Promoter Methylation In Lung Cancer Research Articles

CAVIN2 is frequently silenced by CpG methylation and sensitizes lung cancer cells to paclitaxel and 5-FU.

Aim: To explore the biological functions and clinical significance of CAVIN2in lung cancer. Materials & methods: Methylation-specific PCR was used to measure promoter methylation of CAVIN2. The function of CAVIN2was tested by Cell Counting Kit-8, colony formation, Transwell, flow cytometric analysis, acridine orange/ethidium bromide, chemosensitivity assay and xenograft assay. Results:CAVIN2is significantly downregulated by promoter methylation in lung cancer. CAVIN2overexpression inhibits lung cancer cell migration and invasion. Furthermore, ectopic expression of CAVIN2inhibits cell proliferation in vivo and in vitro by inducing G2/M cell cycle arrest, which sensitizes the chemosensitivity of lung cancer cells to paclitaxel and 5-fluorouracil, but not cisplatin. Conclusion:CAVIN2is a tumor suppressor in non-small-cell lung cancer and can sensitize lung cancer cells to paclitaxel and 5-fluorouracil.

ZAR1 is a novel epigenetically inactivated tumour suppressor in lung cancer

BackgroundLung cancer is the leading cause of cancer-related deaths with 1.8 million new cases each year and poor 5-year prognosis. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby can promote cancer development and progression.ResultsIn this study, we analysed ZAR1 (zygote arrest 1), which has been said to be a maternal-effect gene and its expression mostly limited to certain reproductive tissues. Our study shows that ZAR1 is expressed in normal lung but inactivated by promoter methylation in lung cancer. ZAR1 is hypermethylated in primary lung cancer samples (22% small cell lung carcinoma (SCLC) and 76% non-small cell lung carcinoma (NSCLC), p < 0.001) vs. normal control lung tissue (11%). In lung cancer cell lines, ZAR1 was significantly methylated in 75% of SCLC and 83% of NSCLC vs. normal tissue (p < 0.005/0.05). In matching tumours and control tissues, we observed that NSCLC primary tumour samples exhibited a tumour-specific promoter methylation of ZAR1 in comparison to the normal control lung tissue. Demethylation treatment of various lung cancer cell lines reversed ZAR1 promoter hypermethylation and subsequently re-established ZAR1 expression. In addition, we could show the growth inhibitory potential of ZAR1 in lung cancer cell lines and cancer cell lines. Exogenous expression of ZAR1 not only inhibited colony formation but also blocked cell cycle progression of cancer cell lines.ConclusionsOur study shows for the first time the lung tumour-specific epigenetic inactivation of ZAR1 due to DNA methylation of its CpG island promoter. Furthermore, ZAR1 was characterised by the ability to block tumour growth through the inhibition of cell cycle progression in cancer cell lines. We propose that ZAR1 could serve as an epigenetically inactivated biomarker in lung cancer.

Quantitative assessment of the diagnostic role of CDH13 promoter methylation in lung cancer.

In order to explore the association between cadherin 13 (CDH13) gene promoter methylation and lung carcinoma (LC) risk, we carried out a meta-analysis with searching of PubMed, Web of Science. Ultimately, 17 articles were identified and analysised by STATA 12.0 software. Overall, we found a significant relationship between CDH13 promoter methylation and LC risk (odds ratio=6.98, 95% confidence interval: 4.21-11.56, p<0.001). Subgroup analyses further revealed that LC risk was increased for individuals carrying the methylated CDH13 compared with those with unmethylated CDH13. Hence, our study identified a strong association between CDH13 gene promoter methylation and LC and highlighted a promising potential for CDH13 methylation in LC risk prediction.

Increased methylation of lung cancer-associated genes in sputum DNA of former smokers with chronic mucous hypersecretion

BackgroundChronic mucous hypersecretion (CMH) contributes to COPD exacerbations and increased risk for lung cancer. Because methylation of gene promoters in sputum has been shown to be associated with lung cancer risk, we tested whether such methylation was more common in persons with CMH.MethodsEleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used to profile the sputum of 900 individuals in the Lovelace Smokers Cohort (LSC). Replication was performed in 490 individuals from the Pittsburgh Lung Screening Study (PLuSS).ResultsCMH was significantly associated with an overall increased number of methylated genes, with SULF2 methylation demonstrating the most consistent association. The association between SULF2 methylation and CMH was significantly increased in males but not in females both in the LSC and PLuSS (OR = 2.72, 95% CI = 1.51-4.91, p = 0.001 and OR = 2.97, 95% CI = 1.48-5.95, p = 0.002, respectively). Further, the association between methylation and CMH was more pronounced among 139 male former smokers with persistent CMH compared to current smokers (SULF2; OR = 3.65, 95% CI = 1.59-8.37, p = 0.002).ConclusionsThese findings demonstrate that especially male former smokers with persistent CMH have markedly increased promoter methylation of lung cancer risk genes and potentially could be at increased risk for lung cancer.

