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Basic and Translational Science

Quantitative Assessment ofAKAP12 Promoter Methylationin Human Prostate Cancer UsingMethylation-sensitive High-resolutionMelting: Correlation With Gleason ScoreWeiwei Liu, Jian Gong, Jinghui Hu, Tingting Hu, Yaofei Sun, Junhua Du, Chuanyu Sun,Ming Guan, Haowen Jiang, and Yuan Lu

OBJECTIVES To quantitatively investigate the A kinase anchoring protein 12 (AKAP12) gene promotermethylation and its association with clinicopathologic variables in human prostate cancer (PCa).The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety ofcancers.

METHODS The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatichyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitivehigh-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostatecarcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcrip-tase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis andafter DNA methyltransferase inhibition with 5-aza-2=-deoxycytidine.

ESULTS Methylation (�1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa,5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissuesamples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatichyperplasia or adjacent tissue samples (P � .01). AKAP12 methylation was significantly greaterin the PCa samples with higher Gleason scores (P � .03); however, no correlation was foundwith age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerasechain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaPand 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells.Treatment of LNCaP cells with 5-aza-2=-deoxycytidine markedly decreased the methylationlevels and increased the expression of AKAP12.

ONCLUSIONS The results of the present study have demonstrated that AKAP12 promoter methylation is afrequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker

for predicting the malignancy of PCa. UROLOGY 77: 1006.e1–1006.e7, 2011. © 2011 Elsevier Inc.

as

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Prostate cancer (PCa) has been rapidly increasing inChina and is currently recognized as one of theprincipal medical problems facing the male popu-

ation. The incidence of PCa in Shanghai has beenstimated to have increased from 1.8-2.4/100 000 in 1990

Weiwei Liu and Jian Gong contributed equally to this work.This study was supported by the National Natural Science Foundation (grant

81001059), the Natural Science Foundation of Shanghai (grant 09ZR1405300), agrant from the Shanghai Medical Key Discipline, and the Science and TechnologyCommission of Shanghai Municipality (grant 074119519).

From the Departments of Laboratory Medicine and Urology, Huashan Hospital,Fudan University Shanghai Medical College, Shanghai, China

Reprint requests: Yuan Lu, Professor, Department of Laboratory Medicine, HuashanHospital, Shanghai Medical College Fudan University, 12 Central Urumqi Road,

mShanghai 200040 China. E-mail: yuanlu@hsh.stn.sh.cn

Submitted: June 18, 2010, accepted (with revisions): December 5, 2010

© 2011 Elsevier Inc.All Rights Reserved

to 4.5-7.7/100 000 in 2000 and to about 10.0/100 000 in2004.1 Furthermore, because screening for PCa usingprostate-specific antigen determination and digital rectalexamination are not routine practice in China, mostChinese patients with newly diagnosed PCa will alreadybe symptomatic and have metastatic disease.2

The A kinase anchor protein 12 (AKAP12/gravin)was first isolated as a protein recognized by the serumfrom patients with myasthenia gravis.3 It is an A kinasenchoring protein (AKAP) that belongs to a family ofcaffold proteins and organizes protein kinase A and C.4

It also is an important regulator of the �2-adrenergiceceptor complex, which controls cell signaling, cell ad-esion, mitogenesis, and differentiation.5 DNA hyper-

ethylation of the AKAP12 promoter region and the

0090-4295/11/$36.00 1006.e1doi:10.1016/j.urology.2010.12.010

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resulting underexpression of the corresponding gene hasbeen noted in a variety of human cancers, includinggastric cancer, esophageal cancer, and lung cancer and inmyeloma cells and myeloid malignancies.5-9 Downregu-lation of AKAP12 expression suggests that inactivationof AKAP12 expression could be linked to oncogenesis.

A previous study found that the SSeCKS/gravin (therodent ortholog of human AKAP12) protein and RNAlevels were severely reduced in human and rat PCa celllines. Primary site tumors that progressed lost regulatedSSeCKS expression. SSeCKS/gravin expression was de-tected in benign human prostatic lesions and well-differ-entiated carcinoma, but not in undifferentiated lesionswith a Gleason score (GS) �6. These data suggest a rolefor the loss of SSeCKS/gravin expression in the meta-static progression of PCa.10

We hypothesized that inactivation of the AKAP12gene through CpG methylation could be responsible forthe pathogenesis and progression of PCa. To investigatethis possibility, we analyzed the promoter methylationstatus of the AKAP12 gene in PCa samples and relatedthese findings to the clinical and pathologic outcomes.The study used methylation-sensitive high-resolutionmelting (MS-HRM), which was originally developed forsingle nucleotide polymorphism genotyping.11 MS-HRMoperates on the principle that bisulfite-treated DNA tem-plates with different methylcytosine contents can be re-solved by melting analysis owing to the differences inmelting temperatures.12 HRM has also recently beenroposed as a rapid and sensitive technique for the as-essment of DNA methylation.12-18 We applied MS-RM technology to the detection and quantification ofKAP12 methylation in PCa samples.

MATERIAL AND METHODS

Samples and Clinical CharacteristicsThe tissue samples from 78 patients with PCa and 22 patientswith benign prostatic hyperplasia (BPH) were obtained fromHuashan Hospital, Fudan University. Non-neoplastic prostatictissue samples were obtained from 22 patients with PCa ascontrols. All tissue specimens were frozen immediately aftersurgery and stored at �80°C for additional analysis. All theumors were confirmed to contain �80% tumor cells by histo-ogic examination of sequential sections. All patients providedritten informed consent, and the Ethics Committee ofuashan Hospital approved the study.

Cell Culture Conditions and5-Aza-2=-Deoxycytidine TreatmentIn addition, 4 human PCa cell lines, PC-3, DU145, LNCaP,and 22RV1, were obtained from the American Type CultureCollection (Manassas, VA). The cells were cultured in RoswellPark Memorial Institute 1640 medium and F12K (Gibco-BRL,Grand Island, NY) supplemented with 10% fetal bovine serum(Sigma-Aldrich, St. Louis, MO), and incubated in 5% carbondioxide at 37°C.

The effects of the demethylating agent, 5-aza-2=-deoxycyt-

idine (5-aza-dC), on the AKAP12 gene expression were inves-

1006.e2

tigated in cultured PCa cell lines. The LNCaP cells were treatedwith 1 �mol/L 5-aza-dC (Sigma-Aldrich) for 72 hours, follow-ing a previously described protocol.19 The cells were treated

ith 5-aza-dC daily, and the medium was replaced on treat-ent. Control wells received only the medium with vehicle.he cells were harvested at the end of the treatment and used

or DNA and RNA analysis. cDNA from these cell lines wassed to measure the difference in AKAP12 mRNA expressionevels before and after 5-aza-dC treatment, using quantitativeeverse transcriptase-polymerase chain reaction (RT-PCR).

RNA Extraction and RT-PCRTotal RNA was extracted from each cell line using Trizol(Invitrogen, Carlsbad, CA), according to the manufacturer’sprotocol. First-strand cDNAs were generated from the purifiedmRNA using the SuperScript III First-Strand Synthesis Systemfor RT-PCR and the oligo(dT) primer (Invitrogen). Reversetranscriptase reactions were performed using 1 �g total RNA.

lyceraldehyde 3-phosphate dehydrogenase was used as an in-ernal reference gene. AKAP12 was amplified on the same plates glyceraldehyde 3-phosphate dehydrogenase, using the Hot-tarTaq Plus Master Mix (Qiagen, Hilden, Germany) or Sybrreen PCR Master Mix (Qiagen). The RT-PCR primers were

erformed, as previously described.20

DNA Extraction and Bisulfite TreatmentDNA was isolated from the tissue samples and cell lines usingthe QIAamp DNA minikit (Qiagen), according to the manu-facturer’s instructions. The DNA was quantified using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE). One microgram of DNA was treated withsodium bisulfite using the EZ DNA Methylation Kit (ZymoResearch, Orange, CA), according to the manufacturer’s in-structions.

MS-HRM and EvaluationReal-time PCR followed by HRM was performed in a RotorGene 6000 (Corbett Research, Sydney, Australia). TheAKAP12 MS-HRM assay and evaluation were performed aspreviously described.20 In brief, the reaction mixture consistedof 100 ng of bisulfite-modified template, 1 � PCR Master Mixplus (Qiagen), 200 nmol/L of each primer (as described previ-ously20), and 5 �mol/L of SYTO(R) 9 green fluoresce (Invit-rogen), in a final volume of 20 mL. The reaction cycle startedwith 1 cycle at 95°C for 5 minutes for enzyme activation,followed by 42 cycles of denaturation at 94°C for 30 seconds,annealing at 58°C for 30 seconds, and extension at 72°C for 30seconds, followed by a final extension step at 72°C for 7minutes. The MS-HRM analyses were performed at the tem-perature ramping and fluorescence acquisition settings recom-mended by the manufacturer, ramping from 75° to 90°C, in-creasing by 0.1°C/s. Universal methylated DNA (Chemicon,Millipore, Billerica, MA) and DNA from peripheral bloodmononuclear cells of healthy volunteers were used as fullymethylated positive and unmethylated references, respectively.These 2 controls were mixed in ratios of 0%, 1%, 5%, 10%,20%, 40%, 60%, 80%, and 100% methylated to unmethylatedtemplate after bisulfite modification to create a range of meth-ylated and unmethylated allele dilutions. These standards wereincluded in each experiment. All samples were analyzed in

duplicate. The sensitivity, linearity, and reproducibility of the

UROLOGY 77 (4), 2011

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AKAP12 MS-HRM assay were determined as previously de-scribed.20

Statistical AnalysisThe Mann-Whitney U test was used to compare the AKAP12

ethylation in the PCa, BPH, and adjacent normal tissueamples. The Mann-Whitney U test was also used to compare

AKAP12 methylation with the clinicopathologic features in theCa samples. The Student t test was used to compare the valuesbtained after 5-aza-dC treatment with those from the corre-ponding control experiments. P � .05 was considered statisti-ally significant.

RESULTS

Sensitivity, Linearity, andReproducibility Of AKAP12 MS-HRM AssayThe AKAP12 MS-HRM assay was established in ourprevious study.20 In brief, the assay sensitivity was tested

Figure 1. Verification of AKAP12 MS-HRM assay using seriith serial dilutions of methylated DNA (from 0% to 100%) apecificity of assay. (C) AKAP12 MS-HRM profiles of serialgainst unmethylated DNA (0%). (E) Fluorescence values obNA (from 0% to 100%). (F) Values of fluorescence plotted

ypical standard curve.

y analyzing the consistency of the normalized melting H

UROLOGY 77 (4), 2011

rofiles derived from the samples with the different meth-lated/unmethylated ratios. The obtained amplificationlots indicated that all dilutions were amplified withomparable Cycle threshold (Ct) values (Fig. 1A). Thepecificity of the assay was determined from the meltingurve of the standard dilutions (Fig. 1B). The AKAP12S-HRM assay was able to reproducibly detect 1%ethylated DNA in a background of unmethylated DNA

Fig. 1C). A differential graph was generated by normal-zing each HRM profile against unmethylated DNA (0%ethylated; Fig. 1D). The differential analysis generated

eaks of variable heights because of the differences inuorescence among the dilutions. The values for theifferential fluorescence peaks were obtained using theotor Gene 6000 software (Fig. 1E). These values were

hen plotted against the dilution factor to generate ainear calibration curve (Fig. 1F). The intra- and inter-ssay variability as a coefficient of variation of the MS-

utions of methylated DNA. (A) Amplification plots obtainedplate. (B) Melting curve of standard dilutions used to test

ions. (D) Differential graph of each HRM profile normalizeded at melting temperature for serial dilutions of methylatednst percentage of methylation for each dilution to generate

al dils temdiluttainagai

RM assay were tested by comparing the methylation

1006.e3

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results obtained from 4 results of the same assay and 4independent assays performed on different days. Accord-ing to the methylation levels, the intra-assay variabilitywas 6.05%-9.10%, and the interassay variability was14.50%-17.17% in the present study. The performance ofthe AKAP12 MS-HRM assay was validated and com-pared with the methylation-specific PCR assay for theAKAP12 promoter region, as previously described.20

Methylation levels of �5% were undetectable by themethylation-specific PCR assay (data not shown).

