Promoter Hypermethylation of the ATM Gene as a Novel Biomarker for Breast Cancer

Background: Breast cancer may be induced by activation of protooncogenes to oncogenes and in many cases inactivation of tumor suppressor genes. Ataxia telangiectasia mutated (ATM) is an important tumor suppressor gene which plays central roles in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of double-strand breaks of DNA. In breast cancer, decrease ATM expression correlates with a poor outcome; however, the molecular mechanisms underlying downregulation are still unclear. Promoter hypermethylation may contribute in downregulation. Hence the present investigation was designed to evaluate promoter methylation and expression of the ATM gene in breast cancer cases, and to determine links with clinical and demographic manifestations, in a South Indian population. Methods: Tumor biopsy samples were collected from 50 pathologically confirmed sporadic breast cancer cases. DNA was isolated from tumor and adjacent non-tumorous regions, and sodium bisulfite conversion and methylation-specific PCR were performed using MS-PCR primers for the ATM promoter region. In addition, ATM mRNA expression was also analyzed for all samples using real-time PCR. Results: Fifty eight percent (58%) of cancer tissue samples showed promoter hypermethylation for the ATM gene, in contrast to only 4.44% of normal tissues (p= 0.0001). Furthermore, ATM promoter methylation was positively associated with age (p = 0.01), tumor size (p=0.045) and advanced stage of disease i.e. stages III and IV (p =0.019). An association between promoter hypermethylation and lower expression of ATM mRNA was also found (p=0.035). Conclusion: We report for the first time that promoter hypermethylation of ATM gene may be useful as a potential new biomarker for breast cancer, especially in the relatively young patients.     

SLIT2 is Epigenetically Silenced in Ovarian Cancers and Suppresses Growth when Activated

Objectives: The SLIT2 gene is a novel tumor suppressor gene, whose hypermethylation has been detected inseveral malignances, including breast cancer, colorectal carcinoma and gliomas. In this study, we assessed thestatus of SLIT2 and its functions in ovarian cancers and cell lines. Methods: Methylation-specific PCR was usedto investigate the methylated promoter of SLIT2; the functions of SLIT2 in ovarian cancer cells were measuredby MTT, colony formation assay and flow cytometry. Results: SLIT2 promoter hypermethylation was detected in56 of 66 (84.8%) ovarian cancer samples and downregulation of SLIT2 expression in 52 (78.8%). The decreasedexpression was significantly correlated with SLIT2 promoter hypermethylation (p<0.01). Moreover, reversedexpression of SLIT2 suppressed cell growth, migration, colony formation abilities and induced more apoptosis.Conclusions: These results suggest that SLIT2 is a tumor suppressor in ovarian cancer, and may be a noveltarget for ovarian cancer treatment.     

Aberrant Methylation of RASSF2A in Tumors and Plasma of Patients with Epithelial Ovarian Cancer

Objective: The tumor suppressor gene, Ras-association domain family (RASSF)2A, is inactivated bypromoter hypermethylation in many cancers. The current study was performed to evaluate the methylationstatus of RASSF2A in epithelial ovarian cancer (EOC) tissues and plasma, and correlations with gene expressionand clinicopathologic characteristics. Method: We detected methylation of the RASSF2A gene in tissues andcorresponding plasma samples from 47 EOC patients and 14 patients with benign ovarian tumors and 10 withnormal ovarian tissues. The methylation status was determined by methylation-specific PCR while gene expressionof mRNA was examined by RT-PCR. The EOC cell line, SKOV3, was treated with 5-aza-2’-deoxycytidine (5-azadC).Results: RASSF2A mRNA expression was significantly low in EOC tissues. The frequency of aberrantmethylation of RASSF2A was 51.1% in EOC tissues and 36.2% in corresponding plasma samples, whereas suchhypermethylation was not detected in the benign ovarial tumors and normal ovarian samples. The expression ofRASSF2A mRNA was significantly down-regulated or lost in the methylated group compared to the unmethylatedgroup (p﹤0.05). After treatment with 5-aza-dC, RASSF2A mRNA expression was significantly restored in theSkov3 cell line. Conclusion: Epigenetic inactivation of RASSF2A through aberrant promoter methylation mayplay an important role in the pathogenesis of EOC. Methylation of the RASSF2A gene in plasma may be avaluable molecular marker for the early detection of EOC.     

