cells. In vitro experiments have shown that inhibition of DNA methylation and H3K9 methylation can enhance the activity of the HCN4 promoter activity, and both can affect the binding of the transcription factor NKx2.5to the HCN4 promoter region. Further research on the interaction mechanism between DNA methylation and H3K9me2 in the HCN4 promoter region revealed that the two may be coupled, and that the methylesterase G9a and DNMT1 may directly interact to bind as a complex that affects DNA methylation and H3K9me2 regulation of HCN4 transcription. In conclusion, our studies suggest that the mutual coupling of DNA and H3K9 methylation plays a critical role in regulating the differentiation of BMSCs into pacemaker-like cells from the perspective of interactions between epigenetic modifications, and combined methylation is a promising strategy to optimise pacemaker-like cells for in vitro applications.

Retracted: Correlation Analysis of DNA Methylation in the von Willebrand Factor Promoter Region and the Risk of Unexplained Recurrent Hemophilia: Systematic Review and Meta-Analysis.

[This retracts the article DOI: 10.1155/2022/3977289.].

Chemotherapy-induced executioner caspase activation increases breast cancer malignancy through epigenetic de-repression of CDH12

Cancer relapse and metastasis are major obstacles for effective treatment. One important mechanism to eliminate cancer cells is to induce apoptosis. Activation of executioner caspases is the key step in apoptosis and was considered “a point of no return”. However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process named anastasis. Here we show that breast cancer cells that have survived through anastasis (anastatic cells) after exposure to chemotherapeutic drugs acquire enhanced proliferation and migration. Mechanistically, cadherin 12 (CDH12) is persistently upregulated in anastatic cells and promotes breast cancer malignancy via activation of ERK and CREB. Moreover, we demonstrate that executioner caspase activation induced by chemotherapeutic drugs results in loss of DNA methylation and repressive histone modifications in the CDH12 promoter region, leading to increased CDH12 expression. Our work unveils the mechanism underlying anastasis-induced enhancement in breast cancer malignancy, offering new therapeutic targets for preventing post-chemotherapy cancer relapse and metastasis.

MiR-29b modulates DNA methylation in promoter region of miR-130b in mouse model of Diabetic nephropathy.

The online version contains supplementary material available at 10.1007/s40200-023-01208-2.

Abstract 761: Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer

Abstract Aberrant DNA methylation accumulates in non-malignant gastric mucosa after exposure to environmental pathogens such as H. pylori (HP). To understand how environmental factors and DNA methylation interplay to influence primary gastric cancer (GC) risk, we performed an integrated analysis of clinical factors and DNA methylation data of gastric tissues from a longitudinally monitored cohort in Japan, with validation in a separate cohort from Singapore. The Japanese check-up cohort included 4,234 healthy subjects who underwent gastric mucosal biopsy. The median observation period was 4.2 years, and 77 subjects developed GC. GC incidence correlated with age, drinking, smoking, GC family history, and HP status in the multivariable Cox model. Next, we conducted comprehensive DNA methylation analysis using Infinium MethylationEPIC arrays on gastric tissues (n=164), including (1) mucosal biopsies from subjects who later developed GC (“future GC patients”), (2) mucosal biopsies from HP(+) subjects who did not later develop GC (“future non-GC subjects”), (3) mucosal biopsies from future non-GC HP(−) subjects (“control mucosae”), and (4) GCs and surrounding mucosae. Infinium data of gastric mucosae (n=137) collected in the GCEP cohort (Singapore) were also analyzed. DNA methylation of promoter region observed in GCs, accumulated not only in mucosae adjacent to GCs but also in the biopsy mucosae of future GC patients. Mucosae of future non-GC subjects were more methylated than control mucosae but less methylated than mucosae of future GC patients. Similar findings were observed in the GCEP cohort. DNA methylation levels were associated with clinical factors and histopathological alterations - however, in multivariable analyses, DNA methylation remained an independent GC risk factor. Methylation levels were predictive of not only higher GC risk but also a shorter period to GC incidence. We then focused on the associations between environmental factors and methylation. Heavy drinking and smoking were associated with the accumulation of DNA methylation only in HP(+) subjects. The increases in methylation over time in subjects who quit smoking were significantly attenuated compared to continuous smokers. These results suggest that pro-carcinogenic epigenetic alterations initiated by HP exposure are amplified by unfavorable but modifiable lifestyle choices. Furthermore, target genes methylated by each environmental factor may overlap in part; however, they were not necessarily methylated similarly, suggesting that the best markers for stratifying GC risk may differ for each subgroup classified by exposure to environmental factors. Indeed, candidate markers for stratifying GC risk overlapped among subgroups, whereas markers unique to each subgroup were also identified, highlighting the potential of integrating environmental, lifestyle, and epigenetic information to inform GC precision prevention. Citation Format: Genki Usui, Keisuke Matsusaka, Kie K. Huang, Feng Zhu, Tomohiro Shinozaki, Masaki Fukuyo, Bahityar Rahmutulla, Norikazu Yogi, Tomoka Okada, Motoaki Seki, Eiji Sakai, Kazutoshi Fujibayashi, Stephen K. Tsao, Christopher Khor, Tiing L. Ang, Hiroyuki Abe, Hisahiro Matsubara, Masashi Fukayama, Toshiaki Gunji, Nobuyuki Matsuhashi, Teppei Morikawa, Tetsuo Ushiku, Khay G. Yeoh, Patrick Tan, Atsushi Kaneda. Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 761.

DNA hypomethylation mediates immune response in pan-cancer

ABSTRACT Abnormal DNA methylation is a fundamental characterization of epigenetics in cancer. Here we demonstrate that aberrant DNA methylating can modulate the tumour immune microenvironment in 16 cancer types. Differential DNA methylation in promoter region can regulate the transcriptomic pattern of immune-related genes and DNA hypomethylation mainly participated in the processes of immunity, carcinogenesis and immune infiltration. Moreover, many cancer types shared immune-related functions, like activation of innate immune response, interferon gamma response and NOD-like receptor signalling pathway. DNA methylation can further help identify molecular subtypes of kidney renal clear cell carcinoma. These subtypes are characterized by DNA methylation pattern, major histocompatibility complex, cytolytic activity and cytotoxic t lymphocyte and tumour mutation burden, and subtype with hypomethylation pattern shows unstable immune status. Then, we investigate the DNA methylation pattern of exhaustion-related marker genes and further demonstrate the role of hypomethylation in tumour immune microenvironment. In summary, our findings support the use of hypomethylation as a biomarker to understand the mechanism of tumour immune environment.

Hypermethylation of Smad7 in CD4+ T cells is associated with the disease activity of rheumatoid arthritis.

Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients. Peripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity. Compared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response. DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.

Effect of zebularine on apoptotic pathways in hepatocellular carcinoma cell lines

The alteration of DNA cytosine methylation is one of the most common epigenetic changes that can play a significant role in human cancers. The enzymes involved in DNA methylation of promoter regions of the genes are DNA methyltransferases (DNMTs). The therapeutic activities and apoptotic effects of DNA methyltransferase inhibitors (DNMTIs) have been reported in various cancers. This study was assigned to assess the effect of zebularine on intrinsic and extrinsic pathways, DNAT 1, 3a, and 3b, p21, and p53, viability, and apoptosis in hepatocellular carcinoma (HCC) cell lines.Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, and MHCC97L) were purchased from the National Cell Bank of Iran, Pasteur Institute, treated with zebularine, and the MTT assay was performed. Then, flow cytometry assay and real-time RT-PCR analysis were performed with zebularine. Statistical comparisons between groups were made using GraphPad Prism software version 8.0. A significant difference was considered as P < 0.05.Zebularine up-regulated DR4, DR5, FAS, FAS-L, TRAIL, Bax, Bak, Bim, p21WAF/CIP1 (p21), and p53 and down-regulated DNMTs (DNAT 1, 3a, and 3b), Bcl-2, Bcl-xL, and Mcl-1, significantly resulting in apoptosis induction in HCC cell lines. Maximal and minimal apoptosis was seen in HCCLM3 and MHCC97L cell lines, respectively.Our findings indicated that DNMTI zebularine can induce apoptosis and inhibit cell growth through both pathways (extrinsic and intrinsic) in HCC cell lines HCCLM3, MHCC97H, and MHCC97L.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

VP26, a herpes simplex virus type 1 capsid protein, increases DNA methylation in COASY promoter region

It has been reported that some specific changes in DNA methylation can be due to aging or infection by tumor-related viruses but the effect of herpes simplex virus type 1 (HSV-1) in this regard is unknown. HSV-1 is a well-known virus that causes cold sores. After the primary infection, the virus switches to latent infection and remains in the body for the whole life. As the location of DNA methylation, we focused on the promoter region of the COASY gene, which codes for coenzyme A synthase, because methylation in this region is reportedly associated with Alzheimer's disease (AD). During HSV-1 lytic infection, compared to non-infected cells, COASY DNA methylation decreased but when HSV-1 replication was inhibited by acyclovir, an anti-herpes agent, COASY DNA methylation increased. In addition, for expression of immediate early protein only, there was no significant change in COASY DNA methylation, while for expression of the capsid protein VP26, a late protein known to bind with DNA methyltransferase DNMT3A, in the nucleus only, COASY DNA methylation significantly increased compared to the control, without changes in DNMT3A mRNA. Our results suggested that DNA methylation occurred not due to transcriptional changes in DNMT3A but through translational regulation. In this research, we showed that host COASY DNA methylation is altered by HSV-1 infection, in particular by HSV-1 VP26. It is a potential cause of various diseases, and this is particularly relevant for AD.

PAH-induced metabolic changes related to inflammation in childhood asthma.

Epidemiological studies have shown that PAHs may exert adverse effects on childhood asthma. However, the underlying molecular mechanism remains to be fully elucidated. This study aimed to investigate this process in view of metabolic pathways, especially one-carbon metabolism and tryptophan metabolism. Fifty asthmatic children and 50 control subjects were recruited for this study. Serum IgE and IL-17A levels were detected by ELISA. Serum PAH concentrations were measured by GC-MS. One-carbon-related metabolites and tryptophan metabolites were determined by UPLC-Orbitrap-MS. DNA methylation was analyzed by bisulfite sequencing PCR. ChIP assays were used to examine H3K4me3 enrichment on IL-17A gene. Multivariable linear regression was performed to evaluate the association between PAHs and childhood asthma mediated by intermediators. HE staining in lung tissue, IgE and IL-17A in BALF, metabolic profiles in urine, and Ahr, Il-17a, and Cyp1a1 gene expression were determined in PAH-exposed mice. Serum Fla level was associated with childhood asthma (OR = 1.380, 95% CI: 1.063-1.792), and had a great effect on one-carbon metabolites, especially SAH, SAM, and Ser, which exerted significant mediation effects on the relationship between the Fla concentration and asthma. Moreover, we did find significant mediation effects between serum Fla and asthma by LINE-1 DNA methylation and H3K4me3 levels in the IL-17A promoter region. The differential Trp metabolites, such as Trp, tryptamine, IA, IAA, indole, IAld, and IAAld, indicated that asthmatic children had increased indole-AhR pathway. Mediation analysis failed to show a mediator effect of Trp metabolites in the association between PAHs and childhood asthma. An animal study confirmed that PAH exposure increased methylation levels, and altered Trp metabolite-AhR-IL-17A axis, which may be influenced by gender. PAHs disturbed one-carbon metabolism to influence the methyl group refilling DNA methylation and histone methylation, and disturbed tryptophan metabolism to regulate Th17-cell differentiation, which may elevate serum IL-17A concentration in asthmatic children.