PS02.176: LINE-1 METHYLATION LEVEL AND LOCAL IMMUNE RESPONSE IN ESOPHAGEAL CANCER

Abstract Background In cancer cells, DNA methylation may be altered in two principle ways; global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Since Long interspersed element-1 (LINE-1 or L1; a repetitive DNA retrotransposon) constitutes a substantial portion (approximately 17%) of the human genome, the extent of LINE-1 methylation is regarded as a surrogate marker of global DNA methylation. In previous studies, we demonstrated that LINE-1 hypomethylation was strongly associated with a poor prognosis in esophageal cancer, supporting its potential role as a prognostic marker (Ann Surg 2012). We also found that LINE-1-hypomethylated tumors showed highly frequent genomic gains at various loci containing candidate oncogenes such as CDK6 (Clin Cancer Res 2014). Given that immunotherapy, as represented by PD-1/PD-L1-targeting antibodies, has increasingly gained attention as a novel treatment strategy for esophageal cancer, better understanding of local immune response status in esophageal cancer is important. The aim of this study is to evaluate the relationship between LINE-1 methylation level and local immune response in esophageal cancer. Methods Using a non-biased database of 305 curatively resected esophageal cancers, we evaluated PD-L1 expression and TIL status (CD8 expression) by immunohistochemical analysis (Ann Surg 2017). Results TIL positivity was significantly correlated with longer overall survival (log-rank P < 0.0001). TIL-negative cases demonstrated significantly lower LINE-1 methylation level compared with TIL-positive cases (P = 0.012). This finding certainly supports that LINE-1 methylation level may influence the local immune response status. Conclusion PD-L1 expression was not related with LINE-1 methylation level. Further investigations in this field would provide deeper insights into esophageal tumor immunology and assist the development of new therapeutic strategies against esophageal cancer. Disclosure All authors have declared no conflicts of interest.

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The association between smoking and LINE-1 hypomethylation (global DNA hypomethylation) in normal esophageal epithelium of patients with esophageal squamous cell carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.41 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 41-41

Author(s):

Shiro Iwagami ◽

Yoshifumi Baba ◽

Masayuki Watanabe ◽

Hironobu Shigaki ◽

Keisuke Miyake ◽

...

Keyword(s):

Dna Methylation ◽

Squamous Cell Carcinoma ◽

Esophageal Cancer ◽

Squamous Cell ◽

Methylation Level ◽

Continuous Variable ◽

Smoking History ◽

Esophageal Mucosa ◽

Esophageal Epithelium ◽

Dna Hypomethylation

41 Background: DNA methylation is a major epigenetic mechanism in X-chromosome inactivation, imprinting and repression of transposable elements and endogenous retroviral sequences. Global DNA hypomethylation appears to play an important role in genomic instability, leading to cancer development. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. Smoking and alcohol is extremely important as the etiology of esophageal squamous cell carcinoma. Nonetheless, whether or not smoking and alcohol affect LINE-1 methylation level in normal esophageal epithelium of esophageal cancer patients remains uncertain. Methods: We quantified LINE-1 methylation of normal esophageal mucosa using pyrosequencing technology in 118 resected esophageal squamous cell carcinomas. The data on smoking (Brinkman index, absence or presence) and alcohol amount are available in all cases. We excluded preoperatively treated cases. Results: LINE-1 methylation in normal esophageal epithelium of esophageal cancer patients ranged from 57.1 to 92.8 of 0-100 scale (N=118; mean 81.2; median 80.0; standard deviation 7.2). LINE-1 methylation level (continuous variable) was significantly associated with Brickman index (continuous variable) (r=0.12, p=0.0002); heavy smoker had lower LINE-1 methylation level of normal esophageal mucosa. LINE-1 methylation level was lower in patients with smoking history (mean 79.7) than in patients without smoking history (mean 83.2) (p=0.034). Alcohol assumption was not associated with LINE-1 methylation level (r=0.003, p=0.58). Conclusions: Smoking was associated with LINE-1 hypomethylation in esophageal normal epithelium, suggesting the possibility of epigenetic field effects caused by cigarette in esophageal tumorigenesis. Considering that DNA methylation alterations are reversible and can thus be targets for chemoprevention, our findings may have clinical implication.

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Prognostic significance of LINE-1 methylation level in esophageal squamous cell carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.26 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 26-26 ◽

Cited By ~ 1

Author(s):

Yoshifumi Baba ◽

Shiro Iwagami ◽

Masayuki Watanabe ◽

Hironobu Shigaki ◽

Satoshi Ida ◽

...

