Methylation Of Tumor Suppressor Genes Research Articles

Histone MARylation regulates lipid metabolism in colorectal cancer by promoting IGFBP1 methylation

In the global health community, colorectal cancer (CRC) is a major concern, with a high rate of incidence. Mono-ADP-ribosylation (MARylation) is a type of epigenetics and recognized as one of the causes of CRC development and progression. Although the modification level and target proteins in CRC remain unclear, it has been found that MARylation of arginine-117 of histone 3 (H3R117) promotes the proliferation, upregulates methylation of tumor suppressor gene, and is tightly associated with the metabolic processes in LoVo cells. Lipid metabolism disorder is involved in the development of CRC at the early stage. Our study revealed that MARylation of H3R117 of the LoVo cells modulated lipid metabolism, increased cholesterol synthesis, promoted lipid raft (LR) protein IGF-1R distribution, and inhibited cell apoptosis through IGFBP1. In addition, bioinformatics analyses revealed that IGFBP1 promoter was hypermethylated in CRC when compared to that in normal tissues. Moreover, H3R117 MARylation upregulated the methylation of IGFBP1 promoter through histone H3 citrullination (H3cit) by increasing the H3K9me2, heterochromatin protein1 (HP1), and DNA methyltransferase 1 (DNMT1) enrichment of IGFBP1 promoter. Accordingly, IGFBP1 may function as a tumor suppressor gene, while H3R117 MARylation may promote CRC development. Our study findings enrich the available data on epigenetics of CRC and provide a new idea and experimental basis for H3R117 MARylation as a target in CRC treatment.

Effects of fumonisin B1 on gene-specific DNA methylation related to cancer pathways of p53 signalling and PI3K/Akt signalling in human kidney cells

Abstract Fumonisin B1 (FB1), which is emerged by contamination of corn and similar products with Fusarium species, causes a high risk for human and animal health. FB1 shows hepatotoxic, nephrotoxic, neurotoxic, immunotoxic and teratogenic effects. FB1 has also been linked to carcinogenesis processes that are unrelated to genotoxic pathways. Our goal was to investigate the role of gene-specific DNA methylation and expression of tumour suppressor genes in the toxicity of FB1 in human kidney (HK-2) cells. Using array panels, effects of FB1 exposure (10-100 μM) for 24 h on methylation levels of tumour suppressor genes were investigated. ToppGene Suite (Toppfun) portal was used to perform gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and Reactome enrichment studies. The most of the genes were hypermethylated significantly at 100 μM concentrations of FB1. These results are consistent with the expression results which downregulate the selected genes of CD44, CDKN1A, CDKN2B, CTNNB1, MYC and p16. Functional enrichment analysis further elucidated the molecular mechanisms of FB1 in cancer-related pathways especially p53 signalling and PI3K/Akt signaling. It has been expected that the present study could contribute to the studies on the mechanisms that play a role in FB1 induced carcinogenesis.

Isolation of gDNA From Fecal Samples of Healthy Subjects for Assessing the Influence of Dietary Pulse Consumption on Tumor Suppressor Gene Expression and Methylation Status

Isolation of gDNA From Fecal Samples of Healthy Subjects for Assessing the Influence of Dietary Pulse Consumption on Tumor Suppressor Gene Expression and Methylation Status

Detection of dual-methylated BRCA1/BRCA2 cell-free DNA for ovarian cancer on an aptamer-based integrated microfluidic system

A new screening strategy has been developed to select nucleic acid aptamer probes with an “in vivo-like” systematic evolution of ligands by exponential enrichment (SELEX) process featuring positive, negative selection and in vivo-like selection steps. One methylation-specific aptamer with high affinity and specificity was selected and it could capture methylated cell-free DNA (cfDNA) from unfiltered blood of ovarian cancer (OvCa) patients. This aptamer was conjugated onto magnetic beads and integrated into a new microfluidic chip for rapid and automatic detection and quantification of two prevalent methylated tumor suppressor genes, breast cancer type 1 and 2 susceptibility protein (BRCA 1/2), in the molecular profile of OvCa. The assay was conducted under physiological conditions that mimic the microenvironment of cancer patients at 25–37 °C, with a low pH value (5.5) and/or high blood ion levels, without requiring additional sample preparation steps. The results of this study could be extrapolated to the direct detection of methylated cfDNA at levels as low as 1 pM in blood or plasma. Not only was DNA methylation observed in OvCa patients, but it was also detected in many other types of cancer; it is therefore considered an important factor in carcinogenesis. Hence, it is inferred that this screened aptamer should not only be used for OvCa diagnosis but also for multiple cancer epigenetic detection and drug development research in the future.

