P16 Methylation Research Articles

Epigenetic Characteristics in Primary and Recurrent Glioblastoma-Influence on the Clinical Course.

Epigenetic tumor characteristics are in focus for glioblastoma prognosis. This raises the question if these characteristics present with stable expression during the progression of the disease, and if potential temporal instability might influence their prognostic value. A total of 44 patients suffering from glioblastoma who were treated for their primary and relapse tumors were included in the study. Tumor specimens from the initial and recurrent tumor resection were subjected to evaluation of MGMT, p15, and p16 methylation statuses. MiRNA-21, -24, -26a, and -181d expression was evaluated as well. The stability of these epigenetic markers during the progression of the disease was correlated with further clinical data. A Cancer Genome Atlas (TCGA) dataset of 224 glioblastoma patients was used as an independent cohort to validate the results. Instability was observed in all examined epigenetic markers. MGMT methylation changed in 30% of patients, p15 methylation changed in 35%, and p16 methylation changed in 37.5% of cases. MiRNA expression in corresponding initial and relapse tumor specimens varied considerably in general, individual cases presented with a stable expression. Patients with a decreased expression of miRNA-21 in their recurrence tumor showed significantly longer overall survival. These results are supported by the data from TCGA indicating similar results. Epigenetic characteristics may change during the course of glioblastoma disease. This may influence the prognostic value of derived molecular markers.

Investigation of Genetic Markers for Predicting Oral Cancer Progression and Patient Outcomes.

Genomic methylation being used as a sensitive indicator for identifying oral potentially malignant disorders (OPMDS) and oral cancer could be a viable option. DNA methylation is a potential approach for "cancer therapeutics" and can help researchers truly understand the genomic mechanism that leads to cancer. Investigation of genetic markers for predicting oral cancer progression and patient outcomes. One hundred and twelve patients having oral premalignant lesions and malignant lesions were included in this study. Saliva samples were obtained for analysis of the expression of genetic markers, such as p16, DAP-K, and MGMT. The Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc., USA) would be used to assess genome-wide DNA methylation according to the manufacturer's instructions. Methylation of all the genes, that is, p16, DAP-K, and MGMT, was observed in 12.7% of oral cancer lesions. 41.94% of oral cancer lesions were found to have methylation of two genes. 13.23% cases were found to be associated with methylation of p16 and DAP-K, 12.12% cases were found to have methylation in the p16 and MGMT, and 15.27% cases of oral cancer lesions showed methylation of DAP-K and MGMT. Genetic markers, such as p53, DAP-K, and MGMT, can be used for predicting oral cancer progression and patient outcomes.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

LncRNA DCST1-AS1 drives the malignant progression of pediatric acute myeloid leukemia via regulating p53 methylation.

To uncover the biological role of lncRNA DCST1-AS1 in the process of pediatric AML and its regulatory effect on p53 methylation. Serum level of DCST1-AS1 in AML children and healthy participants was detected by qRT-PCR. The role of DCST1-AS1 in mediating biological functions of AML193 and U937 cells was assessed by functional experiments. p53 methylation level was examined. Through BSP, MSP and dual-luciferase reporter assay, the regulatory effect of DCST1-AS1 on p53 methylation was explored. The correlation between DCST1-AS1 and p53 in the serum of AML children was assessed. Serum level of DCST1-AS1 was higher in AML children than in healthy subjects. Knockdown of DCST1-AS1 decreased proliferative and migratory rates in AML193 and U937 cells. DCST1-AS1 was able to induce methylation of p53 promoter. P53 was markedly upregulated by the knockdown of DCST1-AS1, presenting a negative correlation. LncRNA DCST1-AS1 drives the malignant progression of pediatric AML through inducing methylation of the p53 promoter.

Downregulated lncRNA HOTAIR inhibits the proliferation of granulosa cells in endometriosis by upregulating p21.