Keyword(s):

Dna Methylation ◽

Squamous Cell Carcinoma ◽

Esophageal Cancer ◽

Esophageal Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Methylation Level ◽

Prognostic Significance ◽

Esophageal Mucosa ◽

Dna Hypomethylation

26 Background: Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Esophageal squamous cell carcinoma, the major histological type of esophageal cancer in East Asian countries, is one of the most aggressive malignant tumors. Nonetheless, prognostic significance of LINE-1 hypomethylaiton in esophageal cancer remains uncertain. Methods: Using 217 curatively resected esophageal squamous cell carcinomas, we quantified LINE-1 methylation using bisulfite-PCR-pyrosequencing assay. Cox proportional hazards model was used to calculate mortality hazard ratio (HR), adjusted for clinical, epidemiologic, and pathological variables. Results: Esophageal cancers showed significantly lower LINE-1 methylation level compared to matched normal esophageal mucosa (p<0.0001; N=50). Tumoral LINE-1 methylation ranged from 24.8 to 91.8 of 0-100 scale (N=217; mean 64.5; median 65.0; standard deviation 12.8). LINE-1 hypomethylation was significantly associated with disease-free survival [log-rank p=0.0008; univariate HR= 2.31, 95% confidence interval (CI) 1.38-3.84, p=0.0017; multivariate HR=1.81, 95% CI 1.06-3.05, p=0.031] and overall survival (log-rank p=0.0013; univariate HR=2.21, 95% CI 1.33-3.60, p=0.0026; multivariate HR=1.87, 95% CI 1.12-3.08, p=0.018]. Conclusions: LINE-1 hypomethylation in esophageal cancer is associated with shorter survival, suggesting its role as a prognostic biomarker. Given that epigenetic changes, including DNA methylation alterations, are reversible and can thus be targets for therapy or chemoprevention, our findings may have considerable clinical implications.

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DNA methylation mediated RSPO2 to promote follicular development in mammals

Cell Death and Disease ◽

10.1038/s41419-021-03941-z ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Xiaofeng Zhou ◽

Yingting He ◽

Nian Li ◽

Guofeng Bai ◽

Xiangchun Pan ◽

...

Keyword(s):

Dna Methylation ◽

Wnt Signaling ◽

Signaling Pathway ◽

Methylation Level ◽

Molecular Mechanisms ◽

Cpg Island ◽

Wnt Signaling Pathway ◽

Follicular Development ◽

Expression Level

AbstractIn female mammals, the proliferation, apoptosis, and estradiol-17β (E2) secretion of granulosa cells (GCs) have come to decide the fate of follicles. DNA methylation and RSPO2 gene of Wnt signaling pathway have been reported to involve in the survival of GCs and follicular development. However, the molecular mechanisms for how DNA methylation regulates the expression of RSPO2 and participates in the follicular development are not clear. In this study, we found that the mRNA and protein levels of RSPO2 significantly increased during follicular development, but the DNA methylation level of RSPO2 promoter decreased gradually. Inhibition of DNA methylation or DNMT1 knockdown could decrease the methylation level of CpG island (CGI) in RSPO2 promoter and upregulate the expression level of RSPO2 in porcine GCs. The hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of transcription factor E2F1 and promoted the transcriptional activity of RSPO2. Moreover, RSPO2 promoted the proliferation of GCs with increasing the expression level of PCNA, CDK1, and CCND1 and promoted the E2 secretion of GCs with increasing the expression level of CYP19A1 and HSD17B1 and inhibited the apoptosis of GCs with decreasing the expression level of Caspase3, cleaved Caspase3, cleaved Caspase8, cleaved Caspase9, cleaved PARP, and BAX. In addition, RSPO2 knockdown promoted the apoptosis of GCs, blocked the development of follicles, and delayed the onset of puberty with decreasing the expression level of Wnt signaling pathway-related genes (LGR4 and CTNNB1) in vivo. Taken together, the hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of E2F1 and enhanced the transcription of RSPO2, which further promoted the proliferation and E2 secretion of GCs, inhibited the apoptosis of GCs, and ultimately ameliorated the development of follicles through Wnt signaling pathway. This study will provide useful information for further exploration on DNA-methylation-mediated RSPO2 pathway during follicular development.