Dynamic Changes in Circulating Methylated Markers in Response to Antitumor Therapy of Rectal Cancer.

Rectal cancer (RC) occupies a leading position in the structure of oncological morbidity and mortality. Aberrant methylation of tumor-suppressor genes and hypomethylation of retrotransposons were shown to be detectable in cell-free DNA, circulating in the blood (cfDNA) of cancer patients, indicating the possibility to use them as diagnostic and prognosis markers. Evaluation of the changes in the methylation level of LINE-1 elements and SEPTIN9 and IKZF1 genes in the cell-surface-bound cfDNA (csb-cfDNA) from the blood of RC patients after antitumor therapy at a long-term follow-up. Blood samples were obtained from RC patients (n = 25) before treatment, after preoperative chemotherapy (3 courses according to the XELOX scheme), 10-15days after surgery, and every 3months during 12months of dynamic observation. The methylation level of LINE-1, SEPTIN9, and IKZF1 in the csb-cfDNA was evaluated by quantitative methyl-specific PCR. The LINE-1 methylation level in the csb-cfDNA increased 1.6 times in RC patients after chemotherapy and 3 times after tumor resection versus methylation level before therapy. The SEPTIN9 gene methylation level in the csb-cfDNA decreased by 1.7 times in RC patients after chemotherapy and by 2.3 times after tumor resection compared with the values before the treatment. The IKZF1 gene methylation level decreased by 2 times in RC patients after combined therapy. Notably, all patients with relapses (n = 5) showed an increase in methylation level for the SEPTIN9 and IKZF1 genes and a decrease of methylation level for the LINE-1 elements by 2 times or more in comparison with the level 10-15days after surgery. There were no changes in the circulating SEPTIN9, IKZF1, and LINE-1 methylation levels during the 12-month follow-up period after the combined therapy of RC patients (n = 20) without relapses. The results indicate that SEPTIN9, IKZF1, and LINE-1 methylation levels in the csb-cfDNA are potential markers of the effectiveness of antitumor therapy and early detection of relapse in RC patients.

The Effects of Physical Activity on Gene-Specific Methylation of Antioxidant and Tumour Suppressor Genes in Post-surgery Female Breast Cancer Patients Undergoing Medical Treatment

The Effects of Physical Activity on Gene-Specific Methylation of Antioxidant and Tumour Suppressor Genes in Post-surgery Female Breast Cancer Patients Undergoing Medical Treatment

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Clinical significance of tumor suppressor genes methylation in circulating tumor DNA of patients with pancreatic cancer

BackgroundCirculating tumor DNA (ctDNA) has emerged as a potential diagnostic and prognostic biomarker in various tumors. However, the role of tumor suppressor genes (TSGs) methylation in ctDNA of patients with pancreatic cancer (PC) remains largely unclear. MethodsPatients with PC (n = 43), pancreatic benign diseases (n = 39), and healthy controls (n = 20) were enrolled in the study. Quantitative analysis of methylation pattern of five candidate TSGs including NPTX2, RASSF1A, EYA2, p16, and ppENK in ctDNA was performed by next generation sequencing (NGS). The diagnostic performances of these 5-TSGs methylation were assessed by the operating characteristic (ROC) curve and clinicopathological features correlation analysis. Meanwhile, the changes in methylation levels of these 5-TSGs on the 7th postoperative day were evaluated in 23 PC patients who underwent radical resection. ResultsThe methylation levels of RASSF1A, EYA2, ppENK and p16 genes in patients with PC were significantly higher than those in healthy controls. EYA2, p16 and ppENK genes showed significantly hypermethylation in PC than those in pancreatic benign diseases. NPTX2, RASSF1A, EYA2, p16 and ppENK genes showed significantly hypermethylation in pancreatic benign diseases than those in healthy controls (P < 0.05). The methylation levels of these 5 candidate TSGs were not correlated with the tumor size, nerve invasion, lymph node metastasis and TNM stage of PC. The AUC of these biomarkers for diagnosis of PC ranged from 0.65 to 0.96. The AUC values of these methylated genes and CpG sites for differentiating malignant and benign pancreatic diseases were ranging from 0.68 to 0.92. Combined the hypermethylated genes improved the detective ability of PC than single gene. The methylation levels of NPTX2, EYA2 and ppENK genes were significantly decreased after radical resection of PC. ConclusionQuantitative analysis of methylation pattern of NPTX2, RASSF1A, EYA2, p16 and ppENK in ctDNA by NGS could be a valuable non-invasive tool for detection and monitoring of PC.

