Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer

Early detection of lung cancer is challenging due to a lack of adequate biomarkers. To discover novel tumor suppressor genes (TSGs) silenced by aberrant promoter methylation, we analyzed the gene expression profiles of two lung adenocarcinoma cell lines using pharmacologic-unmasking and subsequent microarray-analysis. Among 617 genes upregulated, we selected 30 genes and investigated the methylation status of their promoters by bisulfite sequencing analysis. Aberrant methylation was detected in four genes (CRABP2, NOEY2, T, MAP2K3) in at least one lung adenocarcinoma cell lines. Furthermore, the T promoter was methylated in 60% of primary lung adenocarcinomas versus 13% of non-malignant lung tissues. Conversely, RT-PCR analysis revealed T expression was low in lung tumors, while high in normal tissues. In addition, no non-synonymous mutations related to gene silencing were found. While further analysis is warranted, our results suggest that T has the potential to be a novel candidate TSG in lung cancer.     

Promoter hypomethylation contributes to the expression of MUC3A in cancer cells

MUC3A is a membrane-bound glycoprotein that is aberrantly expressed in carcinomas and is a risk factor for a poor prognosis. However, the exact mechanism of MUC3A expression has yet to be clarified. Here, we provide the first evidence that MUC3A gene expression is controlled by the CpG methylation status of the proximal promoter region. We show that the DNA methylation pattern is intimately correlated with MUC3A expression in breast, lung, pancreas and colon cancer cell lines. The DNA methylation status of 30 CpG sites from −660 to +273 was mapped using MassARRAY analysis. MUC3A-negative cancer cell lines and those with low MUC3A expression (e.g., MCF-7) were highly methylated in the proximal promoter region, corresponding to 9 CpG sites (−345 to −75 bp), whereas MUC3A-positive cell lines (e.g., LS174T) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC3A-negative cells or those with low MUC3A expression caused elevation of MUC3A mRNA. Our results suggest that DNA hypomethylation in the 5′-flanking region of the MUC3A gene plays an important role in MUC3A expression in carcinomas of various organs. An understanding of epigenetic changes in MUC3A may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with cancer.     

DNA methylation at promoter regions of interleukin 1B, interleukin 6, and interleukin 8 in non-small cell lung cancer

Epidemiologic and experimental evidences support the concept that inflammation promotes the development and progression of cancers. Interleukins (ILs) regulate the expression of several molecules and signaling pathways involved in inflammation. High expression of some ILs in the tumor microenvironment has been associated with a more virulent tumor phenotype. To examine the role of IL-1β, IL-6, and IL-8 in non-small cell lung cancer, we measured mRNA levels and promoter DNA methylation in a panel of cultured human lung cells (n = 23) and in matched pair lung tumor versus adjacent non-tumorous tissues (n = 24). We found that lung cancer cells or tissues had significantly different DNA methylation and mRNA levels than normal human bronchial epithelial cells or adjacent non-tumorous tissues, respectively. High DNA methylation of ILs promoters in lung cancer cells or tissues was associated with low mRNA levels. We found an inverse correlation between DNA methylation of IL1B, IL6, and IL8 gene promoters and their corresponding mRNA levels, such inverse correlation was more significant for IL1B (i.e., all cancer cell lines used in this study had a hypermethylated IL1B promoter which was associated with silencing of the gene). Our results underline for the first time the role of epigenetic modifications in the regulation of the expression of key cytokines involved in the inflammatory response during lung cancer development.     

Promoter hypermethylation and Up-regulation of thyroid-stimulating-hormone-alpha (TSH-α) in thyroid cancer

Thyroid-stimulating-hormone-alpha (TSH-α) is the common subunit of the heterodimeric hormone TSH and also of other glycoprotein hormones. Although both expression and promoter-methylation profiles of the gene have been observed in the pituitary gland and placenta, no observation has been reported in the thyroid gland. We examined TSH-α expression in normal and cancer thyroid tissues. Real-time RT-PCR and immunohistochemistry indicated that TSH-a was repressed in normal tissues while activated in cancer tissues. To identify the epigenetic mechanism of upregulation of TSH-α, the methylation status of the seven CpG sites in the TSH-a promoter was examined in sixty thyroid cancer tissues. Two CpG sites showed remarkably higher levels of methylation in cancer (46 and 45%) than in normal tissues (24 and 23%) (p=0.010 and 0.003). These findings indicate that TSH-α is expressed in the thyroid cancer tissue per se and that its expression can be affected by promoter methylation.     