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Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.2951.2951 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2951-2951

Author(s):

Jun Fan ◽

Asou Norio ◽

Masao Matsuoka

Keyword(s):

Dna Methylation ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Mammalian Cells ◽

Mononuclear Cells ◽

Cpg Island ◽

Methylation Status ◽

Lymphocytic Leukemia ◽

Dna Hypomethylation ◽

Peripheral Blood Mononuclear

Abstract DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress. Figure Figure

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Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽

10.1182/blood.v110.11.492.492 ◽

2007 ◽

Vol 110 (11) ◽

pp. 492-492

Author(s):

Wei-Gang Tong ◽

William G. Wierda ◽

Neby Bekele ◽

Shao-Qing Kuang ◽

Michael J. Keating ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Cpg Island ◽

Trichostatin A ◽

Methylation Status ◽

Cpg Islands ◽

Human Leukemia ◽

Dna Hypomethylation ◽

Line P ◽

Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

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The novel DNA methylation inhibitor zebularine is effective against the development of murine T-cell lymphoma

Blood ◽

10.1182/blood-2005-05-2033 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1174-1177 ◽

Cited By ~ 43

Author(s):

Michel Herranz ◽

Juan Martín-Caballero ◽

Mario F. Fraga ◽

Jesús Ruiz-Cabello ◽

Juana Maria Flores ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cpg Island ◽

Promoter Hypermethylation ◽

Dna Hypomethylation ◽

Chemotherapy Drugs ◽

Positron Emission ◽

Methylation Inhibitor ◽

Dna Methylation Inhibitor ◽

T Lymphomas

AbstractGene silencing by CpG island promoter hypermethylation has awakened the interest for DNA demethylating agents as chemotherapy drugs. Zebularine (1-[β-D-ribofuranosil]-1,2-dihydropyrimidin-2-1) has been recently described as a new DNA methylation inhibitor. Here we have studied its effects in a mouse model of radiation-induced lymphomagenesis using nuclear magnetic resonance (NMR) and positron emission tomography (PET). All control animals presented large thymic T lymphomas and died between 4 and 5.5 months. In contrast, 40% (12 of 30) of zebularine-treated animals were still alive after 1 year (Kaplan-Meier P < .001). NMR and PET imaging showed that surviving animals presented a thymus structure/volume similar to normal mice of the same age. Most important, zebularine demonstrated a complete lack of toxicity in nonirradiated control mice. DNA hypomethylation induced by zebularine occurred in association with depletion in extractable DNA methyltransferase 1 protein. Thus, our data support the role of zebularine as a DNA demethylating agent with antitumor activity and little toxicity.

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Prognostic significance of LINE-1 methylation level in the upper gastrointestinal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10523 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10523-10523

Author(s):

Yoshifumi Baba ◽

Masayuki Watanabe ◽

Shiro Iwagami ◽

Hironobu Shigaki ◽

Takatsugu Ishimoto ◽

...

Keyword(s):

Dna Methylation ◽

Gastrointestinal Cancer ◽

Methylation Level ◽

Prognostic Significance ◽

Disease Free Survival ◽

Dna Hypomethylation ◽

Cancer Specific Survival ◽

Upper Gastrointestinal Cancer ◽

Upper Gastrointestinal

10523 Background: Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in long interspersed nucleotide element-1 (LINE-1) is a good indicator of global DNA methylation level. Although LINE-1 methylation level is attracting interest as a useful marker for predicting cancer prognosis, the prognostic significance of LINE-1 hypomethylaiton in the upper gastrointestinal cancer [i.e., esophageal squamous cell carcinoma (ESCC) and gastric cancer (GC)] remains unclear. Methods: Using 217 ESCC and 207 GC specimens, we quantified the LINE-1 methylation using bisulfite-pyrosequencing technology. During the follow-up, there were a total of 63 ESCC recurrences, 51 ESCC deaths and 56 GC deaths. The median follow-up time for censored patients was 2.8 years. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical, epidemiological, and pathological variables. The term “prognostic marker” is used throughout this study according to the REMARK Guidelines. Results: ESCCs and GCs showed significantly lower LINE-1 methylation levels compared to matched normal mucosa (p<0.0001). In ESCC, LINE-1 hypomethylation was significantly associated with disease-free survival [log-rank p=0.0008; univariate HR= 2.32, 95% confidence interval (CI) 1.38-3.84, p=0.0017; multivariate HR=1.81, 95% CI 1.06-3.05, p=0.031] and cancer-specific survival (log-rank p=0.0020; univariate HR=2.21, 95% CI 1.33-3.60, p=0.0026; multivariate HR=1.87, 95% CI 1.12-3.08, p=0.018]. We found a significant modifying effect of the tumor stage on the relationship between LINE-1 methylation and the recurrence rate (P for interaction = 0.031). In GC, LINE-1 hypomethylation was significantly associated with cancer-specific survival (log-rank p=0.029; univariate HR=2.01, 95% CI 1.09-3.99). Conclusions: LINE-1 hypomethylation is associated with shorter survival in both ESCC and GC, suggesting that it has potential for use as a prognostic biomarker.

