Quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation-sensitive high resolution melting: Correlation with Duke's stage

Abstract

Colorectal cancer is the third most common form of cancer and the second leading cause of cancer mortality in the world 1-3. To establish more effective diagnostic and therapeutic strategies against this deadly disease, it is essential to understand its molecular pathology. One of the key molecular pathogenic elements underlying human cancer is inactivation of tumor suppressor genes 4.A kinase anchor protein 12 (AKAP12/Gravin) was first isolated as a protein recognized by serum from myasthenia gravis patients 5. It is one of the A kinase anchoring proteins (AKAPs), which belongs to a family of scaffolding proteins, and organizes the complex of protein kinase A (PKA) and protein kinase C (PKC) 6. It is also an important regulator of the β2-adrenergic receptor complex, which controls cell signaling, cell adhesion, mitogenesis and differentiation 7. AKAP12 has been mapped to chromosome 6q24–25.2. Accumulating evidence indicates that DNA hypermethylation in the AKAP12 promoter region and concurrent underexpression of the gene occurs in a variety of human cancers, including gastric cancer, esophageal cancer, lung cancer, myeloma cells and myeloid malignancies 7-11. Downregulation of AKAP12 expression suggests that the inactivation of AKAP12 expression may be linked to oncogenesis. A recent report using microarray data for in silico genetic searches suggests that AKAP12 methylation is associated with the downregulation of this gene in colon caner and suggests that AKAP12 may be a potential tumor suppressor gene candidate 4. However, quantitative information regarding the methylation and expression status of this gene and its clinical relevance remain to be determined in this solid tumor. A new approach, methylation-sensitive high resolution melting (MS-HRM), has recently been introduced to quantitatively detect methylation levels 12;13, a method originally developed for single nucleotide polymorphism (SNP) genotyping14. The principle behind MS-HRM is that bisulfite-treated DNA templates with different methyl cytosine contents can be resolved by melting analysis due to differences in melting temperatures15. Recently HRM was also proposed as a rapid and sensitive technique for the assessment of DNA methylation with the use of a new generation of fluorescent dyes.12;13;15-19. Here we have applied MS-HRM technology to detect and quantify AKAP12 methylation and examined the methylation and expression of AKAP12, and the clinical implications of our findings, in colorectal carcinomas and adjacent tissues. We also examined the tumor suppressor activity of AKAP12 in AKAP12-negative cancer cells.