Promoter Methylation of BRCA1, DAPK1 and RASSF1A is Associated with Increased Mortality among Indian Women with Breast Cancer

Background: Promoter methylation has been observed for several genes in association with cancer development and progression. Hypermethylation mediated-silencing of tumor suppressor genes (TSGs) may contribute to breast cancer pathogenesis. The present study was conducted to investigate the promoter methylation status of BRCA1, DAPK1 and RASSF1A genes in Indian women with breast cancer. Materials and Methods: Promoter methylation was evaluated in DNA extracted from mononuclear cells (MNCs) in peripheral blood samples of 60 histopathologically confirmed newly diagnosed, untreated cases of breast cancer as well as 60 age and sex matched healthy controls using MS-PCR. Association of promoter methylation with breast cancer-specific mortality was analyzed with Cox proportional hazards models. Kaplan-Meier survival analysis was performed for overall survival of the breast cancer patients. Results: We observed a significant increase of BRCA1, DAPK1 and RASSF1A promoter methylation levels by 51.7% (P <0.001), 55.0% (P <0.001) and 46.6% (P <0.001), respectively, when compared to healthy controls. A strong correlation was noted between hypermethylation of the tumor suppressor genes BRCA1 (P= 0.009), DAPK1 (P= 0.008) and RASSF1A (P= 0.02)) with early and advanced stages of breast cancer patients. We also found that breast cancer-specific mortality was significantly associated with promoter methylation of BRCA1 [HR and 95% CI: 3.25 (1.448-7.317)] and DAPK1 [HR and 95% CI: 2.32 (1.05-5.11)], whereas limited significant link was evident with RASSF1A [HR and 95% CI: 1.54 (0.697-3.413]. Conclusion: Our results suggest that promoter methylation of BRCA1, DAPK1 and RASSF1A genes may be associated with disease progression and poor overall survival of Indian women with breast cancer.     

Parallel Evaluation of Circulating Tumor DNA and Circulating Tumor Cells in Metastatic Colorectal Cancer

Background

Tissue biopsy is the gold standard for tumor genotyping, but it is an invasive procedure providing a single snapshot into tumor heterogeneity. Liquid biopsy approaches, encompassing the analysis of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), have been proposed as an alternative, with the potential of providing a comprehensive portrait of the tumor molecular landscape. In metastatic colorectal cancer (mCRC), both CTCs and ctDNA analysis have been investigated, but comparative analyses are limited.

Promoter Hypermethylation of LATS2 Gene in Oral Squamous Cell Carcinoma (OSCC) Among North Indian Population.

ABSTRACTLarge Tumor Suppressor (LATS2) gene are Tumor Suppressor gene, linked with epigenetic modifications. LATS2 promoter hypermethylation is an important epigenetic silencing mechanism leading to cancer. Cancer is the most common, vicious and dangerously increasing diseases of the world today, associated with high morbidity and mortality. Oral cancers (OC) are the blazing universal dilemma and is the sixth most frequent cancer observed in Indian population. Tobacco consumption is the main cause of the increase in OSCC. The association between LATS2 in the pathogenesis of cancers propose that their combination might be studied as a possible molecular marker for particular subgroups of patients. Therefore, the present study tried to investigate whether LATS2 promoter methylation was associated with oral squamous cell carcinoma (OSCC) in North Indian subjects. DNA methylation quantitative studies of LATS2 Tumor Suppressor genes were performed by methylation-specific polymerase chain reaction (MSP). 38 out of 70 patients (55 %) were found to be methylated for LATS2 gene, a statistically significant result was obtained (p-value < 0.005) for LATS2 genes. The results suggest that epigenetic changes may be related to the down-regulation of LATS2 expression. It can be concluded that LATS2 gene plays a significant role in the diagnosis of cancer and provide a better alternative as a diagnostic biomarker. Our data infer that a low LATS2 expression due to methylation may contribute to the cancer progression and could be useful for the diagnosis of OSCC. Therefore, investigation of promoter methylation in such genes may provide a biomarker which may prove to be useful in early detection of Oral Cancer.Keywords: Oral squamous cell carcinoma (OSCC), Epigenetic changes, LATS2 gene, Promoter Hypermethylation, Methylation-Specific PCR (MSP), BiomarkersAbbreviations: OSCC- Oral Squamous Cell Carcinoma; DNA- Deoxyribonucleic acid; LATS-Large Tumor Suppressor (gene); MSP-Methylation-Specific Polymerase Chain Reaction     

HE4 as a Serum Biomarker for ROMA Prediction and Prognosis of Epithelial Ovarian Cancer

Background and Purpose: Human epididymis protein 4 (HE4) has been suggested to be a novel biomarkerof epithelial ovarian cancer (EOC). The present study aimed to evaluate and compare HE4 with the commonlyused marker, carbohydrate antigen 125 (CA125), in prediction and therapy-monitoring of EOC. Patients andMethods: Serum HE4 concentrations from 123 ovarian cancer patients and 174 controls were measured by Rocheelectrochemiluminescent immunoassay (ECLIA). Risk of ovarian malignancy algorithm (ROMA) values werecalculated and assessed. In addition, the prospects of HE4 detection for therapy-monitoring were evaluated inEOC patients. Results: The ROMA score could classify patients into high- and low-risk groups with malignancy.Indeed, lower serum HE4 was significantly associated with successful surgical therapy. Specifically, 38 patientswith EOC exhibited a greater decline of HE4 compared with CA125. In contrast, elevation of HE4 better predictedrecurrence (of 46, 11 patients developed recurrence, and with it increased HE4 serum concentrations) and apoor prognosis than CA125. Conclusions: This study suggests that serum HE4 levels are closely associated withoutcome of surgical therapy and disease prognosis in Chinese EOC patients.     

Aberrant Methylation of the 1p36 Tumor Suppressor Gene RIZ1 in Renal Cell Carcinoma

Background: Retinoblastoma protein-interacting zinc finger gene 1(RIZ1) functions as a tumor suppressor.Hypermethylation-mediated RIZ1 silencing has been reported in several cancers, but not in renal cell carcinoma(RCC) yet. Materials and Methods: We examined the RIZ1 expression and methylation in a panel of RCC celllines and 50 primary tumors using semiquantitative/quantitative polymerase chain reaction (PCR), methylationspecific PCR, and bisulfite sequencing genomic. We also explored the relationship between methylation status ofRIZ1 and clinicopathological features in RCC patients. Results: RIZ1 expression was down-regulated or lost inOS-RC-2, 769-P, Caki-1, 786-O and A498 RCC cell lines. Restored expression of RIZ1 was detected after additionof 5-aza-2’-deoxycytidine with/without trichostatin A, suggesting that DNA methylation directly mediates itssilencing. The RIZ1 expression was significantly reduced in RCCs compared to adjacent non-malignant renalsamples (P<0.001). Aberrant methylation was detected in 15 of 50 (30%) RCCs and in 2 of 28 (7%) adjacent nonmalignantrenal samples (P=0.02). No statistically significant correlation between methylated and unmethylatedcases with regard to age, gender, pathological stage and grade was observed. Conclusions: RIZ1 expression isdown-regulated in human RCC, and this down-regulation is associated with methylation. RIZ1 methylation mayplay a role in renal carcinogenesis.     

Epigenetic status of FBXW7 gene and its role in Ovarian cancer pathogenesis

Background: Chromatin immunoprecipitation (ChIP) analysis revealed that the FBXW7 gene and the long non-coding RNA (LINC01588) are potential candidates in epithelial ovarian cancer (EOC) pathogenesis. However, their exact role in EOC is not yet known. Thus, the present study sheds light on the impact of the mutations/ methylation status of the FBXW7 gene. Materials and Methods: We used public databases to assess the correlation between mutations/ methylation status and the FBXW7 expression. Furthermore, we performed Pearson’s correlation analysis between the FBXW7 gene and LINC01588. We performed gene panel exome sequencing and Methylation-specific PCR (MSP) in HOSE 6-3, MCAS, OVSAHO, and eight EOC patients’ samples to validate the bioinformatics results. Results: The FBXW7 gene was less expressed in EOC, particularly in stages III and IV, compared to healthy tissues. Furthermore, bioinformatics analysis, gene panel exome sequencing, and MSP revealed that the FBXW7 gene is neither mutated nor methylated in EOC cell lines and tissues, suggesting alternative mechanisms for FBXW7 gene regulation. Interestingly, Pearson’s correlation analysis showed an inverse, significant correlation between the FBXW7 gene and LINC01588  expression, suggesting a potential regulatory role of LINC01588. Conclusion: Neither mutations nor methylation is the causative mechanism for the FBXW7 downregulation in EOC, suggesting alternative means involving the lncRNA LINC01588.     

循环DNA、循环肿瘤细胞端粒酶与肿瘤早期诊断

肿瘤患者循环DNA水平的增高、肿瘤特征性基因改变及循环肿瘤细胞端粒酶活性的表达,不仅可出现在肿瘤的晚期,也可发生在肿瘤的早期阶段。循环DNA和肿瘤细胞端粒酶的联合检测,对于肿瘤早期诊断有重要意义。     

Promoter Hypermethylation and Expression Changes of BRCA1 Gene in a Cohort of Sporadic Breast Cancer Cases among Pakistani Population

Objective: The purpose of our study was to determine the frequency of BRCA1 promoter hypermethylation and its association with expression changes of BRCA1 and main morphological features in sporadic breast cancer. Methods: A retrospective review of cases was performed to select those with specific morphological features suggestive of breast cancer. BRCA1 promoter hypermethylation and changes in protein expression were evaluated in 30 cancerous and 30 non-cancerous tissue samples. A tissue microarray containing samples from normal and tumor tissue was prepared and stained for BRCA1 protein expression using a commercially available monoclonal antibody against BRCA1 (Ab-1) clone MS110 (mAb). DNA was extracted using modified protocol of Qiagen minikit. DNA was modified using a Bisulfite conversion kit and BRCA1 hypermethylation was detected using a methylation specific PCR. Results: Promoter hypermethylation was negative in 30 non-cancerous samples with retained BRCA1 protein expression. Methylation was positive in 82.6% (n=19/23) of the sporadic cancer samples that had loss of BRCA1 expression and 50% (n=2/4) of the samples with equivocal protein expression. Methylation was negative in all the sporadic breast cancer samples (n=3/3) with retained protein expression. Chi-square analysis showed significant association of BRCA1 promoter methylation with decreased protein expression (P=0.016) and co-existence of loss of BRCA1 and Her2neu at chromosome 17 (P=0.026) respectively. There was no significant association of BRCA1 methylation with morphological features excluding necrosis (P=0.035). Promoter hypermethylation was found to be most common (68.75%) among Triple Negative Breast Cancer (TNBC) females less than 45 years old. Conclusion: Our study suggests that BRCA1 promoter hypermethylation has significant contribution in sporadic breast carcinogenesis. This was our preliminary study in Pakistan. Further studies aimed to determine the in-depth mechanisms of BRCA1 epigenetics in TNBC. BRCAness enriched phenotype in TNBC might be used as a biomarker for the exploitation of therapeutic and clinical implications.     

Promoter Hypermethylation of LATS1 Gene in Oral Squamous Cell Carcinoma (OSCC) among North Indian Population

Background: LATS1 (Large Tumor Suppressor, isoform 1) is a gene that forms a complex with the cyclin-dependent kinase, CDK1, and regulates cell cycle progression. Genetic modifications lead to a loss in the activity of LATS1 gene. OSCC is the most commonly emerging cancer caused by genetic as well as epigenetic changes. Epigenetics changes vary from one population to another because these are influenced by dietary factors and environmental factors.  Tobacco chewing and smoking has been reported as major risk factors in OSCC. No report was found in the previous literature showing promoter hypermethylation of LATS1 gene. Methods: A total of 50 OSCC patients and 20 normal individuals were recruited in this study. Blood samples (50) from OSCC patients and blood samples (20) from healthy individuals as controls were used in the present study. Isolation of genomic DNA was carried out from blood using the standard phenol-chloroform extraction. Further Isolated DNA was modified with sodium bisulfite using the agarose bead method and finally, the methylation studies of LATS1 gene were carried out using Methylation-Specific PCR (MSP-PCR). Results: 19 out of 50 patients (38.0%) were found to be methylated for LATS1 gene.; a statistically significant result was obtained (p -value= < 0.05) with an odds ratio of 0.37 in cases compared to controls. The status of methylation of LATS1 genes was also found to be statistically significantly associated with smokers and tobacco chewers (p-value = < 0.05). The methylation of LATS1 gene showed a significant risk of developing OSCC in patients. Conclusion: These results suggest that the LATS1 gene may provide a better alternative as a diagnostic biomarker. This is the first report on the promoter hypermethylation of LATS1 gene in OSCC patients among the North Indian population.     

