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Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer.

ABSTRACT: Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals.Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals.The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan-Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS.Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

SUBMITTER: Balgkouranidou I

PROVIDER: S-EPMC3992488 | biostudies-other | 2014 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundEarly metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool.MethodsMetastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot.ResultsIn general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05).ConclusionsAKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

Project description:Cervical cancer is the most fourth common cancer in women worldwide. The E6 and E7 high-risk human papillomavirus (HPV) types are the main cause of this cancer. Several studies have revealed that promoter methylation of tumor suppressor genes is induced by HPV E7. Recently, it was found that HPV16-E7 and the DNA methyltransferase 1 complex could bind at the cyclin A1 (CCNA1) promoter, resulting in CCNA1 promoter methylation. Therefore, there is a need to study other tumor suppressor genes for which HPV may induce promoter methylation. The present study investigated whether HPV induced cell adhesion molecule 1 (CADM1) and death associated protein kinase 1 (DAPK1) promoter methylation. C33a (no HPV infection) and SiHa (HPV 16 infection) cell lines were used for methylation status and expression observation. It was found that CADM1 and DAPK1 promoter methylation, no expression of CADM1 and decreased expression of DAPK1, was presented in SiHa cells. While no promoter methylation of these two genes was observed in C33a cells, with positive expression of the genes. It was subsequently investigated whether E6 and/or E7 could induce promoter methylation and decrease the expression of these two genes. Methylation-specific primer PCR and quantitative PCR were performed to elucidate the promoter methylation status and expression of CADM1 and DAPK1 in C33a cells transfected with HPV16 E6-PCDNA3 or HPV16 E7-PCDNA3.1 myc-his, compared to empty vector-transfected cells. The results showed that HPV E7 could induce CADM1 promoter methylation and decrease the gene expression in HPV E7 transfected C33a cells, while HPV E6 could induce DAPK1 promoter methylation and decrease its expression in C33a cells transfected with HPV E6. Finally, the mechanism by which HPV E7 induced CADM1 promoter methylation was observed by performing chromatin immunoprecipitation; the data showed that E7 induced CADM1 methylation by the same mechanism as that for CCNA1, by binding at the CADM1 promoter, resulting in the subsequent reduction of its expression in cervical cancer.

Project description:BackgroundThe number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes.ResultsIn this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters.ConclusionsMethylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

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Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer.

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