Novel miRNA genes methylated in lung tumors

MicroRNAs play an important role in the regulation of expression of many genes and are involved in carcinogenesis. The regulation of miRNA gene expression can involve the methylation of promoter CpG islands. In this work, the methylation of six miRNA genes (mir-107, mir-125b-1, mir-130b, mir-137, mir-375, and mir-1258) in non-small-cell lung cancer (NSCLC) was studied for the first time by methylation-specific PCR using a representative set of specimens (39 cases). Four new genes (mir-125b-1, mir-137, mir-375, and mir-1258) methylated in primary NSCLC tumors were identified with frequencies of 56, 31, 56, and 36%, respectively. The frequencies of miRNA promoter methylation in DNA of tumors and histologically normal tissues differed significantly (P ≤ 0.05 by Fisher’s test). In lung tissues of 20 donors without a history of cancer, these genes were only methylated in a few cases. It was also shown that the previously unstudied promoter CpG islands of mir-107 and mir-130b were not methylated in NSCLC. The frequencies of mir-125b-1 and mir-137 methylation were shown for the first time to correlate with NSCLC progression (clinical stage and metastasis).     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

Expression of the human cancer/testis antigen NY-SAR-35 is activated by CpG island hypomethylation

The novel cancer/testis antigen gene, NY-SAR-35, is expressed exclusively in normal testis and in various histological types of tumor. However, the NY-SAR-35 gene expression is observed to be aberrant in several cancer cell lines and tissues. The analysis of methylation status of the NY-SAR-35 gene promoter in various cancer cell lines showed that its expression was related to methylation of the promoter region. Treatment of human cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine activated the expression of the NY-SAR-35 gene. In addition, transfection experiments on various fragments of the CpG-rich gene promoter indicate that in vitro methylation of the NY-SAR-35 gene promoter results in the loss of promoter activity. The expression of NY-SAR-35 is therefore activated by hypomethylation of the CpG island in the gene promoter.     

Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer

The highly repetitive Alu retroelements are regarded as methylation centres in the genome. Methylation in the gene promoters could be spreading from them. Promoter methylation of MLH1 is frequently detected in cancers, but the underlying mechanism is unclear. The aim of this study is to understand whether the methylation in the Alu elements is associated with promoter methylation in the MLH1 gene. Bisulfite genomic sequencing was used to analyse the CpG sites of the 5' end (promoter, exon 1 and Alu-containing intron 1) of the MLH1 gene in colorectal cancer cells and tissues, and gastric cancer tissues. Hypomethylation in the Alu elements and hypermethylation in the promoters and the regions between the promoters and the Alu elements were detected in two cancer cell lines and seven cancer tissues. However, demethylation or hypomethylation of the MLH1 promoter and regions between promoter and the Alu elements, and hypermethylation in the Alu elements, were identified in the normal tissues. MLH1 promoter methylation may spread from Alu elements that are located in intron 1 of the MLH1 gene. The trans-acting elements binding to the mutation sites could play a role in the methylation spreading.     

Methylation-mediated regulation of the glutathione S-transferase P1 gene in human breast cancer cells

Understanding the mechanisms that regulate the human pi class GST (GSTP1) gene expression in breast cancer cells is of particular importance to the study of breast cancer biology. In cultured human breast cancer cell lines, GSTP1 is exclusively expressed in estrogen receptor-negative (ER−) cells but is undetectable in receptor-positive (ER+) cells. Previously, we examined transiently transfected GSTP1 promoter activities, in vitro GSTP1 promoter–DNA interactions, and GSTP1 mRNA stability. These studies indicated that transiently transfected GSTP1 promoter elements and GSTP1 mRNA stability could only partially explain cell line-specific expression of endogenous GSTP1. In the present study, we examined whether the methylation status of the GSTP1 CpG island plays an important role in GSTP1 regulation. Southern blot analysis revealed that the GSTP1 CpG island is hypermethlyated in ER+, GSTP1 non-expressing cell lines but is undermethylated in ER−, GSTP1 expressing cell lines. Moreover, partial demethylation of the GSTP1 CpG island by treatment with 5-aza-2′-deoxycytidine resulted in de novo gene expression in ER+ cell lines, as detected by RT-PCR, Northern blot and Western blot analyses. Our data strongly indicate that methylation status of the promoter contributes significantly to the levels of GSTP1 expressed in ER− and ER+ breast cancer cell lines.     

Down-regulation of serum/glucocorticoid regulated kinase 1 in colorectal tumours is largely independent of promoter hypermethylation

Background

We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples.

Methodology/Principal Findings

We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG.

Conclusions/Significance

Our results show that even though partial methylation of the promoter region of SGK1 is present, this does not account for the different expression levels seen between normal and tumour tissue.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.