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Showing content from https://pubmed.ncbi.nlm.nih.gov/18308762/ below:

Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung

. 2008 May;29(5):895-904. doi: 10.1093/carcin/bgn017. Epub 2008 Feb 28. Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung

Affiliations

Affiliation

¹ Cancer Biology Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.

PMID: 18308762
PMCID: PMC3312609
DOI: 10.1093/carcin/bgn017

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Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung

Julien D F Licchesi et al. Carcinogenesis. 2008 May.

. 2008 May;29(5):895-904. doi: 10.1093/carcin/bgn017. Epub 2008 Feb 28. Affiliation

¹ Cancer Biology Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.

PMID: 18308762
PMCID: PMC3312609
DOI: 10.1093/carcin/bgn017

Item in Clipboard

Abstract

Background: Atypical adenomatous hyperplasia (AAH) is now recognized as a precursor lesion from which lung adenocarcinomas arise and thus represents an ideal target for studying the early genetic and epigenetic alterations associated with lung tumorigenesis such as alterations of the Wnt pathway.

Methods: We assessed the level of Wnt signaling activity in lung cancer cell lines by determining the level of active beta-catenin and determined the level of expression of Wnt antagonists APC, DKK1, DKK3, LKB1, SFRP1, 2, 4, 5, WIF1 and RUNX3 using reverse transcription-polymerase chain reaction. Using multiplex nested methylation-specific polymerase chain reaction, we analyzed promoter region methylation of these genes in resected lung tissue in the histopathologic sequence of glandular neoplasia (normal lung parenchyma, low-grade and high-grade AAH, adenocarcinoma).

Results: The majority of non-small cell lung cancer cell lines (11 of 16, 69%) have evidence of active Wnt signaling and silencing of Wnt antagonists correlated with promoter hypermethylation. Promoter region methylation of Wnt antagonists was common in primary lung adenocarcinoma and there was a significant increase in the frequency of methylation for Wnt antagonist genes and the number of genes methylated with each stage of tumorigenesis (test for rend P <or= 0.01). Additionally, odds ratios for promoter hypermethylation of individual or multiple Wnt antagonist genes and adenocarcinomas were statistically significantly elevated and ranged between 3.64 and 48.17.

Conclusion: These results show that gene silencing of Wnt antagonists by promoter hypermethylation occurs during the earliest stages of glandular neoplasia of the lung and accumulates with progression toward malignancy.

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Figures
Fig. 1.

Cytoarchitectural atypia in glandular neoplasia…

Fig. 1.

Cytoarchitectural atypia in glandular neoplasia of the lung. ( A ) Histologically normal…

Fig. 1.

Cytoarchitectural atypia in glandular neoplasia of the lung. (A) Histologically normal lung parenchyma. (B) A LG-AAH characterized by scattered atypical cuboidal epithelial cells lining delicate septa. (C) In this HG-AAH, the atypical cells are more crowded and there is increased fibrosis of the interstitium but without overt invasion of the lung parenchyma. (D) The periphery of this adenocarcinoma shows growth of large atypical cells along intact alveolar walls. More central areas of the tumor showed frank stromal infiltration.

Fig. 2.

The Wnt pathway is active…

Fig. 2.

The Wnt pathway is active in NSCLC cell lines and promoter hypermethylation of…

Fig. 2.

The Wnt pathway is active in NSCLC cell lines and promoter hypermethylation of Wnt antagonists correlates with gene repression. (A) Active Wnt signaling in NSCLC cell lines. Whole-cell lysate for NSCLC's cell lines was analyzed by western blot using the active β-catenin antibody that has previously been established as a readout for canonical Wnt pathway. The highest level of β-catenin/TCF-signaling activity was detected in H1666, H1435 and H358 cell lines. KRAS codon 12 mutation was detected in H358 (indicated by *), A549 and H157 (data not shown). For loading control, the membrane was stripped and reprobed with a β-actin antibody. (B) Gene reexpression detected using RT–PCR and (C) promoter demethylation (MSP analysis) of Wnt antagonists following 5-aza-2′-deoxycitidine treatment in NSCLC cell lines. NSCLC cell lines were treated for 96 h with either 2 μM of 5-aza-2′-deoxycitidine (AZA) or dimethyl sulfoxide (Mock). Colon cancer cell lines HCT116, RKO and DKO were also screened by RT–PCR and MSP and were used as controls since the expression and methylation profiles of these genes were known in these cell lines. The APC gene was not expressed at baseline in H1435 and H838. Additionally, APC promoter was fully methylated at baseline in H1435 as detected by a methylated band only, and H838 was highly methylated (Figure 2B and C). Treatment with 5-aza-2′-deoxycitidine led to reexpression of APC in both cell lines which correlated with APC promoter demethylation as seen by the appearance of strong unmethylated amplification. Among the NSCLC cell lines screened, only H838 showed no expression of DKK1 together with complete methylation of DKK1 promoter at baseline. Following drug treatment, DKK1 promoter became demethylated as seen by the appearance of a faint unmethylated band, and DKK1 transcript was reexpressed (Figure 2B and C). Complete methylation of RUNX3 promoter was observed at baseline and correlated with gene silencing in H460. Drug treatment of H460 led to both gene expression and promoter demethylation as shown by the appearance of an unmethylated band along with the methylated band (Figure 2C). RUNX3 was partially methylated in H1666 and H838 mock-treated cells and both cell lines expressed RUNX3, suggesting that partial methylation of RUNX3 promoter allows for gene expression in these cell lines. (D) Summary of gene expression and methylation analysis in NSCLC cell lines. Black boxes represent absence of expression or fully methylated promoter; gray boxes, weak basal expression or partial methylation and white boxes, expression and no methylation of the promoter.