Loss of heterozygosity of the Mutated in Colorectal Cancer gene is not associated with promoter methylation in non-small cell lung cancer

‘Mutated in Colorectal Cancer’ (MCC) is emerging as a multifunctional protein that affects several cellular processes and pathways. Although the MCC gene is rarely mutated in colorectal cancer, it is frequently silenced through promoter methylation. Previous studies have reported loss of heterozygosity (LOH) of the closely linked MCC and APC loci in both colorectal and lung cancers. APC promoter methylation is a marker of poor survival in non-small cell lung cancer (NSCLC). However, MCC methylation has not been previously studied in lung cancer. Therefore, we wanted to determine if MCC is silenced through promoter methylation in lung cancer and whether this methylation is associated with LOH of the MCC locus or methylation of the APC gene. Three polymorphic markers for the APC/MCC locus were analysed for LOH in 64 NSCLC specimens and matching normal tissues. Promoter methylation of both genes was determined using methylation specific PCR in primary tumours. LOH of the three markers was found in 41–49% of the specimens. LOH within the MCC locus was less common in adenocarcinoma (ADC) (29%) than in squamous cell carcinoma (SCC) (72%; P=0.006) or large cell carcinoma (LCC) (75%; P=0.014). However, this LOH was not accompanied by MCC promoter methylation, which was found in only two cancers (3%). In contrast, 39% of the specimens showed APC methylation, which was more common in ADC (58%) than in SCC (13%). Western blotting revealed that MCC was expressed in a subset of lung tissue specimens but there was marked variation between patients rather than between cancer and matching non-cancer tissue specimens. In conclusion, we have shown that promoter methylation of the APC gene does not extend to the neighbouring MCC gene in lung cancer, but LOH is found at both loci. The variable levels of MCC expression were not associated with promoter methylation and may be regulated through other cellular mechanisms.

Promoter methylation of glutathione S‐transferase π1 and multidrug resistance gene 1 in bronchioloalveolar carcinoma and its correlation with DNA methyltransferase 1 expression

The presence of glutathione S-transferase (GST) pi1 (GSTP1) or multidrug resistance gene 1 (MDR1) promoter methylation in lung cancer was studied for the first time to the authors' knowledge; and, to date, the clinical significance of methylation is not clear. The objective of the current study was to determine the promoter methylation status of GSTP1 and MDR1, which encode GST-pi and P-glycoprotein (Pgp), respectively, in patients with bronchioloalveolar carcinoma (BAC) and to investigate whether methyltransferase 1 (DNMT1)-mediated GSTP1 or MDR1 methylation are responsible for disease progression and prognosis in patients with BAC. Protein expression levels of DNTM1, GST-pi, and Pgp were determined by immunohistochemistry in samples from 36 patients with BAC. Promoter methylation status of the GSTP1 and MDR1 genes was determined by using methylation-specific polymerase chain reaction analysis. The results demonstrated a significant correlation between the methylation of the GSTP1 or MDR1 promoters and negative expression of their respective proteins in BAC (P < .05). A significant correlation also was demonstrated between GSTP1 methylation and recurrence-free and overall survival of patients with BAC. DNMT1 protein expression levels were correlated with GSTP1 promoter methylation and patient prognosis (P < .05). However, no correlation was observed between DNMT1 expression and MDR1 methylation. GSTP1 promoter methylation mediated by DNMT1 may promote BAC progression and could serve as a poor prognostic indicator for patients with this disease. DNMT1 protein expression also may be considered as a prognostic indicator. Methylation of the MDR1 promoter may be mediated through pathways other than DNMT1 in BAC and does not appear to be associated with disease progression or patient prognosis.

ΔDNMT3B Variants Regulate DNA Methylation in a Promoter-Specific Manner

DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, DeltaDNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. DeltaDNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of DeltaDNMT3B4 in primary lung cancer, suggesting a role of DeltaDNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of DeltaDNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of DeltaDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected DeltaDNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and DeltaDNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation.

A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies

BackgroundPromoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.Methods and FindingsIn an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.ConclusionsBy using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

AKAP12α is AssoCiated with Promoter Methylation in Lung Cancer

Promoter methylation is an important mechanism for silencing tumor-suppressor genes in cancer and it is a promising tool for the development of molecular biomarkers. The purpose of the present study was to investigate whether inactivation of the A Kinase Anchoring Protein 12 (AKAP12) gene is assoCiated with promoter methylation in lung cancer. The AKAP12 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) in ten lung cancer cell lines. The methylation status of the AKAP12alpha promoter was analyzed by performing bisulfite sequencing analysis in ten lung cancer cell lines, twenty four lung tissues and matched normal tissues. The AKAP12alpha expression was reduced in 6 of 10 (60%) lung cancer cell lines, whereas the AKAP12beta expression was absent in 1 of 10 (10%) lung cancer cell lines. The AKAP12alpha expression was restored after treatment with the demethylating agent 5-aza-2'-deoxycytidine in three lung cancer cell lines. Methylation of CpG island 1 in the AKAP12alpha promoter was detected in 30% of the lung cancer cell lines, whereas methylation of CpG island 2 in the AKAP12alpha promoter was observed in the immortalized bronchial cell line and in all the lung cancer cell lines. In lung tumors, the CpG island 1 in the AKAP12alpha promoter was infrequently methylated. However, CpG island 2 in the AKAP12alpha promoter was highly methylated in lung tumors compared with the surrounding normal tissues, and this was statistically significant (p=0.0001). Our results suggest that inactivation of the AKAP12alpha expression is assoCiated with DNA methylation of the promoter region in lung cancer, and that AKAP12alpha may play an important role in lung cancer carcinogenesis.

A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies

A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies