Promoter Methylation Levels Research Articles

Reactivation of methylation-silenced PAX1 inhibits cervical cancer proliferation and migration via the WNT/TIMELESS pathway.

Although aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation-specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p < 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune-infiltration via public databases. Then, CRISPR-based methylation perturbation tools (dCas9-Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain- and loss-of-function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co-immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR-based PAX1 demethylation editing might be a promising therapeutic strategy for CC.

Abstract PO4-15-09: Unraveling the influence of progesterone receptor status on endocrine therapy sensitivity: insight from epigenetic and fragmentomics ctDNA profiling in estrogen receptor positive, HER2 negative metastatic breast cancer (MBC)

Abstract Background: In patients (pts) with estrogen receptor (ER) positive (pos), HER2 negative (neg) MBC, the absence of progesterone receptor (PR) expression is generally associated with a poorer endocrine therapy (ET) response and an unfavorable prognosis. However, real-time molecular changes during ET according to PR status are still not fully understood. The aim of this study was to examine the prognostic impact of PR and its association with the methylation levels of ESR1 promoters A (promA) and B (promB) at different timepoints in pts with ERpos HER2neg MBC who received first-line ET. Methods: Pts were enrolled in the prospective multicenter MAGNETIC.1 trial (CRO-2018-56) between January 2018 and January 2023. As first-line ET, pts received either fulvestrant or AIs with or without CDK4/6 inhibitors (CDK4/6i) as first-line ET. ctDNA samples were collected at baseline (T0) and at 3-month intervals (T3 and T6) and analyzed through methylation-specific (MS) droplet digital PCR (ddPCR), ddPCR fragmentomics and next generation sequencing (NGS). Clinico-pathological differences across the PR expression spectrum were analyzed using Fisher’s exact test. Matched pairs variations across timepoints (T0, T3, T6) of ACTBfragments distribution and ESR1 promA and promB were tested through Wilcoxon signed rank test. Results: Out of the 111 enrolled pts, 28 had PRneg MBC, of which 20 had an invasive ductal carcinoma (IDC) histotype, and 10 were diagnosed as de novo disease. Bone was the most common metastatic site both in the PRpos and PRneg subgroups (75% vs 73%), most pts received ET+CDK4/6i (96% vs 95%). ESR1 and PIK3CA mutations were respectively detected in 8% and 6%, and in 23% and 25% pts respectively for PRpos and PRneg. The prognostic impact of PR status was evident for both PFS (median 38 mos and 14 mos respectively for PRpos and PRneg, P = 0.0048) and OS (median not reached and median 33 mos respectively for PRpos and PRneg, P= 0.001). A significant increase in promB was observed at T3 vs T0 and at T6 vs T3 in the PRpos subgroup (respectively P=0.0158 and P&amp;lt; 0.001) but not in the PRneg. When comparing T6 to T0, no significant differences were observed regardless of PR status. At T3, significant decreases were observed in both the PRpos and PRneg populations for ACTBshort (P &amp;lt; 0.001 and P=0.03, respectively), ACTBmedium (P=0.0049 and P= 0.035, respectively) and ACTBlong(P&amp;lt; 0.001 and P&amp;lt; 0.001, respectively), compared to T0. In the comparison of T6 to T3, a significant variation in ACTBshort levels was observed in both subgroups (P&amp;lt; 0.001 for PRpos and P=0.012 for PRneg). while a significant reduction in ACTBmedium levels was observed only in the PRpos population when comparing T6 to T0 (P&amp;lt; 0.001). Conclusions: PRneg status was confirmed as an unfavorable prognostic factor with respect to PRpos both for PFS and OS, potentially due to different ET sensitivity. Epigenetic ctDNA profiling revealed different dynamics of ESR1 promB between PR-positive and PR-negative MBC, suggesting a distinct role of ESR1 in the onset of ET resistance within the two subgroups. Citation Format: Lorenzo Foffano, Elisabetta Molteni, Arianna Dri, Alessandra Franzoni, Linda Cucciniello, Lucia Da Ros, Silvia Buriolla, Silvia Bolzonello, Claudia Noto, Stefania Russo, Simon Spazzapan, Elena Nascimbeni, Brenno Pastò, Giada Targato, Serena Della Rossa, Marta Bonotto, Alessandro Marco Minisini, Giuseppe Damante, Barbara Belletti, Lorenzo Gerratana, Fabio Puglisi. Unraveling the influence of progesterone receptor status on endocrine therapy sensitivity: insight from epigenetic and fragmentomics ctDNA profiling in estrogen receptor positive, HER2 negative metastatic breast cancer (MBC) [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 PO4-15-09.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Hypomethylation-associated LINC00987 downregulation induced lung adenocarcinoma progression by inhibiting the phosphorylation-mediated degradation of SND1.

DNA methylation, an epigenetic regulatory mechanism dictating gene transcription, plays a critical role in the occurrence and development of cancer. However, the molecular underpinnings of LINC00987 methylation in the regulation of lung adenocarcinoma (LUAD) remain elusive. This study investigated LINC00987 expression in LUAD patients through analysis of The Cancer Genome Atlas data sets. Quantitative real-time polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization assays were used to assess LINC00987 expression in LUAD. The bisulfite genomic sequence PCR (BSP) assay was used to determine the methylation levels of the LINC00987 promoter. The interaction between LINC00987 and SND1 was elucidated via immunoprecipitation and RNA pull-down assays. The functional significance of LINC00987 and SND1 in Calu-3 and NCI-H1688 cells was evaluated in vitro through CCK-8, EdU, Transwell, flow cytometry, and vasculogenic mimicry (VM) tube formation assays. LINC00987 expression decreased in LUAD concomitant with hypermethylation of the promoter region, while hypomethylation of the LINC00987 promoter in LUAD tissues correlated with tumor progression. Treatment with 5-Aza-CdR augmented LINC00987 expression and inhibited tumor growth. Mechanistically, LINC00987 overexpression impeded LUAD progression and VM through direct binding with SND1, thereby facilitating its phosphorylation and subsequent degradation. Additionally, overexpression of SND1 counteracted the adverse effects of LINC00987 downregulation on cell proliferation, apoptosis, cell migration, invasion, and VM in LUAD in vitro. In conclusion, this pioneering study focuses on the expression and function of LINC00987 and reveals that hypermethylation of the LINC00987 gene may contribute to LUAD progression. LINC00987 has emerged as a potential tumor suppressor gene in tumorigenesis through its binding with SND1 to facilitate its phosphorylation and subsequent degradation.

Abstract CT230: Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments

Abstract CheckMate 548 (NCT02667587, CM548) is a phase III, randomized trial evaluating first line nivolumab (NIVO) in combination with standard of care (SOC) [surgical resection followed by radiotherapy and temozolomide (TMZ)] versus placebo plus SOC in patients with newly diagnosed glioblastoma (GBM) positive for promoter methylation of O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation status and PD-L1 expression predict better outcomes in GBM patients treated with TMZ and in patients treated with NIVO in other indications, respectively. However, the impact of varying levels of MGMT promoter methylation and PD-L1 expression on patient outcome in GBM remains unclear. In CM548, MGMT promoter methylation levels were quantified through a central lab assay (LapCorp) and PD-L1 expression was determined by percentage of detectable membrane staining of tumor cells. MGMT methylation and PD-L1 data were prospectively generated at the time of enrolment. Our analyses showed that increasing levels of MGMT promoter methylation level were associated with longer overall survival in patients treated with SOC alone (n = 349). However, no survival benefit was observed with increasing levels of MGMT methylation in patients treated with NIVO in combination with SOC (n = 353). In addition, our data showed a non-linear relationship between PD-L1 expression and overall survival across both arms. In patients with intermediate PD-L1 expression (≥5%, &amp;lt;20%), longer survival was observed in the nivolumab + SOC arm compared to SOC alone. This benefit was not observed in patients with low (≥1%, &amp;lt;5%) and high (&amp;gt;20%) PD-L1 expression. Potential mechanisms driving these observed associations are currently explored using genomic data generated from patient samples. MGMT promoter methylation and PD-L1 expression are potential clinically relevant predictive biomarkers of response to treatments administered in the CM-548; however, the binary classification of positive/negative status of these markers does not fully capture their relationship with efficacy in CM548. Data generated from our exploratory analyses suggests that new, refined criteria are needed for better patient outcomes prediction. Citation Format: Yu-Han Hung, Aparna Chhibber, Michael Lim, Michael Weller, David A. Reardon, Antonio Omuro, Keith L. Ligon, Ana Lako, Ann Forslund. Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT230.