This study was carried out to elucidate the biological function of HOTAIR in granulosa cells of endometriosis and the underlying mechanism. Granulosa cells were extracted from endometriosis patients and subjects with fallopian tube factor alone who received IVF-ET. Relative levels of HOTAIR and p21 in the extracted granulosa cells were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, HOTAIR level in endometriosis patients in stage I-II or III-IV was determined. Regulatory effects of HOTAIR on the proliferation of KGN cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 5-Ethynyl-2'- deoxyuridine (EdU) and colony formation assay. Flow cytometry was conducted to evaluate the potential influence of HOTAIR on apoptosis of KGN cells. The interaction between HOTAIR and EZH2, SUV12 was detected by RNA binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assay. Finally, the potential role of the HOTAIR/p21 axis in mediating cellular behaviors of KGN cells was explored. HOTAIR was downregulated in granulosa cells extracted from endometriosis patients relative to those with fallopian tube factor alone who received IVF-ET. Knockdown of HOTAIR suppressed the proliferative ability and induced apoptosis of KGN cells. RIP and ChIP assay showed that silence of HOTAIR released EZH2 to suppress the DNA methylation of p21. Knockdown of p21 could reverse the regulatory effect of HOTAIR on the proliferative change of KGN cells. Downregulated HOTAIR suppresses the proliferative ability and induces apoptosis of granulosa cells in endometriosis by upregulating p21.

Elucidating the role of nicotinamide N-methyltransferase-p53 axis in the progression of chronic kidney disease.

Chronic kidney disease (CKD) is a significant global health issue characterized by progressive loss of kidney function. Renal interstitial fibrosis (TIF) is a common feature of CKD, but current treatments are seldom effective in reversing TIF. Nicotinamide N-methyltransferase (NNMT) has been found to increase in kidneys with TIF, but its role in renal fibrosis is unclear. Using mice with unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblast cells (NRK-49F) stimulated with transforming growth factor-β1 (TGF-β1), we investigated the function of NNMT in vivo and in vitro. We performed single-cell transcriptome sequencing (scRNA-seq) on the kidneys of mice and found that NNMT increased mainly in fibroblasts of UUO mice compared to sham mice. Additionally, NNMT was positively correlated with the expression of renal fibrosis-related genes after UUO injury. Knocking down NNMT expression reduced fibroblast activation and was accompanied by an increase in DNA methylation of p53 and a decrease in its phosphorylation. Our findings suggest that chronic kidney injury leads to an accumulation of NNMT, which might decrease p53 methylation, and increase the expression and activity of p53. We propose that NNMT promotes fibroblast activation and renal fibrosis, making NNMT a novel target for preventing and treating renal fibrosis.

ABERRANT METHYLATION OF CANCER-RELATED GENES IN VIETNAMESE BREAST CANCER PATIENTS: ASSOCIATIONS WITH CLINICOPATHOLOGICAL FEATURES.

Epigenetic alteration is one of the most common molecular changes identified in the progression of breast cancer (BC). To study the frequency and relation between methylation of BRCA1, MLH1, MGMT, GSTP1, APC, RASSF1A, p16, WIF, and EGFR and the clinicopathological features in Vietnamese BC patients. Methylation-specific polymerase chain reaction (MS-PCR) and SPSS 20.0 software were utilized in order to identify methylated frequency as well as evaluate its relationship with the patient's clinical features. In 162 BC cases, the methylation rates of the selected genes were 53.7%, 22.8%, 38.9%, 34.6%, 29.0%, 46.3%, 20.4%, 18.5%, and 28.4% respectively. In 32 cases of benign breast diseases (BBD) - 12.5%, 15.6%, 6.3%, 3.1%, 12.5%, 21.9%, 3.1%, 15.6% and 3.1%. BC samples displayed higher BRCA1, MGMT, GSTP1, APC, RASSF1A, WIF1, and p16 methylation levels than BBD samples (p < 0.001). Hypermethylation of BRCA1, GSTP1, and RASSF1A was predominant in the invasive ductal carcinoma, while hypermethylation of BRCA1, GSTP1, RASSF1A, WIF-1, and p16 was found to significantly correlate with lymph node metastasis (p < 0.05). Hypermethylation of BRCA1, MGMT, and GSTP1 was more common in stage III (p < 0.05) than in stages I/II, whereas MLH1 methylation was predominant in stage I and APC methylation was less common in stage III (p = 0.03). In addition, methylation of RASSF1A and EGFR was more frequent in younger patients (p < 0.01) than in elder patients. These data suggest that a gene panel (BRCA1/MGMT/GSTP1) can be used to support the diagnosis and screening of Vietnamese patients' BC with a sensitivity of 70%, and a specificity of 85%.

P11.23.A GLIOBLASTOMA MOLECULAR CORRELATES OF SEIZURE OCCURRENCE/RECURRENCE AFTER SURGERY: THE ROLE OF P53 MUTATION

Abstract BACKGROUND The integration of molecular data and histology changed the diagnostic approach and prognostic characterization for patients with brain tumors. The aim of this study is identify molecular risk factors associated with seizure occurrence and/or recurrence after surgery in patients with brain glioblastoma. MATERIAL AND METHODS Patients with brain glioblastoma were consecutively enrolled and underwent brain surgery, followed by histopathological and molecular evaluation. Immuno-histochemical, molecular (synaptophysin, GFAP, ATRX, p53, ki67 index and MGMT gene promoter methylation) and clinical (age, sex and seizure occurrence/recurrence before and/or after surgery) data were collected. Histopathological and molecular features were compared between patients with and without seizure occurrence/recurrence after surgery. A step-backward logistic regression was performed to investigate the association between molecular features and seizure occurrence/recurrence. RESULTS 100 patients (age 59.51±1.23, 65 males) were enrolled. Forty-nine patients had seizures before surgery. After surgery, 40 patients (age 59.43±1.64, 27 males) had seizure occurrence/recurrence, while 60 (age 59.57±1.75, 38 males) had not. Patients with epilepsy (i.e., seizures before and/or after surgery) had lower synaptophysin (33.33%, p=0.008) and higher p53 (85%, p=0.038) mutation rate compared with patients without epilepsy. In the whole sample, seizure occurrence/recurrence after surgery was significantly associated with p53 mutation (OR: 3.9, CI 1.12-13.50, p=0.032) and seizure occurrence before surgery (OR: 5.3, CI 2.15-13.08, p&amp;lt;0.001). In patients without seizures before surgery, p53 mutation perfectly predicted seizure occurrence, therefore the OR was not computable. In fact, all patients without p53 mutation didn’t have seizure after surgery, while all patients that had seizure after surgery, also had p53 mutation (p=0.028). However, to note 71% patients with p53 mutation didn’t have seizure after surgery. CONCLUSION The p53 mutation is related with a high risk of seizure occurrence/recurrence after surgery in brain glioblastoma, even in patients that didn’t have seizures before surgery. Conversely, the absence of p53 mutation appears to protect against the occurrence of seizures. If confirmed, this result may help in the management of anti-seizure treatment after surgery in brain glioblastoma patients.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Organochlorine pesticides and epigenetic alterations in thyroid tumors.

Cancer incidence depends on various factors e.g., pesticide exposures which cause epigenetic alterations. The present research aimed to investigate the organochlorine pesticides (OCPs) impacts on promoter methylation of three tumor-suppressor genes and four histone modifications in thyroid nodules in 61 Papillary thyroid carcinoma (PTC) and 70 benign thyroid nodules (BTN) patients. OCPs were measured by Gas chromatography. To identify promoter methylation of TSHR, ATM, and P16 genes, the nested-methylation-specific PCR (MSP) was utilized, and histone lysine acetylation (H3K9, H4K16, and H3K18) and lysine methylation (H4K20) were detected by performing western blot analysis. Further TSHR methylation and less P16 methylation were observed in PTC than in BTN. No substantial difference was detected for ATM methylation between PTC and BTN groups. Also, OCP dramatically increased the odds ratio of TSHR (OR=3.98, P=0.001) and P16 (OR=5.65, P<0.001) methylation while confounding variables reduced the chances of ATM methylation arising from 2,4-DDE and 4,4-DDT influence. Hypomethylation of H4K20 and hypo-acetylation of H3K9, H4K16, and H3K18 (P<0.001) were observed in PTC samples than BTN. Furthermore, OCPs substantially decreased the odds ratio of H3K9 (OR=3.68, P<0.001) and H4K16 (OR=6.03, P<0.001) acetylation. The current research indicated that OCPs could contribute to PTC progression by TSHR promoter hypermethylation and decreased acetylation of H3K9 and H4K16. In addition, in PTC patients, assessing TSHR promoter methylation and acetylation of H3K9 and H4K16 could have predictive values.

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.

Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria

ABSTRACT Background Urothelial bladder carcinoma (UBC) is usually detected during work-up for hematuria. Cystoscopy and/or contrast-enhanced imaging are the gold standard tools for UBC diagnosis, despite limited by being invasive, expensive and low yield in small flat tumors. Objectives To assess the diagnostic performance of urine-based DNA methylation of six genes (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in patients with hematuria. Patients and methods Voided urine was collected from consecutive patients presented with hematuria for urine cytology and DNA methylation assay of the assigned genes using methylation-specific Polymerase Chain Reaction (PCR). Further assessment by office cystoscopy and imaging with subsequent inpatient cystoscopic biopsy for positive findings was done. The diagnostic characteristics of DNA methylation and urine cytology were assessed based on its capability to predict UBC. Results We included 246 patients in the study with identified macroscopic hematuria in 204 (82.9%) patients. Positive cytology was found in 78 (31.7%) patients. DNA methylation of GATA4, P16, P14, APC, CDH1 and CD99 genes was identified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 (43.1%), 90 (36.6%) and 71 (28.9%) patients, respectively. The sensitivity of the assigned genes for UBC detection ranges from 35% (95%CI: 31–39) to 83% (95%CI: 79–87). Optimal specificity (SP) (100%) was noted for P16, APC and CDH1 genes. While for the other genes (GATA4, P14 and CD99), the SP was 95% (95%CI: 92–98), 96% (95%CI: 92–99) and 97% (95%CI: 93–99), respectively. On multivariate logistic regression analysis, all genes exclusively demonstrated independent prediction of UBC. On receiver operator characteristic (ROC) analysis, all tested genes methylation showed superior area under the curve (AUC) when compared to urine cytology. Conclusions We have developed a novel urine-based DNA methylation assay for detection of UBC in patients with hematuria with superior diagnostic performance and independent predictive capacity over urine cytology.

Helicobacter pylori and Epstein-Barr Virus Co-Infection in Gastric Disease: What Is the Correlation with p53 Mutation, Genes Methylation and Microsatellite Instability in a Cohort of Sicilian Population?

Genetic predisposition, environmental factors, and infectious agents interact in the development of gastric diseases. Helicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection has recently been shown to be correlated with these diseases. A cross-sectional study was performed on 100 hospitalized Italian patients with and without gastric diseases. The patients were stratified into four groups. Significant methylation status differences among CDH1, DAPK, COX2, hMLH1 and CDKN2A were observed for coinfected (Hp-EBV group) patients; particularly, a significant presence of COX2 (p = 0.0179) was observed. For microsatellite instability, minor stability was described in the Hp-HBV group (69.23%, p = 0.0456). Finally, for p53 mutation in the EBV group, exon 6 was, significantly, most frequent in comparison to others (p = 0.0124), and in the Hp-EBV group exon 8 was, significantly, most frequent in comparison to others (p < 0.0001). A significant positive relationship was found between patients with infection (Hp, EBV or both) and p53 mutation (rho = 0.383, p = 0.0001), methylation status (rho = 0.432, p < 0.0001) and microsatellite instability (rho = 0.285, p = 0.004). Finally, we observed among infection and methylation status, microsatellite instability, and p53 mutation a significant positive relationship only between infection and methylation status (OR = 3.78, p = 0.0075) and infection and p53 mutation (OR = 6.21, p = 0.0082). According to our analysis, gastric disease in the Sicilian population has different pathways depending on the presence of various factors, including infectious agents such as Hp and EBV and genetic factors of the subject.

The intra-tumoral heterogeneity in glioblastoma - a limitation for prognostic value of epigenetic markers?

Epigenetic tumor features are getting into focus as prognostic markers in glioblastoma. Whether intra-tumoral heterogeneity in these epigenetic characteristics may influence prognostic value remains unclear. Of 154 patients suffering from glioblastoma, 120 patients served as reference collective, while 34 patients were compiled as test collective. MGMT, p15, and p16 promoter methylation and miRNA expression levels (miRNA-21, miRNA-24, miRNA-26a, and miRNA-181d) were measured in each tumor specimen. Serving as a statistical baseline, epigenetic heterogeneity between tumors (inter-tumoral) was estimated within a triplet of three tumor specimens from three different reference patients. For estimation of epigenetic heterogeneity within a tumor (intra-tumoral), previous results were compared to three tumor specimens within one glioblastoma of patients of the test collective. Resulting levels of heterogeneity were then correlated with survival and validated by an external TCGA data set. Heterogeneity in MGMT promoter methylation occurred less likely in the test group compared to the reference group. No difference in heterogeneity was observed between test and reference group regarding p15 and p16 methylation. Intra-tumoral heterogeneity within the test group regarding miRNA-21, miRNA-24, miRNA-26a, and miRNA-181d expression was not distinguishable from inter-tumoral heterogeneity. A homogenously increased miRNA-21 expression was associated with reduced overall survival in the test collective. The findings could be validated by comparison with TCGA datasets. Heterogeneity of epigenetic characteristics in one glioblastoma may be of the same magnitude as heterogeneity between different patients. Not only the extent of epigenetic characteristics but also the extent of intra-tumoral heterogeneity may influence survival in glioblastoma.

Abstract 6528: HPV status and epigenetic profiling of saliva from a cancer-free population

Abstract Identifying environmental factors and their relationship with epigenetic alterations is essential for understanding cancer, a leading cause of death. Currently, cancer screening and early detection development efforts are ongoing and epigenetics-based tests are one of the top choices as strategies. The understanding of how these alterations behave in cancer-free (CF) populations with well annotated data is essential. We randomly selected 601 saliva specimens from two Brazilian countrywide epidemiological studies POP-Brazil and SMESH. Extracted DNAs were sodium bisulfite converted and analyzed by Quantitative Methylation-Specific PCR (QMSP). Five genes previously reported as DNA methylation prone in cancer were selected (ITGA4, EDRNB, PAX5, p16, CCNA1). All participants were interviewed and provided sociodemographic and sexual behavior information as well as genital (gn) and oral biological samples. Variables were categorized and Fisher’s exact test was performed using SAS 9.4. Surprisingly, methylation in 3 cancer associated genes showed considerable frequencies in this CF group. DNA methylation was observed for EDNRB in 75/601 samples (12.5%), PAX5 in 55/601 (9.2%), and ITGA4 in 20/601 (3.3%). 12 samples displayed concomitant methylation on 2 of these 3 genes. p16 and CCNA1 methylation were only present in 1 sample each. Preliminary analyses suggest that specific genes methylation are observed with the presence of low (LR) and high-risk (HR) HPV types. EDNRB methylation groups with the presence of HR HPV type 16, 31, 39, 51, 58 and 59 from gn samples and LR types 6 and 42. ITGA4 methylation and HR HPV types 16 and 59 were detected in oral samples, and types 52, 56 and 68 in gn samples, as well as LR HPV types 40 and 42. In people with early onset of smoking, EDNRB and PAX5 seem to be more frequently methylated. The positivity of PAX5 was higher in participants who smoke, use alcohol and presented a positive diagnosis of syphilis and/or HIV. PAX5 methylation was more frequent in HR HPV 33, 58, 68 and 62 and LR type 6. Further analyses are ongoing. Our study shows methylation in cancer related genes in a CF population. The variables collected include some proven cancer risk factors and others still unknown, thus lead us to uncover directions to identify main cancer related players in a Brazilian population. The presence of cancer related alterations may indicate premalignant stages, exposure and lifestyle consequences, both at the same time or separately. In our pilot analysis, different types of HPV seem to correlate with epigenetic changes, this could bring attention to less studied HPV types and their role in carcinogenesis. Additional analyses of the current data and larger prospective, population-based studies, assessing the use of salivary DNA methylation in cancer-free and cancer groups of subjects are needed to explore the current findings and dissect the potential of saliva as a means of tracking cancer related changes. Citation Format: Ana Paula Muterle Varela, Dieila Giomo De Lima, Laura Palmieri, Juliana Comerlato, Fernando Hayashi Sant’Anna, Isabel C Bandeira, Marina Bessel, Pedro Isaacsson Velho, Eliana M. Wendland, Mariana Brait. HPV status and epigenetic profiling of saliva from a cancer-free population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6528.

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.

Associations between DNA methylation and genotoxicity among lead-exposed workers in China

Studies have shown that lead (Pb) exposure caused genotoxicity, however, the underlying mechanisms remain unclear. A mechanism may be via DNA methylation which is one of the most widely studied epigenetic regulations for cellular activities. Whether this is involved in Pb-induced genotoxicity has rarely been studied. Our study aimed to examine whether DNA methylation was associated with Pb exposure and genotoxicity, and to explore its potential mediating roles. A total of 250 Pb-exposed workers were enrolled. Blood lead levels (BLLs) and genotoxic biomarkers (Micronuclei and Comet) were analyzed. Methylation levels at CpG sites of LINE1 and Alu and promoter region of P53, BRCA1, TRIM36 and OGG1 were measured by pyrosequencing. Generalized linear model (GLM) combined with restricted cubic splines (RCS) were used to analyze relationships between Pb exposure, DNA methylation and genotoxicity. Mediation effect was used to explore mediating roles of DNA methylation. The distribution of BLLs was right-skewed and showed wide ranges from 23.7 to 636.2 μg/L with median (P25, P75) being 218.4 (106.1, 313.9) μg/L among all workers. Micronuclei frequencies showed Poisson distribution [1.94 ± 1.88‰] and Comet tail intensity showed normal distribution [1.69 ± 0.93%]. GLM combined with RCS showed that Alu methylation was negatively associated with BLLs, while P53 and OGG1 methylation were positively associated with BLLs. Micronuclei were negatively associated with Alu and TRIM36 methylation but positively with P53 methylation. Comet was positively associated with P53 and BRCA1 methylation. Mediation effect showed that Alu methylation mediated 7% effects on association between Pb exposure and micronuclei, whereas, P53 methylation mediated 14% and BRCA1 mediated 9% effects on association between Pb exposure and Comet. Our data show that Pb exposure induced changes of global and gene-specific DNA methylation which mediated Pb-induced genotoxicity.

Ginsenoside Rg3 inhibits renal cell carcinoma cell migration, invasion, colony formation, and tube formation and enhances apoptosis through promoting the DNA demethylation and histone acetylation.

This study explored the effect and mechanism of Rg3 on renal cell carcinoma (RCC) progression. RCC cells were treated with different concentrations of Rg3, 5-Aza-dc (a methyltransferase inhibitor) or TSA (a deacetylase inhibitor). Rg3-induced cytotoxicity, migration, invasion, colony formation, tube formation and apoptosis of RCC cells were evaluated by CCK-8, wound healing, Transwell, colony formation, tube formation and flow cytometry assays, respectively. Methylation and expressions of p53, p21 and p16, and expressions of methylation-related genes and histone deacetylases and histone acetylation-related genes (H3 (acetyl K14), H3 (acetyl K9), H4 (acetyl K12), H4 (acetyl K5) and H4 (acetyl K16)) were analysed by qRT-PCR and western blot. Rg3 dose-dependently decreased the viability, inhibited migration, invasion, colony formation and tube formation, and enhanced apoptosis of RCC cells. Rg3 enhanced the demethylation levels and expressions of p53, p21 and p16 as well as the expressions of histone acetylation-related genes, but repressed the expressions of methylation-related genes and histone deacetylases. Rg3 had the same effect as 5-Aza-dc and TSA did on the above-mentioned cellular changes. Rg3 restrains RCC cell migration, invasion, colony formation and tube formation, yet enhances apoptosis through promoting demethylation of p53, p21 and p16, and histone acetylation.

Contribution of DNA methylation to the risk of hepatitis C virus-associated hepatocellular carcinoma: A meta-analysis.

DNA methylation is a crucial epigenetic modification in hepatocellular carcinoma (HCC), and hepatitis C virus (HCV) can induce hepatocarcinogenesis. Nevertheless, the interaction mechanism between DNA methylation and HCV infection in HCC is still ambiguous. In this study, we performed a comprehensive meta-analysis to assess the contribution of DNA methylation in HCV-associated HCC. After four steps of literature screening, we finally obtained 33 qualified case-control studies for this meta-analysis. These studies consisted of 587 HCV-positive cancer tissues and 326 HCV-negative cancer tissues. Our results revealed that four genes (p16, GSTP1, APC, and RUNX3) were more hypermethylated in the HCV-positive liver cancer tissues than in the HCV-negative liver cancer tissues. In addition, the p16 gene was more hypermethylated in the HCV-positive paracancerous tissues than in the HCV-negative paracancerous tissues. Subgroup meta-analysis by geographical populations showed that p16 methylation was significantly higher in HCV-positive cancerous tissues from Japanese and Chinese. Besides, p16 methylation was significantly higher among patients (>60 years) but not among the others (≤60 years). However, there was no obvious association between DNA methylation and other clinicopathological characteristics, including gender, tumor size, differentiation, and clinical stage. Our study suggested that DNA methylation could become potential biomarkers for HCV-associated HCC. DNA methylation contributed to the risk of HCV-associated HCC.