AKAP12alpha is associated with promoter methylation in lung cancer

Abstract

Purpose: 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.

Materials and methods: 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.

Results: 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).

Conclusion: 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.

Keywords: AKAP12α; Lung neoplasms; Promoter methylation.

Fig. 1

Expression profiles of the AKAP12 gene in lung cancer cell lines.

Fig. 2

The restoration of the AKAP12α expression by 5-aza-2'-deoxycytidin.

Fig. 3

A map of the CpG islands of the AKAP12α gene spanning 1 kb upstream of the transcription start site. The 5' region of the AKAP12α gene contains two large CpG islands 1 and 2 (gray area), which encompass a putative promoter region.

Fig. 4

Methylation status of the AKAP12α promoter in lung cancer cell lines. (A) The frequency of CpG island 1 methylation for each sample is indicated by the methylatied CpG site/total CpG sites (%). (B) The frequency of CpG island 2 methylation for each sample is indicated bymethylated CpG site/total CpG sites (%).

Fig. 5

Methylation status of the AKAP12α promoter in lung cancer tissues and the matched normal tissues. (A) The frequency of CpG island 1 methylation for each sample is indicated by methylated CpG site/total CpG sites (%). (B) The frequency of CpG island 1 methylation for each sample is indicated by methylated CpG site/total CpG sites (%). p-value (p<0.05) computed from a paired sample t-test.

Fig. 6

Percentage of methylated USF binding sites in CpG island 2 of the AKAP12α promoter. The frequency of methylation for each sample is indicated by the methylated USF binding site/total USF binding CpG sites (%).