Cyclin B1: A potential prognostic and immunological biomarker in pan-cancer.

Cyclin B1 (CCNB1) encodes a regulatory protein essential for the regulation of cell mitosis, particularly in controlling the G2/M transition phase of the cell cycle. Current research has implicated CCNB1 in the progression of various types of cancer, including gastric cancer, breast cancer, and non-small cell lung cancer. In this study, we conducted a pan-cancer analysis of CCNB1 to investigate its prognostic significance and immunological aspects. Our comprehensive investigation covered a wide range of analyses, including gene expression, promoter methylation, genetic alterations, immune infiltration, immune regulators, and enrichment studies. We utilized multiple databases and tools for this purpose, such as The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, the Human Protein Atlas (HPA), the University of Alabama at Birmingham CANcer data analysis Portal (UALCAN), the Gene Expression Profiling Interactive Analysis (GEPIA), the DNA Methylation Interactive Visualization Database (DNMIVD), the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Sangerbox, and cBioPortal. Data analyses were executed using GraphPad Prism software, R software, and various online tools. Our findings demonstrated a significant increase in CCNB1 expression across 28 cancer types. Elevated CCNB1 expression correlated with decreased overall survival (OS) in 11 cancer types and disease-free survival (DFS) in 12 cancer types. Additionally, DNA promoter methylation levels were significantly decreased in 14 cancer types. Furthermore, the study verified a significant association between CCNB1 expression and immune infiltration, immune modulators, microsatellite instability (MSI), and tumor mutational burden (TMB). Enrichment analysis indicated that CCNB1 is involved in multiple cellular pathways. Collectively, our results suggested that CCNB1 has the potential to serve as a valuable prognostic biomarker and may be a promising target for immunotherapy in various cancer types.

PAX6 gene promoter methylation is correlated with myopia in Chinese adolescents: a pilot sutdy

ABSTRACT Purpose A large number of epidemiological studies have shown that myopia is a complex disease involving genetic, environmental, and behavioral factors. The purpose of this study was to explore the role of PAX6 gene methylation in myopia in Chinese adolescents. Methods Eighty junior high school students were divided into four groups based on their vision test results: mild myopia, moderate myopia, severe myopia, and non-myopia control. The methylation level of PAX6 gene promoter was detected by bisulfate pyrosequencing. Results The methylation level of PAX6 gene in myopia group (8.06% ± 1.43%) was slightly but significantly higher than that in non-myopia controls (7.26% ± 1.17%). In addition, PAX6 gene methylation levels presented a decreasing pattern along with the aggravation of myopia. Post-hoc analysis indicated significant inter-group differences for the mild myopia group and other groups (All p < .05). In the subgroup analysis by gender, the methylation level of PAX6 gene promoter in girls was higher than that in boys (p = .023). The ROC curves showed a high accuracy of PAX6 gene methylation to predict mild myopia (AUC (95% CI) = 0.828 (0.709–0.947), p < .001). Conclusions The methylation of PAX6 gene might play a role in the onset and progression of myopia in Chinese adolescents. And this could potentially explore the potential molecular mechanisms of juvenile myopia in the future.

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells

BackgroundA decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear. MethodsAged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes. ResultsMSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth. ConclusionMSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

Plasma Cell-Free Adenomatous Polyposis Coli Gene Promoter Methylation as a Prognostic Biomarker for Hepatocellular Carcinoma

Introduction: Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Lack of biomarkers for follow-up after treatment is a clinical challenge. DNA methylation has been proposed to be a potential biomarker in HCC. However, there is still a lack of evidence of its clinical use. This study aimed to evaluate the value of using plasma Adenomatous Polyposis Coli promoter methylation level (APC-MET) as a potential biomarker in HCC treatment. Method: A total of 96 patients with HCC at BCLC stage B who underwent local tumor ablation treatment were prospectively included in this study. APC-MET was examined in the plasma of each patient before and 1 month after treatment. The prediction value of APC-MET for survival outcome and disease status after treatment was analyzed and adjusted with alpha-fetoprotein and protein induced by vitamin K absence-II using Cox regression analysis. Results: Univariate Cox regression analysis showed preoperative APC-MET >0 (HR, 2.9, 95% CI: 1.05–8.05, p = 0.041) and postoperative APC-MET >0 (HR, 3.47, 95% CI: 1.16–10.4, p = 0.026) were both predictors of death, and preoperative APC-MET >0 was a predictor of disease progression after treatment (HR, 2.04, 95% CI: 1.21–3.44, p = 0.007). In multivariate models, preoperative APC-MET >0 was a significant predictor of disease progression after adjusting with the other two traditional biomarkers (HR, 1.82, 95% CI: 1.05–3.17, p = 0.034). Conclusions: Hypermethylation of APC promoter appears to be a potential biomarker that could predict patient survival and disease progression outcomes in patients with intermediate-stage HCC after local ablation treatment.

Abstract 1426: Lipid-mediated epigenetic activation of PPAR-gamma signaling promotes immune-checkpoint resistance in hepatocellular carcinoma

Abstract Background: Although use of immune-checkpoint inhibitors (ICI) has yielded impressive results in cancer patients, resistance to ICI therapies has increasingly been observed, especially in hepatocellular carcinoma (HCC). Materials and Methods: We generated a novel anti-programmed death-ligand 1 (PD-L1) treatment resistant mouse hepatoma cell Hepa1-6 (PD-L1R) by 7-generation of in vivo selection. To dissect tumor cell-intrinsic resistant mechanism, we performed single-cell RNA-sequencing (scRNA-seq) from anti-PD-L1-treated tumors generated from parental or PD-L1R cells. Expression level of target genes was detected by QPCR, western blot and immunohistochemical analyses. Lipid content and fatty acid uptake was examined by Oil Red O and BIODIPY staining. Peroxisome proliferator-activated receptor gamma (PPARγ) promoter methylation level was analyzed by bisulfite pyrosequencing. To investigate the tumor cell-extrinsic factors, we profiled the myeloid and lymphoid lineages by flow cytometry. Results: ScRNA-seq revealed significant gene enrichments in lipid metabolism and PPARγ signaling pathway in PD-L1R tumors, which could be verified in both mRNA and protein levels. Moreover, we observed evident lipid accumulation in the PD-L1R tumors when compared to the parental tumors by Oil Red O staining. Consistently, PD-L1R tumor cells exhibited higher lipid content and enhanced fatty acid uptake in vitro, as demonstrated by BODIPY493/503 and BODIPY-C16 analyses. Of note, palmitic acid could dose-dependently increase Pparg mRNA expression in parental cells to a similar level with the PD-L1R cells, supporting an important role of lipids in inducing PPARγ expression. Pyrosequencing analysis indicated that PD-L1R cells exhibited lower PPARγ methylation level compared with parental cells. Notably, 5-Azacytidine treatment in parental cells significantly reduced the promoter DNA methylation and increased Pparg mRNA expression to similar extent with the PD-L1R tumor cells, thus indicating DNA hypomethylation-driven PPARγ up-regulation. Flow cytometry analysis showed that ICI resistance was associated with lower cytotoxic CD8+ T cells but higher intermediate Th17 cells, myeloid-derived suppressor cells and T regulatory cells in the tumor microenvironment (TME). Conclusions: Our study suggested that lipid exposure may epigenetically up-regulate PPARγ transcription in the development of anti-PD-L1 resistance. Detailed investigation on the crosstalk between aberrant lipid metabolism and immunosuppressive TME is ongoing. Acknowledgment: This study is supported by RGC General Research Fund (14120621 and 14119023), Li Ka Shing Foundation and CUHK Strategic Seed Funding for Collaborative Research Scheme. Citation Format: Zhewen Xiong, Stephen Lam Chan, Jingying Zhou, Xuezhen Zeng, Weiqin Yang, Haoran Wu, Jianquan Cao, Jie-Ting Low, Michael Wing-Yan Chan, Kevin Yuk-Lap Yip, Joseph Jao-Yiu Sung, Alfred Sze-Lok Cheng. Lipid-mediated epigenetic activation of PPAR-gamma signaling promotes immune-checkpoint resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1426.

Abstract 2120: Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population

Abstract Asparaginase is a key component of the therapeutic regimen used to treat patients with acute lymphoblastic leukemia (ALL). Calaspargase pegol (Asparlas) is composed of E. coli L-asparaginase and is PEGylated with approximately 5000 Da polyethylene glycol (PEG). Asparlas depletes asparagine (Asn) by deamination into aspartic acid. Asn synthetase (ASNS) is the key enzyme responsible for de novo Asn synthesis. In the absence of Asn in their direct environment, normal cells having adequate ASNS activity can survive with their own Asn supply while cancer cells lacking ASNS activity undergo apoptosis as they depend on extracellular sources of Asn. The ASNS gene has a typical CpG island in its promoter and its methylation status in tumor cells has been reported to be associated with asparaginase sensitivity clinically in T-ALL and in solid tumors preclinically. In this study we have investigated ASNS promoter hypermethylation as a biomarker for Asparlas activity in solid tumors and explored the feasibility of translating these results into the clinic. Analyses of the TCGA datasets indicated that a relatively high fraction of hepatocellular carcinoma (HCC) and gastric cancers are hypermethylated at the ASNS promoter. Testing Asparlas across a panel of HCC cell lines demonstrated that ASNS promoter-hypermethylated cell lines with lowest ASNS mRNA expression are the most responsive to Asparlas treatment in vitro. In vivo efficacy studies in mice bearing human ASNS promoter-hypermethylated xenograft models of HCC and gastric cancer showed that Asparlas treatment inhibited tumor growth in a dose-dependent manner and resulted in tumor rejections at the highest doses. Data from the HCC model was used to build a PK/PD efficacy model to project efficacious concentrations in patients. A panel of HCC PDX models with different levels of ASNS promoter methylation was evaluated to better understand the relationship between this biomarker and response to Asparlas. Treatment with Asparlas resulted in complete depletion of Asn in plasma and a partial depletion of Asn in the tumor for all models tested and strong anti-tumor activity against a model that had high methylation of the ASNS promoter. This activity was lost in models with lesser degrees of methylation indicating a strict threshold of promoter methylation for efficacy. These data confirm and extend previous findings that hypermethylation of the ASNS promoter has a synthetic lethal relationship with Asn-depleting therapies including Asparlas and highlight the need to carefully define the minimum threshold of hypermethylation to select HCC patients who could potentially benefit from Asparlas treatment. Citation Format: Ethika Tyagi, Magdalena Pohorecka, Valérie Duvivier, Aurelie Studeny, Damien Valour, Natacha Moulharat, Hélène Darville, Nicolas Provost, Pierre Barbier Saint Hilaire, Eef Hoeben, Donald Simons. Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2120.

The Role of Epstein-Barr Virus (EBV) Infected Gastric Cancer in Increasing microRNA124 (miR-124) Promoter Methylation and Enhancer of Zeste Homolog 2 (EZH2) Gene Expression.

The tumor suppressor microRNAs, miR-21, miR-124, and miR-494, participate in the controlling several cellular processes. To assess target miRNAs promoter methylation levels, we investigated 304 pairs of gastric cancer (GC) tissues and non-tumor tissues. We used a commercial real-time polymerase chain reaction (RT-PCR) for Epstein-Barr virus (EBV) and Helicobacter pylori kit to detect EBV and H. pylori DNA in GC tissues. After finding hypermethylation in the promoter of the miR-124 gene, we evaluated its expression level using quantitative PCR (qPCR). Bioinformatics analysis confirmed miR-124 as a target of enhancer of Zeste homolog 2 (EZH2). Additionally, qPCR confirmed the association between EZH2 and miR-124. EBV and H. pylori DNA were detected in 9.5% and 15.1% of GC patients, respectively. Our findings also revealed significant differences in the miR-124 methylation levels among EBV-infected GC patients, H. pylori infected GC patients, GC patients without EBV and H. pylori infection, and non-tumor tissue. Bioinformatics and qPCR assays suggested an inverse relationship between the expression levels of EZH2 and miR-124 in EBV-infected GC patients. Our data revealed hypermethylation of the miR-124 promoter and significant reduction in its expression in EBV-infected GC tissues. It is possible that miR-124 may target EZH2 by binding to the 3'-UTR of the EZH2 gene, thus potentially contributing to the development of EBV-infected GC.

Significant heightened methylation levels of RUNX3 gene promoter in patients with systemic lupus erythematosus.

Researchers are actively investigating new diagnostic and prognostic biomarkers that offer improved sensitivity and specificity for systemic lupus erythematosus (SLE). One area of interest is DNA methylation changes. Previous studies have shown a connection between the RUNX3 gene dysfunction and SLE. In this study, the focus was on examining the methylation level of the RUNX3 promoter in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy individuals. A total of 80 individuals diagnosed with SLE from Iran, along with 77 healthy individuals, were included. The methylation levels of the RUNX3 gene in the extracted DNA were evaluated using the MethyQESD method. To determine the diagnostic effectiveness of the RUNX3 promoter methylation level, a receiver operating characteristic (ROC) curve was generated. The methylation of the RUNX3 promoter was found to be significantly higher in patients with SLE compared to healthy individuals (p < .001). This difference in methylation levels was observed between SLE patients and healthy individuals and between SLE patients with renal involvement and those without renal involvement (86.29 ± 10.30 vs 40.28 ± 24.21, p < .001). ROC analyses revealed that the methylation level of the RUNX3 promoter had a diagnostic power of 0.769 [95% CI (0.681-0.814)] for SLE. Additionally, there was a positive correlation between the RUNX3 methylation level and levels of creatinine and C4. The findings of this study emphasize the potential use of RUNX3 methylation levels in PBMCs of SLE patients as biomarkers for diagnosing the disease, predicting renal damage, and assessing disease activity.

Hypermethylation of the glutathione peroxidase 4 gene promoter is associated with the occurrence of immune tolerance phase in chronic hepatitis B

BackgroundHepatitis B virus (HBV) infection is a public health problem that seriously threatens human health. This study aimed to investigate the clinical significance of glutathione peroxidase 4(GPX4) in the occurrence and development of chronic hepatitis B (CHB).MethodsA total of 169 participants including 137 patients with CHB and 32 healthy controls (HCs) were recruited. We detected the expression of GPX4 and stimulator of interferon genes (STING) in peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (RT-qPCR). The methylation level of GPX4 gene promoter in PBMCs was detected by TaqMan probe-based quantitative methylation-specific PCR (MethyLight). Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum levels of GPX4, IFN-β, oxidative stress (OS) related molecules, and pro-inflammatory cytokines.ResultsThe expression levels of GPX4 in PBMCs and serum of CHB patients were lower than those of HCs, but the methylation levels of GPX4 promoter were higher than those of HCs, especially in patients at the immune tolerance phase. STING mRNA expression levels in PBMCs and serum IFN-β levels of patients at the immune activation phase and reactivation phase of CHB were higher than those at other clinical phases of CHB and HCs. GPX4 mRNA expression level and methylation level in PBMCs from patients with CHB had a certain correlation with STING and IFN-β expression levels. In addition, the methylation level of the GPX4 promoter in PBMCs from patients with CHB was correlated with molecules associated with OS and inflammation.ConclusionsGPX4 may play an important role in the pathogenesis and immune tolerance of CHB, which may provide new ideas for the functional cure of CHB.

