AKAP12 Promoter Hypermethylation And Inhibitory Effect On The Tumor Progression And Metastasis In Human Colorectal Carcinoma

Colorectal cancer is the third most common form of cancer and the second leading cause of cancer mortality in the world.[2;14;27]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.A kinase anchor protein 12(AKAP12/AKAP250/Gravin) was first isolated as a protein recognized by serum from myasthenia gravis patients.It is one of the A kinase anchoring proteins(AKAPs),which belongs to a family of scaffolding proteins, and organizes the complex of PKA and PKC.It is also an important regulator of theβ₂-adrenergic receptor complex,which controls cell signaling,cell adhesion, mitogenesis and differentiation.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 was noted in a variety of human cancers,including gastric cancer,esophageal cancer,lung cancer, myeloma cells and myeloid malignancies[10;29-32]Downregulation of AKAP12 expression suggests that the inactivation of AKAP12 expression may be linked to oncogenesis.A recent report using microarray data in silico genetic searches suggests that AKAP12 methylation is associated with the downregulation of AKAP12 in colon caner and suggests that AKAP12 may be a potential tumor suppressor gene candidate.However more information regarding the methylation and expression status of this gene and it's inhibiting progression and metastasis remains to be determined in this solid tumor.(1) AKAP12 expression and promoter methylation in colorectal cancer tissuesTo determine whether AKAP12 is downregulated in colorectal cancer tissues,we examined the expression of this gene.AKAP12 expression levels were determined by real-time RT-PCR in 45 tumor-normal pairs.AKAP12 mRNA was decreased in 31 of 45(68.9%) colorectal cancer tissues.The frequency of AKAP12 downregulation did not correlated with patients' age,gender,Duke's stage and differentiation.To determine whether the decreasing of AKAP12 expression was mediated by promoter hypermethylation,the methylation status of the 5'CpG island of AKAP12 gene was determined by MSP.The CpG islands of AKAP12 were methylated in 26 of 45(57.8%) tumor cases.Hypermethylation of AKAP12 mRNA was significantly associated with more advanced Duke's stage(P=0.02).The frequency of AKAP12 downregulation did not correlated with hypermethylation.To gain more information on the methylation status,particularly for the upstream region of the basic promoter and around the translation start sites,a 228bp fragment of the AKAP12 promoter region,containing 17 CpG dinucleotides,was sequenced.(2) AKAP12 expression and promoter methylation in colorectal cancer cell linesBy RT-PCR,we first determined the expression of AKAP12 gene in five colorectal cell lines.Complete loss of AKAP12 transcript occurred in SW480,LoVo and Colo320 cells.In contrast,AKAP12 transcript remained in LS174T and Colo205 cells.Consistent with RT-PCR assay,Western blot analysis showed the lack of AKAP12 protein expression in SW480,LoVo and Colo320 and positive expression in Colo205 and LS174T.To determine if methylation was responsible for down regulation of AKAP12 in carcinoma cancer cell lines,we performed MSP analysis. Only methylated DNA was seen in LoVo and Colo320 cells whereas LS174T, Colo205 and SW480 showed partial methylation.To assess whether methylation of AKAP12 promoter was associated with transcriptional silencing,we analyzed AKAP12 mRNA expression in the presence or absence of the demethylating agent 5-Aza-dC and the histone deacetylase inhibitor TSA.Treatment of Colo320,LoVo and SW480 cells harboring extensive methylation with 5-Aza-dC and TSA upregulated the expression of AKAP12 by 5.4-fold,6.3-fold,3.6-fold and 6.0-fold, 9.3-fold,21.7fold,respectively.Interestingly the highest levels(35.2-fold,13.8-fold and 120.9-fold) of gene expression were observed when the cell lines were cultured with both agents,suggesting that aberreant histone deacetylation is also an important mechanism for inactivation of this gene and 5-aza-dC and TSA act synergistically to enhance transcriptional reactivation. (3) AKAP12 inhabit the progression growth and invasion in vitro and in vivoCell proliferation and apoptosis assays were performed by WST and flow-cytometric analysis.AKAP12 inhibited colorectal cells growth,proliferation and apoptosis.We also to detect the local invasion of the cell lines and the results suggested that AKAP12 could decrease the invasive,migratory and transformation capacity of the colorectal cells in vitro.We have found that AKAP12 have the inhibitory ability of growth and invasion on colorectal cancer in vitro,we considered that if the function of AKAP12 could be maintained in vivo? So we conducted successfully the subcutaneous xenotransplantation of colorectal in nude severe combined immunodeficient mice. Tumor size of the LoVo-AKAP12 animals was significantly smaller than that of animals from control groups(P＜0.01).The results also suggested that AKAP12 could decrease the metastasis capacity by detection the Alu sequence.(4) Rapid determination of AKAP12 methylation levels in peripheral blood using methylation-sensitive high resolution melting(MS-HRM) analysisThe aim of this study was to apply methylation specific high resolution melting (MS-HRM) technology to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers and in three colorectal cancer cell lines.In this study 38 of the 80 colorectal cancer samples (47.5%) were found to be methylated at the AKAP12 promoter region.AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation(P=0.03).We also compared the results generated by MS-HRM with a traditional methylation-specific PCR(MSP) assay.We found that intra-assay variability ranged from 6.14-9.90%and inter-assay variability ranged from 14.5-17.2%.The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA,whereas the MSP method was unable to detect less than 10% methylation in our this study.We demonstrate here for the first time,the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples.