Identification of a Candidate Tumor-Suppressor Gene Specifically Activated during Ras-Induced...

GENES, CHROMOSOMES & CANCER 48:10–21 (2009)

Identification of Candidate Tumor Suppressor GenesInactivated by Promoter Methylation in Melanoma

Vanessa F. Bonazzi, Darryl Irwin, and Nicholas K. Hayward*

Oncogenomics Laboratory,Queensland Institute of Medical Research, 300 Herston Rd,Herston,QLD 4006,Australia

Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation.

To identify novel methylated TSGs in melanoma, we carried out global mRNA expression profiling on a panel of 12 mela-

noma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Tri-

chostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays.

After qRT-PCR confirmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C,

RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-specific cleaved ampli-

fication products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed

reduced mRNA expression in 35–59% of the melanoma cell lines compared to melanocytes and which was correlated

with a high proportion of promoter methylation (>40–60%). The same genes also showed extensive promoter methylation

in 6–25% of the tumor samples, thus confirming them as novel candidate TSGs in melanoma. VVC 2008 Wiley-Liss, Inc.

INTRODUCTION

Melanoma represents a significant public

health burden in all white-skinned populations

and its incidence is rising faster than that of

almost all other cancer types, e.g., 4% increase

each year for the past 30 years in the U.S. (Bed-

dingfield, 2003; Coups et al., 2008). Queensland,

Australia has the highest incidence of melanoma

in the world, with 34,000 residents in a popula-

tion of �4 million people being diagnosed with

the disease in the last two decades (Coory et al.,

2006). The 5-year survival rate for metastatic ma-

lignant melanoma is less than 5% as the tumors

are largely refractory to existing therapies (Cum-

mins et al., 2006).

The etiology of melanoma is complex, involv-

ing both genetic and environmental components.

The CDKN2A locus accounts for susceptibility in

�25% of all melanoma families (Bishop et al.,

2002), whereas mutations in the CDK4 oncogene

are rare (Zuo et al., 1996; Soufir et al., 1998;

Molven et al., 2005). In addition to germline

changes, somatic mutations of both CDKN2A and

CDK4 occur during melanoma development (Cas-

tellano et al., 1997; Whiteman et al., 1997; Mon-

zon et al., 1998; Walker et al., 1998; Auroy et al.,

2001). Apart from CDK4, other oncogenes thus faridentified to play a significant role in melanoma

tumorigenesis encode components of the mito-

gen-activated protein kinase (MAPK) pathway.

Somatic mutations of the BRAF gene have been

shown to be the principal mechanism by which

the MAPK pathway is activated in melanomas

(Brose et al., 2002; Davies et al., 2002; Gorden

et al., 2003; Satyamoorthy et al., 2003; Pavey et al.,

2004) and benign melanocytic nevi (Dong et al.,

2003; Pollock et al., 2003). In the majority of mel-

anomas that lack BRAF mutations, the MAPK

pathway is activated through mutation of mem-

bers of the RAS proto-oncogene family, particu-

larly NRAS (van Elsas et al., 1996; Brose et al.,

2002). Other oncogenes downstream of the

MAPK pathway, such as MYC (Kraehn et al.,

2001; Pastorino et al., 2003) and CCND1 (Sauter

et al., 2002) have also been shown to be activated

in melanoma via amplification and over-

expression.

In relation to tumor suppressor genes (TSGs),

CDKN2A ranks as the gene most commonly

deleted in melanoma, after which the PTEN(Packer et al., 2006), TP53 (Giglia-Mari and

Sarasin, 2003) and APAF1 (Dai et al., 2004;

Fujimoto et al., 2004; Mustika et al., 2005) genes

Additional Supporting Information may be found in the onlineversion of this article.

*Correspondence to: Nicholas K. Hayward, Oncogenomics Lab-oratory, Queensland Institute of Medical Research, 300 HerstonRd, Herston, QLD 4029, Australia.E-mail: [email protected]

Received 20 May 2008; Accepted 9 August 2008

DOI 10.1002/gcc.20615

Published online 19 September 2008 inWiley InterScience (www.interscience.wiley.com).

VVC 2008 Wiley-Liss, Inc.

show the highest frequency of inactivation in spo-

radic melanomas, principally through deletion,

mutation and hypermethylation, respectively.

Clearly, additional melanoma TSGs exist, and a

large number of chromosomal loci showing loss of

heterozygosity (LOH) have been implicated in

melanoma development, highlighting widespread

chromosomal instability (Curtin et al., 2005). How-

ever, many TSGs are not primarily inactivated

through mutation or deletion, but rather through

epigenetic changes such as promoter methylation,

which down-regulate gene expression and lead to

inactivation of TSGs which play a role in progres-

sion to malignancy (Rothhammer and Bosserhoff,

2007). Previous studies assessing genome-wide

methylation in cutaneous melanoma following

treatment with the DNA methyltransferase inhibi-

tor 5-Aza-2-deoxycytidine (5AzadC) have identi-

fied TSPY, HOXB13 and SYK as novel TSGs (van

der Velden et al., 2003; Gallagher et al., 2005;

Muthusamy et al., 2006).

In the current study we sought to identify

additional melanoma TSGs silenced by promoter

methylation by carrying out an array-based analy-

sis in a well-annotated panel of cell lines after

combined treatment with 5AzadC and an inhibi-

tor of histone deacetylase, Trichostatin A (TSA).

Follow-up analysis of some of the candidate

TSGs was carried out in a larger panel of mela-

noma cell lines as well as a panel of fresh tumors

and melanocyte cultures using the highly sensi-

tive, specific method of mass spectrometry of

base-specific cleaved amplification products (i.e.,

the Sequenom Epityper assay) (Ehrich et al.,

2005; Coolen et al., 2007). Correlations were then

assessed between the degree of promoter methyl-

ation and the mRNA levels in the melanoma cell

lines.

MATERIALS AND METHODS

Cell Culture

A panel of 12 melanoma cell lines derived

from primary cutaneous melanomas or their me-

tastases were used (Supp. Info. Table S1). All

cell lines were cultured in RPMI 1640 supple-

mented with 10% fetal bovine serum as described

earlier (Castellano et al., 1997). Primary human

melanocytes were obtained from neonatal fore-

skins and cultured in 10% heat-inactivated FCS

(CSL, Melbourne, Australia) in RPMI 1640 me-

dium supplemented with 100 U/ml penicillin,

100 lg/ml streptomycin, 3 mM HEPES with the

addition of 6 ng/ml cholera toxin and 16.2 nM

phorbol 12-myristate 13-acetate (PMA) (Sigma

Chemical, St. Louis, Missouri) as described ear-

lier (Leonard et al., 2003).

5AzadC and TSA Treatment of Cells and RNA

Extraction

Cells were split to 20% confluence in a T25

flask (7% for MM96L) 24 hr before treatment.

Cells were then treated for 3 days with 5 lM5AzadC (Sigma) from 100 mM 50% acetic acid

dissolved stock or were mock treated with the

same volume of phosphate buffered saline (PBS)/

50% acetic acid. The 3-day 5AzadC incubation

was followed by a 4-hr incubation with 300 nM

TSA (Sigma). RNeasy Midi-kits (Qiagen) were

used to extract total RNA from cells in log phase

growth according to the manufacturer’s instruc-

tions, with on-column DNase digestion (Qiagen

RNase-Free DNase Set). All RNA samples were

run on an Agilent Bioanalyzer (Agilent, CA) using

an RNA 6000 Nano LabChip kit to check for

RNA integrity, purity and concentration. Only

samples with an RNA integrity number (RIN) of

>8.0 were used for microarray analysis.

Preparation of cRNA and Illumina Array

Hybridization

Biotinylated cRNA were prepared from 500 ng

of total RNA using an Illumina TotalPrep RNA

Amplification Kit (Ambion, TX) and cRNA yields

were quantified using an ND-1000 spectropho-

tometer (Nanodrop Technologies). cRNA (1500

ng) were hybridized to Sentrix Human-6 Expres-

sion version 2 BeadChips (Illumina) containing

46,000 human genes using the hybridization solu-

tion supplied by the manufacturer. All reagents

and procedures for washing, detection, and scan-

ning were performed according to the Bead-

Station 500X system protocols.

Quantitative RT-PCR

To confirm the validity of the microarray

expression data, the mRNA levels were assessed

by quantitative reverse transcriptase-polymerase

chain reaction (qRT-PCR) (see Supp. Info. Table

S2 for primer sequences). First-strand cDNA syn-

thesis was performed with 3 lg total RNA for

each sample in a total volume of 20 ll using

Superscript III reverse transcriptase (Invitrogen,

CA) and random primers. Subsequent PCR reac-

tions were carried out on a Corbett RotorGene

EPIGENETIC GENE SILENCING IN HUMAN MELANOMA 11

Genes, Chromosomes & Cancer DOI 10.1002/gcc

6000 (Corbett Research, Australia) using SYBR

Green RT-PCR Master Mix (Applied Biosystems,

Foster City, California). CLTA (clathrin light

chain mRNA) was chosen as the normalization

control transcript, as it showed minimal variation

in the microarray hybridizations (within 0.7 to

1.3-fold of the reference value in all samples).

Specificity of PCR products obtained was

assessed by melting curve analysis.

DNA Extraction, Bisulfite Conversion, and PCR

Qiagen DNeasy Blood and Tissue Kits were

used to isolate genomic DNA from cells in log

phase growth as per the manufacturer’s instruc-

tions. All samples were run on an Agilent Bioana-

lyzer (Agilent, CA) using a DNA 12000 Nano

LabChip kit to check for DNA integrity, purity

and concentration. EZ-96 DNA methylation kits

(Zymo Research, CA) were used for bisulfite

treatment of 1 lg of genomic DNA from 46 mel-

anoma cell lines, 2 colorectal cancer cell lines

(Co115 and LIM 2405), 2 esophageal cancer cell

lines (OE19 and OE33), 2 glioma cell lines (T46

and T50), and 16 fresh melanoma tumors. DNA

from two pools of melanocytes was used as refer-

ence. Each gene promoter was divided into sev-

eral amplicons (Supp. Info. Table S3). The target

regions were then amplified using the primer

pairs and annealing temperatures defined by the

MethPrimer program. These primers contain a

T7-promoter tag (forward: 50-AGGAAGAGAG-fw

primer-30, reverse: 50-CAGTAATACGACTCAC

TATAGGGAGAAGGCT-rev primer-30) to allow

further in vitro transcription. One microliter of

modified DNA was used for the PCR reactions

carried out in a total volume of 5 ll. Unincorpo-

rated dNTPs were dephosphorylated by incuba-

tion at 37�C for 40 min in the presence of shrimp

alkaline phosphatase (SAP) (Sequenom).

In Vitro Transcription and EPITYPER Assay

Two microliters of this SAP-treated PCR mix-

ture were used as template in a 7 ll transcriptionreaction containing RNase A and T7 polymerase

[Sequenom, (Ehrich et al., 2005; Coolen et al.,

2007)]. Transcription and digestion were per-

formed simultaneously at 37�C for 3 hr. After the

addition of 20 ll H2O and 6 mg CLEAN resin

(Sequenom), 22 nl of the cleavage reactions were

dispensed onto silicon chips preloaded with ma-

trix (SpectroCHIPS, Sequenom). Mass spectra

were collected using a MassARRAY mass spec-

trometer (Bruker-Sequenom) and analyzed using

proprietary peak picking and signal-to-noise

calculations.

Statistical Analysis

Pearson correlation coefficients were used to

correlate global promoter methylation with the

1st CpG methylation for PPP1R3C (Fig. 3) and

Ectodermal Neural Cortex 1 (ENC1) (Fig. 6).

Spearman test was applied for the correlation

between mRNA expression and methylation of

the entire region. A t test (t ¼ r/Sr) was per-

formed to obtain the significance (Sr ¼ (1�r2)/n)of the Spearman coefficient.

RESULTS

5AzadC Treatment and mRNA Expression

Profiling of Melanoma Cell Lines

The initial set of cell lines was chosen to be

representative of the different range of muta-

tional profiles occurring in melanoma (Supp. Info.

Table S1). As a first step toward identifying novel

TSGs involved in the development of melanoma

we carried out global gene expression analysis of

12 melanoma cell lines before and after treatment

with 5AzadC and TSA. Some trial tests were

done to determine the appropriate duration of

TSA exposure. FACS analysis indicated a clear

increase of the S-phase population and a drop in

the G2-phase population after 6-hr incubation

(data not shown), therefore we chose a 4-hr incu-

bation with TSA to minimize effects of cell cycle

dysregulation on mRNA levels. Expression pro-

files were generated for each cell line before and

after drug treatment using Illumina Sentrix

Human-6 Expression version 2 BeadChips com-

prising �48,000 probe sets. Analysis showed that

across the entire panel of 12 cell lines a total of

8,144 nonredundant genes were re-expressed

with >2 fold-change after treatment (between

1,457 and 3,386 genes in individual samples).

Genes reactivated in all 12 cell lines were

removed from further analysis since they are

likely responding to drug treatment as part of the

‘‘cellular stress response,’’ or due to promoter

demethylation of genes normally silenced in the

melanocytic lineage. Genes were further filtered

to identify those with an average of >4-fold

increased expression in at least four samples in

the panel of 12 lines and >10-fold increase in at

least one of the cell lines. From this set of 670

genes, we selected AKAP12, ARHGEF16,

12 BONAZZI ET AL.


ARHGAP27, ENC1, PPP1R3C, PPP1R14C,RARRES1, TP53INP1 for initial follow-up since

they had not previously been documented to be

involved in melanoma. Table S4 (Supp. Info.)

shows the relative fold-change in expression for

these genes before and after drug treatment in

each melanoma cell line used for the microarray

study. Transcript levels of these eight genes were

assessed by qRT-PCR in the four cell lines that

showed the highest expression differences before

and after drug treatment. There was generally

good agreement between the microarray and

qRT-PCR results, although the absolute value of

the fold-changes varied somewhat randomly

between the two methodologies (Fig. 1). How-

ever, for each of these genes both techniques

showed a >5-fold average change in expression

after drug treatment.

Identification of Candidate Gene Promoter CpG

Islands Methylated in Melanoma Cell Lines

At this juncture, three different methods could

have been used to confirm the methylation status

of the candidate TSG promoters. Methylation-

specific PCR and sequencing of multiple clones

following bisulphite treatment both present some

limitations and are long processes. In contrast,

quantitative DNA methylation analysis using

mass spectrometry of base-specific cleaved ampli-

fication products (Sequenom Epityper assay) is a

rapid and comprehensive current-generation tech-

nology. The Epityper assay allows assessment of

methylation differences across a wide number of

samples and large regions of genes. It specifies

which CpGs in the gene are methylated and

quantifies the degree to which they are methyl-

ated. This technique was thus chosen to follow-

up these eight candidate genes in a large and

well-annotated panel of 46 melanoma cell lines,

as well as cultured melanocytes for comparison as

the nonmalignant control cell type. Each gene

promoter was divided into one or more ampli-

cons, which were then amplified by PCR (Supp.

Info. Table S3) and subjected to Epityper assay.

For PPP1R3C, ENC1, RARRES1, and TP53INP1,the degree of methylation and corresponding

level of expression of the gene, as assessed by

previous Affymetrix microarray analysis (Johans-

son et al., 2007), were well correlated, details of

which follow.

The CpG island in the 50UTR of PPP1R3Ccould be assessed in a single amplicon (nucleo-

tide positions �342 to þ252). This region showed

extensive methylation over the entire amplicon

(Fig. 2) with methylation of the first CpG reflect-

ing the global methylation of this region (Pearson

correlation ¼ 0.94) (Fig. 3A). Overall, there was a

good correlation between the levels of PPP1R3Cpromoter methylation and mRNA expression

(Spearman coefficient ¼ �0.823, P < 0.0005).

More than half of the melanoma cell line panel

(26/46; 57%) had a high degree of PPP1R3Cmethylation (>50% of all CpGs), which is mark-

edly different to that seen in melanocytes (�6%),

and which corresponds with low levels of tran-

script (Fig. 4). In contrast, all of the melanoma

cell lines with PPP1R3C expression higher than

melanocytes had a low (<10%) level of methyla-

tion, similar to melanocytes.

The ENC1 CpG island was divided into four

amplicons covering the 50UTR region (position

�710 to þ582) around the transcription start site.

Although Amplicons 3 and 4 did not show any

Figure 1. Comparison between microarray and qRT-PCR expression data for 8 candidate genes. Plot-ted are the mean fold-change values for the four cell lines showing the greatest differences after drugtreatment.



differential methylation between melanoma cell

lines and melanocytes (Supp. Info. Figure S1A),

Amplicons 1 and 2 presented specific profiles of

methylation in melanoma cell lines, inversely

correlated with mRNA expression (Supp. Info.

Figure S2A). Here again, the first CpG of

Figure 2. Epityper results for the PPP1R3C promoter in melano-cytes, 46 melanoma cell lines, 16 fresh melanoma tumors and celllines from other tumor types (colon, esophageal and glioma). Thesoftware uses a color coding to show the range of methylation: red

to yellow for 0 to 100% of methylation. While the melanocytes showno methylation across the amplicon, the melanoma cell lines and freshtumors present different patterns of methylation. The patterns in theother tumor types are different to that of the melanoma cell lines.