High expression of UBE2S promotes progression of hepatocellular carcinoma by increasing cancer cell stemness

To investigate the expression of the ubiquitination enzyme UBE2S in different cell types in hepatocellular carcinoma (HCC) microenvironment and its impact on proliferation and stemness of HCC cells. TCGA and CPTAC database were used to analyze the transcriptional and promoter methylation levels and protein expressions of UBE2S in HCC. Specific expression patterns of UBE2S, intercellular communication and key transcription factors in different cell types were analyzed based on single-cell sequencing data from TISCH website. We further examined UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells using immunohistochemistry and immunofluorescence staining. We also tested the effects of UBE2S knockdown on stemness of HCC-LM3 and HepG2 cells using clone formation experiments and sphere formation assay. Analysis based on TCGA database suggested significant overexpression of UBE2S in both paired and non-paired tumor tissues (P < 0.001), and its transcriptional level increased with tumor grades. The methylation level of UBE2S promoter was significantly decreased in HCC (P < 0.001), and its transcription level increased obviously in HCC with TP53 mutation (P < 0.001). Analysis of CPTAC database also demonstrated overexpression of UBE2S protein in HCC tissues (P < 0.001). Three prognostic models suggested that HCC patients with high UBE2S expression had poorer prognosis (P < 0.001). Single-cell sequencing data analysis revealed high expressions of UBE2S in T cells and high intensities of interaction between endothelial cells, epithelial cells and fibroblasts in HCC microenvironment. Immunohistochemistry and immunofluorescence staining demonstrated high UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells. In HCC-LM3 and HepG2 cells, UBE2S knockdown significantly inhibited cell clone formation and tumor sphere formation (P < 0.05). UBE2S is highly expressed in T cells in HCC microenvironment in close correlation with a poor prognosis. High UBE2S expression promotes the stemness of HCC cells.

Exploring the Role of PCDHGB4 in the Occurrence of Lung Squamous Cell Carcinoma Based on Bioinformatics Analysis

Lung squamous cell carcinoma (LUSC) is a subtypes of non-small cell lung cancer (NSCLC). It has been reported that members of the protocadherin γ family can regulate tumor cell growth by inhibiting the Wnt signaling pathway. Protocadherin-gamma subfamily B4 (PCDHGB4) as a family member in LUSC was rarely reported. The aim of this study was to investigate the role and potential prognostic value of PCDHGB4 in the development of LUSC using bioinformatics methods. The Cancer Genome Atlas (TCGA), cBioPortal and UALCAN databases were used to analyze the expression, prognosis, clinicopathological features, immune cell infiltration, immune regulatory genes, immune checkpoint inhibitors (ICIs), and methyltransferases of PCDHGB4 in LUSC. At the single cell level, we analyzed the clustering results of cell subtypes and the expression of PCDHGB4 in different immune cell subpopulations. In addition, we compared the promoter methylation levels of PCDHGB4 in LUSC tissues and normal tissues and performed protein-protein interaction and mutation analysis. Finally, enrichment analysis was performed based on the differentially expressed genes. Bioinformatics analysis results showed that the expression level of PCDHGB4 in LUSC tissues was lower than that in normal tissues. Survival analysis showed that increased PCDHGB4 expression was associated with poor prognosis. Single-cell sequencing analysis showed that PCDHGB4 was expressed in T cells, monocytes or macrophages, and dendritic cells. It was further found that PCDHGB4 played an important role in tumor immunity and confirmed that PCDHGB4 was associated with immune checkpoints, immune regulatory genes, and methyltransferases. Besides, enrichment analysis revealed that PCDHGB4 was involved in multiple cancer-related pathways. The expression of PCDHGB4 was low in LUSC. PCDHGB4 was related to the poor prognosis of patients, and PCDHGB4 was closely related to the infiltration and pathway of tumor immune cells. PCDHGB4 may be a potential prognostic marker and a new target for immunotherapy in LUSC.

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.

Promoter of lncRNA MORT is aberrantly methylated in colorectal cancer

Aberrant DNA methylation plays essential roles in the colorectal cancer (CRC) carcinogenesis and has been demonstrated as a promising marker for cancer early detection. In this project, methylation status of the MORT promoter was studied in CRC and their marginal tissues using qMSP assay. Furthermore, we investigated the molecular function of MORT in CRC progression using computational analysis. The results showed a high methylation level of MORT promoter in CRC tissues. By in silico analysis, we found that MORT downregulation could promote the proliferation of CRC cells via sponging of has-miR-574-5p and has-miR-31-5p, and alteration of their targets expression pattern such as MYOCD and FOXP2. In conclusion, based on our results, promoter hypermethylation of MORT might be considered as a potential biomarker for CRC detection.

