METH-2 silencing and promoter hypermethylation in NSCLC

Abstract

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT-PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.

Figure 1

Relative expression levels of METH-2 in paired normal and tumour lung tissues. Patient numbers are shown. The heavier band represents the control gene (O28) and the lower band represents METH-2. A remarkable reduction of METH-2 transcript is observed in all tumour samples. N: normal; T: tumour.

Figure 2

Immunohistochemical detection of METH-2 protein in lung tissues. (A, B) Normal lung. Positive staining is observed in ciliated bronchial epithelial cells (arrows). (C, D) Lung adenocarcinoma showing weakly positive tumour cells (T) and moderately positive staining of adjacent normal bronchial epithelium (N). Counterstained with H&E. Magnification: (A, D) × 10; (B, C) × 20.

Figure 3

(A) METH-2 cMSP analysis on lung tumour DNAs. The presence of the methylation-independent control fragment (299 bp) confirms sufficient amount of converted DNA in the reaction, while the presence of the methylation-specific product (169 bp) demonstrates the existence of methylated copies. DNAs from tumours 341, 371 and 180 show no methylation of METH-2, while those from 314, 308, 124, 177, 161 and 153 demonstrate methylation of the METH-2 promoter. N: negative (unmethylated) control; P: positive (methylated with SssI methylase) control; M: X174/HaeIII. (B) Capillary electrophoretic analysis of METH-2 cMSP products on Agilent Bioanalyser 2100 DNA chip. The presence of the methylation-independent control peak (299 bp) indicates sufficient amount of converted DNA, while the presence of the methylation-specific product peak (169 bp) demonstrates the existence of methylated copies.

Figure 4

Allelic imbalance analysis of METH-2 using an intragenic SNP marker. Five heterozygotes are demonstrated. Sample pairs 151, 353 and 124 show no AI. Sample pairs 341 and 308 show AI in the tumour tissue (indicated by the disturbance of the allele ratios compared to the normal).