Epigenetic Regulation in Oral Squamous Cell Carcinoma Microenvironment: A Comprehensive Review

Oral squamous cell carcinoma (OSCC) is a prevalent and significant type of oral cancer that has far-reaching health implications worldwide. Epigenetics, a field focused on studying heritable changes in gene expression without modifying DNA sequence, plays a pivotal role in OSCC. Epigenetic changes, encompassing DNA methylation, histone modifications, and miRNAs, exert control over gene activity and cellular characteristics. In OSCC, aberrant DNA methylation of tumor suppressor genes (TSG) leads to their inactivation, subsequently facilitating tumor growth. As a result, distinct patterns of gene methylation hold promise as valuable biomarkers for the detection of OSCC. Oral cancer treatment typically involves surgery, radiation therapy, and chemotherapy, but even with these treatments, cancer cells cannot be effectively targeted and destroyed. Researchers are therefore exploring new methods to target and eliminate cancer cells. One promising approach is the use of epigenetic modifiers, such as DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors, which have been shown to modify abnormal epigenetic patterns in OSCC cells, leading to the reactivation of TSGs and the suppression of oncogenes. As a result, epigenetic-targeted therapies have the potential to directly alter gene expression and minimize side effects. Several studies have explored the efficacy of such therapies in the treatment of OSCC. Although studies have investigated the efficacy of epigenetic therapies, challenges in identifying reliable biomarkers and developing effective combination treatments are acknowledged. Of note, epigenetic mechanisms play a significant role in drug resistance in OSCC and other cancers. Aberrant DNA methylation can silence tumor suppressor genes, while alterations in histone modifications and chromatin remodeling affect gene expression related to drug metabolism and cell survival. Thus, understanding and targeting these epigenetic processes offer potential strategies to overcome drug resistance and improve the efficacy of cancer treatments in OSCC. This comprehensive review focuses on the complex interplay between epigenetic alterations and OSCC cells. This will involve a deep dive into the mechanisms underlying epigenetic modifications and their impact on OSCC, including its initiation, progression, and metastasis. Furthermore, this review will present the role of epigenetics in the treatment and diagnosis of OSCC.

Circulating free DNA integrity index and promoter methylation of tumor suppressor gene P16, DAPK and RASSF1A as a biomarker for oropharyngeal squamous cell carcinoma.

Circulating free DNA (cfDNA) is in use for the non-invasive diagnosis of tumors. Methylation of tumor suppressor genes (TSGs) is an early event in carcinogenesis and may serve as tumor biomarker. We have investigated cfDNA integrity and methylation of tumor suppressor genes P16, DAPK and RASSF1A in serum cfDNA of oropharyngeal squamous cell carcinoma (OPSCC) comparing paired serum and tumor tissue samples to evaluate their diagnostic use. Prospective case-control study, paired serum and tissue samples from 56 OPSCC, and 15 normal controls (NC). Sybr green Quantitate real time PCR was used for cfDNA quantification through amplification ALU 115 and 247 fragments. Promoter methylation of was analyzed in paired samples using methylation specific PCR. There was significantly high cfDNA integrity in OPSCC compared to normal control (p=<0.0001). The cfDNA integrity values were significantly higher and associated with nodal status (p=0.016). The AUC for cfDNA integrity was 0.967. The P16, DAPK and RASSF1 promoters were significantly hypermethylated in serum of OPSCC compared to NC with high concordance in tissue (up to 96.55%). The gene promoter methylation of P16 was associated with smoking (p=0.030), RASSF1A with stage (p=0.011). The combination of ALU115 with cfDNA integrity and combination of gene methylation increases diagnostic sensitivity. In followup samples the cfDNA change was not different. Liquid biopsy approach including cfDNA integrity, methylation profiling in cfDNA, in combination or separately can assist in the diagnosis of OPSCC along with radio diagnostic scan. Serum changes represent changes in tissue with very high concordance.

Tazemetostat, a selective EZH2 inhibitor, with pembrolizumab as treatment of anti-PD-1 resistant head and neck squamous-cell carcinoma (HNSCC): A phase 1-2 trial.