AKAP12 Expression and PromoterMethylation and Effects Of 5-Aza-Dc in Pca Cell LinesRT-PCR showed that the PCa PC-3 and DU-145 celllines expressed AKAP12 RNA (Fig. 2A) and LNCaP and22RV1 did not. The AKAP12 locus was methylated inLNCaP and 22RV1 cells (Fig. 2B). However, the per-centage of methylation differed between the 2 cell lines.LNCaP cells showed extensive methylation (84.6%), and22RV1 showed less methylation (13.2%). The expressionlevel of AKAP12 mRNA was increased 9.3-fold after-aza-dC treatment in LNCaP cells (Fig. 2C). TheKAP12 methylation levels were reduced from 84.6% to

Figure 2. Expression and methylation status of AKAP12 in 4(A) Expression of AKAP12 (165 bp) in 4 prostate cancer c3-phosphate dehydrogenase (226 bp) analyzed as internal ccell lines (PC-3, DU-145, LNCaP, and 22RV1) normalizeexpression by 5-aza-dC. Expression of AKAP12 gene in PCa(1 �mol/L) for 72 hours. Expression level represented as x-frror of 3 independent experiments. *P � .05. (D) Differentio 5-aza-dC for 72 hours normalized against unmethylated

.51% (Fig. 2D) after treatment with 5-aza-dC. These f

1006.e4

esults suggest that demethylation restored AKAP12 genexpression in PCa cell lines.

linicopathologic Correlations WithKAP12 Promoter Methylation in Prostatic Tissueshe level of DNA methylation was detected in 78 PCa,2 BPH, and 22 normal adjacent tissue samples using theKAP12 MS-HRM assay. A total of 47 (60.2%) of the8 PCa samples, 5 (22.7%) of the 22 BPH samples, and(9.1%) of the 22 adjacent tissue samples showed �1%ethylation of the AKAP12 promoter region. The me-

ian methylation value of AKAP12 in 78 PCa tissues was5.4% (range 1.0%-82.1%). Figure 3A presents theKAP12 MS-HRM results of 3 representative samples

PCa, BPH, and adjacent tissues samples). The Mann-hitney U test revealed that AKAP12 methylation was

ignificantly greater in PCa tissues than in the BPH ordjacent normal tissues (Fig. 3B, P � .01, for both).owever, a significant difference was also seen inKAP12 methylation between the BPH and adjacentormal tissue samples (P � .04).Within the PCa tissue samples, no correlations were

state cancer cell lines (PC-3, DU-145, LNCaP, and 22RV1).ines analyzed using RT-PCR. Expression of glyceraldehydel. (B) Differential fluorescence signals of 4 prostate cancerainst unmethylated control. (C) Restoration of AKAP12line LNCaP after exposure to demethylating agent 5-aza-dCcreases over untreated control cell. Bars indicate standard

orescence signals of LNCaP cells before and after exposurerol.

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ound between age, pT category, or serum prostate-spe-

UROLOGY 77 (4), 2011

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cific antigen level and methylation status (Table 1). Thedegree of AKAP12 methylation increased as the GSincreased (P � .03). The AKAP12 methylation levelswere 1%-20% in 24 (51.1%), 20%-60% in 18 (38.3%),and 60%-100% in 5 (10.6%) of the 47 PCa samples.

COMMENTAccumulating evidence has indicated that DNA hyper-methylation in the AKAP12 promoter region and con-urrent underexpression of the gene occurs in a variety ofuman cancers. One study found that SSeCKS/gravinrotein and RNA levels were severely reduced in humannd Rat PCa cell lines. They could be detected in benignuman prostatic lesions and well-differentiated carci-oma but not in undifferentiated lesions with a GS �6,uggesting a role for the loss of SSeCKS/gravin expressionn the metastatic progression of human PCa.10 However,

Figure 3. AKAP12 MS-HRM curves showing methylation starostate samples (PCa, BPH, and adjacent normal tissuetween prostatic tissue group and concentration of methy

Table 1. Comparison of human AKAP12 methylation strat-ified by patient characteristics

VariablePatients

(n)

AKAP12 Methylation (%)

Median (Range)P

Value

Total 78 15.4 (1.0-82.1)Gleason score .03

4-7 39 9.8 (1.0-66.2)8-10 39 22.2 (7.8-82.1)

pT stage .27pT1-T2 40 18.7 (1.0-69.8)pT3-T4 38 11.9 (2.6-82.1)

Age (y) .51�70 50 17.8 (1.0-61.4)�70 28 11.1 (2.6-82.1)

Preoperative serumPSA (ng/mL)

.38

�10 29 12.3 (1.0-69.3)�10 49 17.2 (2.6-82.1)

AKAP12, A kinase anchoring protein 12; PSA, prostate-specificantigen.

transcriptional regulation of AKAP12 gene expression

UROLOGY 77 (4), 2011

through epigenetic mechanisms has not been investi-gated in PCa. Furthermore, the relationships betweenAKAP12 methylation levels and clinicopathologic vari-ables in PCa remain to be elucidated. Promoter methyl-ation of cancer-related genes is a frequent event inPCa21,22 and is a promising tool for early cancer detec-ion.22 We sought to determine the promoter methyl-

ation status of AKAP12 in tissue specimens from primaryPCa, BPH, and normal adjacent tissue, as well as in 4PCa cell lines, using MS-HRM, a highly sensitive andspecific method for the detection and quantitation of DNAmethylation. The AKAP12 promoter was hypermethylatedin PCa (P � .01), providing evidence to support theAKAP12 epigenetic-mediated silencing hypothesis.