Lack of significance of the BRCA2 promoter methylation status in different genotypes of the MTHFR a1298c polymorphism in ovarian cancer cases in Iran

Objective: Promoter methylation, which can be regulated by MTHFR activity, is associated with silencingof genes. In this study we evaluated the methylation status (type) of the BRCA2 promoter in ovarian cancerpatients carrying different genotypes of the MTHFR gene (A or C polymorphisms at position 1298). Methods:The methylation type of the BRCA2 promoter was evaluated using bisulfate-modified DNA in methylationspecificPCR and the MTHFRa1278c polymorphism was assessed by PCR-RFLP. Results: Analysis of theBRCA2 promoter methylation type of cases showed that 7 out of 60 cases (11.7%) were methylated while theremaining 53 (88.3%) were unmethylated. In methylated cases, one out of the 7 cases had a CC genotype andthe remaining 6 methylated cases had an AC genotype. The AA genotype was absent. In unmethylated cases,34, 18, and one out of these had AC, AA and CC genotype, respectively. Conclusion: There was no significantrelationship between the methylation types of the BRCA2 promoter in different genotypes of MTHFRa1298cpolymorphism in ovarian cancer; p=0.255. There was no significant relation between the methylation types ofthe BRCA2 promoter in different genotypes of the MTHFRa1298c polymorphism in ovarian cancer.     

Association between Cigarette Smoking and RASSF1A Gene Promoter Hypermethylation in Lung Cancer Patients: a Metaanalysis

Objectives: Epidemiological studies have shown that molecular mechanisms underlying the development oflung cancers differ between smokers and unsmokers. Aberrant promoter methylation in some tumor suppressorgenes is frequent in lung tumors from smokers but rare in those from non-smokers. Recently, many studies haveinvestigated the association between cigarette smoking and RASSF1A gene promoter hypermethylation in lungcancer patients, but a unanimous conclusion could not be reached. We therefore performed this meta-analysisto derive a more precise estimation of any association. Study Design: An electronic search of PubMed andChinese Biomedicine databases was conducted to select studies. A total of 19 case-control studies were chosen,and odds ratios (ORs) with confidence intervals (CIs) were used to assess the strength of associations. Results:The case-control studies covered 2, 287 lung cancer patients: 63.4%(1449) of the patients were smokers, 36.6%(838) were unsmokers. The overall results suggested that smokers with lung cancer had a 1.297-fold (95% CI:1.066~1.580, p=0.010, p=0.087) higher risk for RASSF1A gene hypermethylation than the non-smokers. In thestratified analysis, an increased risk of RASSF1A gene hypermethylation in smokers than in non-smokers wasfound in Asian (OR=1.481, 95%CI: 1.179~1.861, p=0.001, p=0.186). Conclusions: This meta-analysis supportsthe idea that RASSF1A gene hypermethylation is associated with cigarette smoking-induced lung cancer.     

Circulating Hypermethylated RASSF1A as a Molecular Biomarker for Diagnosis of Hepatocellular Carcinoma

Background: Detection of circulating DNA can be applied for the diagnosis of many malignant neoplasms, including the hepatocellular carcinoma (HCC). The molecular pathogenesis of HCC is complex, involving different genetic and epigenetic alterations, chromosomal aberrations, gene mutations and altered molecular pathways. RASSF1A is a well-established tumor suppressor gene which suffers frequent inactivation due to promoter hypermethylation of CPG islands in multiple tumors including HCC, resulting in the reduction or loss of gene expression. Objective: To examine the role of circulating RASSF1A as a non-invasive diagnostic marker for HCC. Participant and Methods: A total of 45 HCC patients with a background of HCV infection, 40 cases of HCV infection without tumours and 40 apparently healthy controls were subjected to full history taking, clinical examination, routine laboratory investigations, assessment of serum AFP and detection of circulating hypermethylated RASSF1A gene by methylation-sensitive restriction enzyme digestion and real-time PCR. Results: The level of hypermethylated RASSF1A was significantly elevated in the HCC group as compared to the HCV and control groups (p=0.001 for both). Copy number in serum was associated with increased tumor size (p value <0.001). On the other hand, no significant correlation was observed between RASSF1A and AFP (p=0.5). Using ROC curve analysis, the best cut-off for circulating serum RASSF1A to differentiate the HCC group was 8 copies/μl. Conclusion: The presence of hypermethylated RASSF1A in serum may be a useful and informative biomarker for HCC diagnosis and might be introduced as a screening method for populations at risk of HCC development.