6022 Background: EZH2 is the catalytic subunit of PRC2, which catalyzes H3K27me3 of target gene promotors and silences transcription of several cancer-related genes. EZH2 overexpression is common in HNSCC and correlated with methylation of several tumor suppressor genes. EZH2 siRNA downregulated EZH2 mRNA and protein and reduced colony formation in an EZH2-expressing HNSCC cell line. EZH2 inhibitors decreased H3K27me3 in HPV + and - cell lines. Tazemetostat, an EZH2 inhibitor, upregulated expression of HLA class I molecules in anti-PD-1-resistant HNSCC cell lines and mouse models, and increased antigen-specific CD8+ T cell proliferation, IFN-γ production, CXCL10 expression, and tumor cytotoxicity. In an anti-PD-1-resistant HNSCC model, tazemetostat and an anti-PD-1 suppressed tumor growth. The primary aims of this phase 1-2 trial were to establish 1) the recommended phase 2 dose (RP2D) of tazemetostat given with pembrolizumab and 2) the objective response rate of this regimen among patients (pts) with anti-PD-1 resistant, PD-L1 positive HNSCC. We report the results of Phase 1. Methods: In phase 1, eligible pts had recurrent/metastatic (RM) HNSCC, adequate performance status and organ function, and ability to swallow study drug. A 3+3 dose-escalation phase 1 design was used to assess 3 dose-levels of tazemetostat (400, 600, and 800 mg orally twice daily) with pembrolizumab (200 mg IV). Cycle 1 was 35 days, with tazemetostat given on days 1-35 and pembrolizumab on day 15. Subsequent cycles were 21 days, with tazemetostat given on days 1-21 and pembrolizumab on day 1. Dose-limiting toxicity (DLT), assessed during cycle 1, was defined as grade 4 neutropenia/thrombocytopenia, grade 3 febrile neutropenia, or study drug-related grades 3-4 non-hematologic adverse events (AEs). Pts must have completed cycle 1 to be evaluable for DLT assessment; otherwise, an equal number of additional pts were enrolled. The RP2D was defined as the highest dose level in which 0 of the first 3 or &lt;1 of 6 pts experienced a DLT. Results: 12 pts enrolled to phase 1: 3 each on the 400 and 600 mg dose-levels of tazemetostat and 6 on the 800 mg dose-level. Key tumor characteristics included primary site (larynx: 4, oral cavity: 3, oropharynx: 3, nasopharynx: 1, cutaneous: 1), HPV status (+: 1, -: 11), and number of lines of prior systemic treatment for RM disease (1-2: 6, &gt;3: 6). Nine pts were evaluable for DLT; 3 pts on the 800 mg dose-level did not complete cycle 1 and were not evaluable for DLT. DLT events and grade 4-5 AEs did not occur. Grade 3 AEs that occurred after cycle 1 included anemia (2 pts, both at 800 mg dose) and fatigue (1, at 400 mg dose). Across all cycles, the most common AEs included anemia (10 pts), fatigue (9), rash (7), and myalgia (4). Conclusions: Among pts with RM-HNSCC, the RP2D of tazemetostat was 800 mg twice daily when given with pembrolizumab. Enrollment to phase 2 is ongoing. Clinical trial information: NCT04624113 .

Methylated tumor suppressor gene SCARA5 inhibits the proliferation, migration and invasion of nasopharyngeal carcinoma.

Background: SCARA5 may play an important role in nasopharyngeal carcinoma. Materials & methods: PCR and immunohistochemistry were used to detect the expression and promoter methylation of SCARA5. Cell proliferation assays, spheroid culture, flow cytometry analysis, Transwell assays and xenotransplantation tests were utilized to determine the functional effects of SCARA5. RNA-sequencing, western blotting, immunofluorescence and dual-luciferase reporter assays were used to assess SCARA5-mediated outcomes. Results: SCARA5 was downregulated by promoter methylation. Overexpression of SCARA5 inhibited cell migration, invasion and proliferation. SCARA5 enhanced nasopharyngeal carcinoma cell sensitivity to chemotherapy with cisplatin and 5-fluorouracil. SCARA5 drives tumor apoptosis by downregulating HSPA2. Conclusion: SCARA5 may be a useful clinical marker innasopharyngeal carcinoma.

The Mechanism of DNA Methylation and miRNA in Breast Cancer.

Breast cancer is the most prevalent cancer in the world. Currently, the main treatments for breast cancer are radiotherapy, chemotherapy, targeted therapy and surgery. The treatment measures for breast cancer depend on the molecular subtype. Thus, the exploration of the underlying molecular mechanisms and therapeutic targets for breast cancer remains a hotspot in research. In breast cancer, a high level of expression of DNMTs is highly correlated with poor prognosis, that is, the abnormal methylation of tumor suppressor genes usually promotes tumorigenesis and progression. MiRNAs, as non-coding RNAs, have been identified to play key roles in breast cancer. The aberrant methylation of miRNAs could lead to drug resistance during the aforementioned treatment. Therefore, the regulation of miRNA methylation might serve as a therapeutic target in breast cancer. In this paper, we reviewed studies on the regulatory mechanisms of miRNA and DNA methylation in breast cancer from the last decade, focusing on the promoter region of tumor suppressor miRNAs methylated by DNMTs and the highly expressed oncogenic miRNAs inhibited by DNMTs or activating TETs.

Methylation study of tumor suppressor genes in human aberrant crypt foci, colorectal carcinomas, and normal colon.

Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls. In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed. %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation. Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.

Promoter methylation levels of RASSF1 and ATIC genes are associated with lung cancer in Iranian patients.

Epigenetic alterations like methylation of tumor suppressor genes or oncogenes, in respiratory epithelium have been associated with lung cancer. Hypermethylation of genes promoter is an epigenetic event, and is responsible to tumor suppressor genes inactivation as well as oncogenes activation. This study aimed to assess the role of methylation status in promoter of RASSF1 and ATIC genes their potential implication in the pathogenesis of lung tumor in Iranian patients. In this study, we collected 100 tissue samples (50lung cancer tissues and 50 adjacent non-cancerous lung tissues) from Iranian lung cancer patients. The genomic DNA was extracted, and methylation status of both RASSF1 andATIC genes was investigated by methylation-sensitive high-resolution melting (MS-HRM) assay technique and Real-Time PCR. Cancer Genome Atlas (TCGA) dataset wasalso analyzed for further validation of the gene's methylation. Methylation of RASSF1 gene promoter was significantly higher in lung tumor tissues. However, promoter methylation levels of ATIC gene was significantly lower in lung tumor tissues. These results were additionally confirmed by TCGA analysis. Promoter methylation of both RASSF1 and ATIC genes was significantly associated with lymph node metastasis, and clinical stage of lung cancer. Thereceiver operating characteristic (ROC) curve analysis indicated a high accuracy of promoter methylation in these genes as a diagnostic biomarker for lung cancer. Methylation levels of both RASSF1 and ATIC genes promoters were associated with lung cancer pathogenesis in Iranian population, and may be a suitable biomarker for diagnosis and prognosis of lung cancer in early stage of tumorigenesis.

Global and promoter specific hypermethylation of tumor suppressor genes P16, SOCS1, and SHP1 in oral squamous cell carcinoma and oral submucous fibrosis.

Aberrant methylation pattern leads to altered gene expression, that is, involved in the transformation of various cancers, including oral squamous cell carcinoma (OSCC). In the present study, an attempt has been made to examine the association of global and promoter-specific methylation of tumor suppressor genes in patients with OSCC and oral submucous fibrosis (OSMF). Promoter-specific methylation of tumor suppressor genes P16, SOCS1, and SHP1 had been studied earlier for their aberrant methylation patterns in other cancers; however, these studies were mainly conducted in-vitro or in animal models, and as such, only a few studies are available on human samples. In the present study evaluation of promoter-specific methylation of genes P16, SOCS1, and SHP1 in 76 patients' blood and tissue samples was done and compared with methylation of 35 healthy control samples using qPCR. Further, these samples were analyzed for global methylation patterns using ELISA. The results have shown a significant decreasing trend of promoter methylation (OSCC > OSMF > Controls); the methylation indices (MI) were significantly higher in OSCC than in the controls. The median MI of three genes for OSCC were P16MI (0.96), SHP1MI (0.79), and SOCS1 (0.80). Similarly, median MIs for OSMF were P16MI (0.18), SHP1 MI (0.19), and SOCS1 MI (0.5) against controls with MI (0) for each of the three genes. The global methylation %mC values were 1.9, 0.5, and 0.1, respectively. The values of MI and %mC were found to correlate with various risk factors such as tobacco, smoking, and alcohol consumption, which are positively involved in OSMF pathogenesis followed by oral cancer progression. Further, the methylation trend in tissue was reflected in blood samples, proving a window for methylation load to be used as a lesser invasive biomarker. The sensitivity and specificity of methylation load were also found reasonable. Therefore, the current study suggests that there may be a role of global and promoter-specific methylation load in the transition of OSMF to OSCC.