Fig. 3.

Multiplex nested MSP analysis of…

Fig. 3.

Multiplex nested MSP analysis of Wnt antagonists in clinical samples with 2% agarose…

Fig. 3.

Multiplex nested MSP analysis of Wnt antagonists in clinical samples with 2% agarose gel showing the resolution of multiplex nested MSP products in normal lung (normal-adjacent lung), LG-AAH, HG-AAH and adenocarcinoma (AdenoC) patient samples. Each specimen is identified by its tissue type and sample number ( Supplementary Table 2 is available at Carcinogenesis Online). For each multiplex nested MSP experiment, methylated and unmethylated controls were included. These include the normal human bronchoepithelial cell line, a non-immortalized lung cell lines, normal lymphocytes and DKO. Additionally, other cell lines such as DLD-1, H69, which have a known methylation profile for some of the genes analyzed, as well as the in vitro methylated DNA IVD were also used as controls. Examples of multiplex nested MSP results are shown for genes antagonizing Wnt ligands and frizzled receptors (SFRP4, SFRP5 and WIF1), LRP5/6 (DKK1 and DKK3) and β-catenin (RUNX3). SFRP4 promoter hypermethylation was detected in AAH39 but not in AAH40 or AAH41 of patient 12. Additionally, SFRP4 methylation was present in the adenocarcinoma sample (AdenoC14) of the same patient, whereas the normal-adjacent specimens (normal-adjacent 29 and 30) showed absence of methylation for that gene. SFRP5 was methylated in AAH48 and adenoC16 samples but not in the normal-adjacent 38 or 39 of patient 13. Some genes such as WIF1 were found methylated in the normal-adjacent samples. Indeed, the normal-adjacent 37 which was sampled from the same slide as AAH41, from patient 12, also shows evidence of WIF1 promoter hypermethylation as seen by the presence of both a methylated and unmethylated band in these samples. Promoter hypermethylation was also frequently detected for DKK1, DKK3 and RUNX3.

Fig. 4.

Increases in the frequency and…

Fig. 4.

Increases in the frequency and prevalence of methylation of Wnt antagonist genes parallels…

Fig. 4.

Increases in the frequency and prevalence of methylation of Wnt antagonist genes parallels histological progression. (A) The increase in the frequency of methylation across tissue types was statistically significant for all the genes but WIF1 (test for trend P ≤ 0.01). For example, SFRP1 was methylated in 2% of normal lung specimens from cancer patients (normal adjacent), 11% of LG-AAH, 14% of HG-AAH and 58% of adenocarcinoma samples. The normal lung specimens obtained postmortem from disease-free patients were all unmethylated for each of the Wnt antagonists analyzed. The difference in frequency of methylation between HG-AAH and LG-AAH was statistically significant for DKK1 and RUNX3 (P value ≤ 0.05) ( Supplementary Table 3 is available at Carcinogenesis Online). (B) The percentage of samples according to number of genes methylated presented for normal lung (normal-adjacent lung), LG-AAH, HG-AAH and adenocarcinoma samples. The mean number of genes methylated was 1.63 for normal adjacent, 1.72 for LG-AAH, 3.29 for HG-AAH and 6.42 for adenocarcinoma, with this trend of increasing number of genes methylated with grade reaching statistical significance (test for trend P ≤ 0.01). Complete statistical analysis is presented in Supplementary Table 4 is available at Carcinogenesis Online).