AKAP12 methylation levels were also significantly as-sociated with the GS. The methylation levels were sig-nificantly greater in GS 8-10 tumors, indicating thatAKAP12 methylation could be a useful marker for mon-itoring the progression of carcinogenesis in those withPCa. It is possible that the increased level of methylationmight be related to the progressive accumulation of cellswith AKAP12 promoter methylation during prostate car-cinogenesis in these patients. AKAP12-mediated sup-pression of tumor growth and metastatic colonization canbe achieved through direct or indirect interactions withmultiple proteins involved in apoptosis, angiogenesis,and associated signaling pathways in cancer cells.23,24

These included several signaling molecules that partici-pate in cell proliferation and cytoskeletal organization, aswell as protein kinase C, protein kinase A, cyclin D,calmodulin, and vascular endothelial growth fac-tor.10,25,26 These functions of AKAP12 mean that neo-plastic cells might obtain a growth advantage throughAKAP12 silencing, explaining the high prevalence ofpromoter methylation at this locus in PCa specimens.Additional studies are needed to determine the basis forthis observation.

Regarding the PCa cell lines, LNCaP cells showed

of PCa samples. (A) Differential fluorescence signals of 3ormalized against unmethylated control. (B) Association

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extensive methylation at the AKAP12 locus, and 22RV1

1006.e5

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cells displayed much lower methylation rates. Further-more, AKAP12 gene expression was increased 9.3-fold inthe LNCaP cells after treatment with the demethylatingagent, 5-aza-dC. The mechanisms responsible for thedownregulation of AKAP12 expression could differ be-tween PCa cell lines, although the level of promotermethylation was lower than that in LNCaP cells. Thesediscrepant results might be explained by other factors,such as histone deacetylase.

The AKAP12 promoter was methylated in 22.7% ofthe BPH samples, a proliferative benign lesion of theprostate. This level of methylation was significantly lowerthan that of the PCa tissues (P � .01). These resultssupport the hypothesis that AKAP12 acts as a tumorsuppressor gene. However, immunohistochemistry forAKAP12 was not performed in the BPH specimens in thepresent series; therefore, no direct comparisons could bemade. Nonetheless, previous studies reported AKAP12promoter methylation in normal tissues and showed thatthis epigenetic alteration failed to completely abrogategene expression.10

The principle of MS-HRM analysis is that PCR prod-ucts generated from bisulfite-treated DNA templates withdifferent methylcytosine contents have different meltingtemperatures, which can be resolved by melting analysisin a thermal cycler coupled with a fluorometer.18,27

Quantification can be performed by interpolation on astandard curve generated from serial dilutions of methyl-ated and unmethylated DNA. The efficiency of bisulfiteconversion was tested by sequencing cloned PCR frag-ments derived from bisulfite-treated CpGenome Univer-sal Methylated DNA (Zymo Research, Irvine, CA), andall the CpG sites were shown to be fully methylated. Thesensitivity of detection with MS-HRM has been reportedto be equivalent to that of the MethyLight assay, withboth assays providing reproducible results at levels as lowas 0.1% methylation.14 In our study, AKAP12 MS-HRMould reproducibly detect 1% methylated DNA in aackground of unmethylated DNA, which methylation-pecific PCR was able to reproducibly detect 10% meth-lation in our previously described study.20 After chang-

ing the cutoff value to �5%, the AKAP12 methylationwas still significantly greater in the PCa tissues than inthe BPH specimens (P � .01). The reason for setting theutpoint at �1% was to highlight the high sensitivity ofhe quantitative MS-HRM assay in detecting theKAP12 promoter methylation. However, MS-HRM

nalysis demonstrated important advantages over theethyLight assay used in our previous study.20

We have concluded that AKAP12 promoter methyl-ation is a frequent event in human PCa and represents apotential molecular biomarker for predicting the malig-nancy of PCa.

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