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Characterization of SIRT1/DNMTs Functions and LINE-1 Methylation in Patients with Age-Related Macular Degeneration

Journal of Clinical Medicine ◽

10.3390/jcm8020159 ◽

2019 ◽

Vol 8 (2) ◽

pp. 159 ◽

Cited By ~ 11

Author(s):

Andrea Maugeri ◽

Martina Barchitta ◽

Matteo Fallico ◽

Niccolò Castellino ◽

Michele Reibaldi ◽

...

Keyword(s):

Dna Methylation ◽

Macular Degeneration ◽

Methylation Level ◽

Surrogate Marker ◽

Dna Methyltransferases ◽

Sirtuin 1 ◽

Age Related Macular Degeneration ◽

Clinical Biomarkers ◽

Age Related

Previous studies proposed the application of DNA methylation signatures as clinical biomarkers of age-related macular degeneration (AMD). However, the characterization of Long Interspersed Nuclear Element-1 (LINE-1) methylation levels—a surrogate marker of global DNA methylation—in AMD patients has not been investigated so far. In the present study, we first characterized DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions in blood samples of 40 AMD patients and 10 age- and sex-matched controls. Then, we evaluated whether changes in DNMTs functions were associated with different LINE-1 methylation levels in leukocyte DNA. We demonstrated that total DNMTs activity was 48% higher in AMD patients than in controls (p = 0.005). AMD patients also exhibited up-regulation of DNMT1 and DNMT3B expression (FC = 2.6; p = 0.003 and FC = 2.4; p = 0.018, respectively). In line with increased DNMTs functions, the LINE-1 methylation level was higher in AMD patients than in controls (mean = 69.10%; SE = 0.68 vs. mean = 65.73%; SE = 0.59; p = 0.020). All p-values were adjusted by Bonferroni correction. In AMD patients, LINE-1 methylation level was positively associated with total DNMTs activity (r = 0.694; p < 0.001), DNMT1 (r = 0.579; p < 0.001), and DNMT3B (r = 0.521; p = 0.001) expression. Our results encourage further large-size prospective research to understand the relationship between LINE-1 methylation and AMD aetiology, and its usefulness in the clinical setting.

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Perinatal Lead (Pb) Exposure and Cortical Neuron-Specific DNA Methylation in Male Mice

Genes ◽

10.3390/genes10040274 ◽

2019 ◽

Vol 10 (4) ◽

pp. 274 ◽

Cited By ~ 5

Author(s):

John F. Dou ◽

Zishaan Farooqui ◽

Christopher D. Faulk ◽

Amanda K. Barks ◽

Tamara Jones ◽

...

Keyword(s):

Dna Methylation ◽

Intensity Ratio ◽

Cpg Island ◽

Epigenetic Changes ◽

Male Mice ◽

Dna Hypomethylation ◽

Neurodevelopmental Outcomes ◽

Ratio Difference ◽

Wide Range ◽

Pb Exposure

: Lead (Pb) exposure is associated with a wide range of neurological deficits. Environmental exposures may impact epigenetic changes, such as DNA methylation, and can affect neurodevelopmental outcomes over the life-course. Mating mice were obtained from a genetically invariant C57BL/6J background agouti viable yellow Avy strain. Virgin dams (a/a) were randomly assigned 0 ppm (control), 2.1 ppm (low), or 32 ppm (high) Pb-acetate water two weeks prior to mating with male mice (Avy/a), and this continued through weaning. At age 10 months, cortex neuronal nuclei were separated with NeuN+ antibodies in male mice to investigate neuron-specific genome-wide promoter DNA methylation using the Roche NimbleGen Mouse 3x720K CpG Island Promoter Array in nine pooled samples (three per dose). Several probes reached p-value < 10-5 , all of which were hypomethylated: 12 for high Pb (minimum false discovery rate (FDR) = 0.16, largest intensity ratio difference = −2.1) and 7 for low Pb (minimum FDR = 0.56, largest intensity ratio difference = −2.2). Consistent with previous results in bulk tissue, we observed a weak association between early-life exposure to Pb and DNA hypomethylation, with some affected genes related to neurodevelopment or cognitive function. Although these analyses were limited to males, data indicate that non-dividing cells such as neurons can be carriers of long-term epigenetic changes induced in development.

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