A noninvasive method for predicting clinically significant prostate cancer using magnetic resonance imaging combined with PRKY promoter methylation level: a machine learning study

BackgroundTraditional process for clinically significant prostate cancer (csPCA) diagnosis relies on invasive biopsy and may bring pain and complications. Radiomic features of magnetic resonance imaging MRI and methylation of the PRKY promoter were found to be associated with prostate cancer.MethodsFifty-four Patients who underwent prostate biopsy or photoselective vaporization of the prostate (PVP) from 2022 to 2023 were selected for this study, and their clinical data, blood samples and MRI images were obtained before the operation. Methylation level of two PRKY promoter sites, cg05618150 and cg05163709, were tested through bisulfite sequencing PCR (BSP). The PI-RADS score of each patient was estimated and the region of interest (ROI) was delineated by 2 experienced radiologists. After being extracted by a plug-in of 3D-slicer, radiomic features were selected through LASSCO regression and t-test. Selected radiomic features, methylation levels and clinical data were used for model construction through the random forest (RF) algorithm, and the predictive efficiency was analyzed by the area under the receiver operation characteristic (ROC) curve (AUC).ResultsMethylation level of the site, cg05618150, was observed to be associated with prostate cancer, for which the AUC was 0.74. The AUC of T2WI in csPCA prediction was 0.84, which was higher than that of the apparent diffusion coefficient ADC (AUC = 0.81). The model combined with T2WI and clinical data reached an AUC of 0.94. The AUC of the T2WI-clinic-methylation-combined model was 0.97, which was greater than that of the model combined with the PI-RADS score, clinical data and PRKY promoter methylation levels (AUC = 0.86).ConclusionsThe model combining with radiomic features, clinical data and PRKY promoter methylation levels based on machine learning had high predictive efficiency in csPCA diagnosis.

CLCN3 in mediating the proliferation of human ovarian cancer cells.

Chloride channel-3 (CLCN3), a crucial component of the voltage-gated chloride channel family, is implicated in numerous physiological and pathophysiological processes. This study aimed to investigate the characteristics of CLCN3 in pancancer and its influence on the immune response through the use of a range of databases. Concurrently, we assessed the impact of CLCN3 on the proliferation of ovarian cancer (OC) cells and explored its potential mechanisms. We employed the Tumor Immune Estimation Resource (TIMER) 2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the messenger RNA (mRNA) and the protein expression of CLCN3 across various cancers. The prognostic significance of CLCN3 was evaluated using the Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) database. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) facilitated the analysis of CLCN3 promoter methylation levels. The association between CLCN3 expression and tumor-infiltrating immune cells was investigated using various algorithms. The cBioportal database facilitated the analysis of CLCN3 mutations and mutation sites across various cancers. The Tumor-Immune System Interactions Database (TISIDB) database was employed to explore the correlation between CLCN3 expression and immune or molecular subtypes across a variety of cancer types. We collected ovarian tissue samples, encompassing both normal ovarian and OC tissues. The human OC cell lines, SKOV3 cells and OVCAR433 cells, were cultured. CLCN3 expression was determined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR), while phosphatidylinositol 3-kinase/Akt kinase (PI3K/AKT) expression was detected using Western blot. We utilized small interfering RNA (siRNA) technology to suppress CLCN3 expression. The proliferative capacity of SKOV3 and OVCAR433 cells was assessed using the Cell Counting Kit 8 (CCK-8) assay. CLCN3 demonstrated an aberrant expression in a number of cancer types and was markedly reduced in OC tissues. Poor prognosis in cervical squamous cell cancer and myeloid leukemia was linked to excessive expression of CLCN3. The examination of immune cell infiltration, which included CD8+ T cells, B cells, T regulatory cells, and cancer-associated fibroblast cells, showed a strong association with aberrant CLCN3 expression. Following the use of siRNA technology, the ability of the ovarian carcinoma cell line SKOV3 and OVCAR433 to proliferate as well as the expression of PI3K/AKT both increased. CLCN3 is a possible biomarker for immune-related processes and the prognosis of cancer, and the PI3K/AKT signaling pathway may affect OC cells' ability to proliferate.