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.

Constitutional BRCA1 Methylation and Risk of Incident Triple-Negative Breast Cancer and High-grade Serous Ovarian Cancer

About 25% of all triple-negative breast cancers (TNBCs) and 10% to 20% of high-grade serous ovarian cancers (HGSOCs) harbor BRCA1 promoter methylation. While constitutional BRCA1 promoter methylation has been observed in normal tissues of some individuals, the potential role of normal tissue methylation as a risk factor for incident TNBC or HGSOC is unknown. To assess the potential association between white blood cell BRCA1 promoter methylation and subsequent risk of incident TNBC and HGSOC. This case-control study included women who were participating in the Women's Health Initiative study who had not received a diagnosis of either breast or ovarian cancer before study entrance. A total of 637 women developing incident TNBC and 511 women developing incident HGSOC were matched with cancer-free controls (1841 and 2982, respectively) in a nested case-control design. Cancers were confirmed after central medical record review. Blood samples, which were collected at entry, were analyzed for BRCA1 promoter methylation by massive parallel sequencing. The study was performed in the Mohn Cancer Research Laboratory (Bergen, Norway) between 2019 and 2022. Associations between BRCA1 methylation and incident TNBC and incident HGSOC were analyzed by Cox proportional hazards regression. Of 2478 cases and controls in the TNBC group and 3493 cases and controls in the HGSOC group, respectively, 7 (0.3%) and 3 (0.1%) were American Indian or Alaska Native, 46 (1.9%) and 30 (0.9%) were Asian, 1 (0.04%) and 1 (0.03%) was Native Hawaiian or Pacific Islander, 326 (13.2%) and 125 (3.6%) were Black or African, 56 (2.3%) and 116 (3.3%) were Hispanic, 2046 (82.6%) and 3257 (93.2%) were White, and 35 (1.4%) and 35 (1.0%) were multiracial. Median (range) age at entry was 62 (50-79) years, with a median interval to diagnosis of 9 (TNBC) and 10 (HGSOC) years. Methylated BRCA1 alleles were present in 194 controls (5.5%). Methylation was associated with risk of incident TNBC (12.4% methylated; HR, 2.35; 95% CI, 1.70-3.23; P < .001) and incident HGSOC (9.4% methylated; HR, 1.93; 95% CI, 1.36-2.73; P < .001). Restricting analyses to individuals with more than 5 years between sampling and cancer diagnosis yielded similar results (TNBC: HR, 2.52; 95% CI, 1.75-3.63; P < .001; HGSOC: HR, 1.82; 95% CI, 1.22-2.72; P = .003). Across individuals, methylation was not haplotype-specific, arguing against an underlying cis-acting factor. Within individuals, BRCA1 methylation was observed on the same allele, indicating clonal expansion from a single methylation event. There was no association found between BRCA1 methylation and germline pathogenic variant status. The results of this case-control suggest that constitutional normal tissue BRCA1 promoter methylation is significantly associated with risk of incident TNBC and HGSOC, with potential implications for prediction of these cancers. These findings warrant further research to determine if constitutional methylation of tumor suppressor genes are pancancer risk factors.

SHF Acts as a Novel Tumor Suppressor in Glioblastoma Multiforme by Disrupting STAT3 Dimerization.

Sustained activation of signal transducer and activator of transcription 3 (STAT3) is a critical contributor in tumorigenesis and chemoresistance, thus making it an attractive cancer therapeutic target. Here, SH2 domain‐containing adapter protein F (SHF) is identified as a tumor suppressor in glioblastoma Multiforme (GBM) and its negative regulation of STAT3 activity is characterized. Mechanically, SHF selectively binds and inhibits acetylated STAT3 dimerization without affecting STAT3 phosphorylation or acetylation. Additionally, by blocking STAT3‐DNMT1 (DNA Methyltransferase 1) interaction, SHF relieves methylation of tumor suppressor genes. The SH2 domain is documented to be essential for SHF's actions on STAT3, and almost entirely replaces the functions of SHF on STAT3 independently. Moreover, the peptide C16 a peptide derived from the STAT3‐binding sites of SHF inhibits STAT3 dimerization and STAT3/DNMT1 interaction, and achieves remarkable growth inhibition in GBM cells in vitro and in vivo. These findings strongly identify targeting of the SHF/STAT3 interaction as a promising strategy for developing an optimal STAT3 inhibitor and provide early evidence of the potential clinical efficacy of STAT3 inhibitors such as C16 in GBM.