14 BONAZZI ET AL.


amplicon 1 seemed to reflect the methylation pat-

tern of the entire region (Fig. 6A). 17/46 (37%)

melanoma cell lines had a high degree of ENC1methylation (>60% of CpGs), whereas, in mela-

nocytes it was �7%. Methylation showed a good

inverse correlation (Spearman coefficient ¼�0.313, P < 0.025) with mRNA levels. In seven

(15%) melanoma cell lines, mRNA expression

was higher than melanocytes (average fold

change ¼ 1.4, range 1.05–2.22) but some of these

Figure 3. Correlation between PPP1R3C global methylation and CpG1 in (a) melanoma cell lines andmelanocytes (av. mel) (Pearson correlation: r ¼ 0.94) and (b) melanoma fresh tumors and cell lines fromother tumor types.

Figure 4. Correlation between methylation status of PPP1R3C and mRNA levels in 46 melanoma celllines and melanocytes. The vertical bar represents the separation between low level (>50% methylation)and high level (>80% methylation) in melanoma cell lines. (Spearman correlation: r¼�0.82, P< 0.0005).



cell lines still had a high level of global methylation

(up to 45% of CpGs). Comparing their methylation

profiles we observed that the first CpG (CpG1) of

amplicon 1 was not methylated in these cell lines,

highlighting the importance of this site for silencing

ofENC1 transcription in melanoma.

The 50UTR regions of both RARRES1 (�302

to þ965) and TP53INP1 (�901 to þ253) were di-

vided in to three amplicons (Supp. Info. Figures

S1–S2, B–C). These two genes showed a global

sweep of methylation throughout the region with

no specific CpG correlating with gene silencing.

Respectively, 27/46 (59%) and 16/46 (35%) mela-

noma cell lines showed low mRNA expression of

RARRES1 and TP53INP1 which was inversely

correlated with a high methylation level (>40%)

(in both cases, Spearman correlation ¼ �0.7, P <0.0005). For both genes, melanoma cell lines with

higher expression than melanocytes had a meth-

ylation profile similar to melanocytes (<22%).

The methylation status of each potential CpG

methylation site for these four genes in all mela-

noma cell lines and melanocytes is summarized

in Table 1.

Confirmation of Candidate Gene Promoter

Methylation in Fresh Tumors

To confirm that methylation of the candidate

genes was relevant to melanomagenesis in vivo

and did not occur solely as a consequence of in

vitro cell culture, we repeated the Epityper

assays on 16 fresh melanoma samples (Supp.

Info. Table S5 lists the patient information). No

genotyping has been done on these samples.

Since mRNA levels were not assessed in the

fresh tumor samples, we cannot correlate the pro-

portion of methylation with expression. However,

we used the same methylation cut offs as for the

melanoma cell lines to apply to the fresh tumors

to determine the proportion of the latter samples

that were methylated. Four (25%) of the mela-

noma tumors showed >50% CpG methylation of

the PPP1R3C promoter (Fig. 5). This frequency

is conservative because it does not take into con-

sideration stromal contamination in the primary

tumors which will likely have the effect of

decreasing the observed overall percentage of

methylation of the DNA assessed. The same cor-

relation between methylation of CpG1 and the

Figure 5. Analysis of PPP1R3C methylation status in 16 melanoma tumors compared to melanocytes.

TABLE 1. Summarization of the Proportion of Methylation Status for ENC1, PPP1R3C, RARRES1, and TP53INP1 in 46Melanoma Cell Lines and 16 Melanoma Fresh Tumors

Gene

Melanoma cell lines Melanoma fresh tumors

Number (%)

% Methylation mRNA (arbitrary units) % Methylation

Melanocytesvalue (%) Range (%)

Melanocytesvalue Range Number (%) Range (%)

ENC1 17 (37) 6.50 64.6–100 13.82 0–844.2 1 (6.25) 58.40PPP1R3C 26 (57) 5.90 55.9–95.8 485.3 2.5–271.9 4 (25) 50–64RARRES1 27 (59) 29.70 43.7–96.25 37.5 0.3–18.9 2 (12.5) 40–73TP53INP1 16 (35) 23 41.7–89.5 407.5 1.1–216.9 3 (18.75) 40–55

16 BONAZZI ET AL.


whole amplicon methylation was observed (Pear-

son correlation ¼ 0.95).

The other three candidate TSGs also showed

promoter methylation in some of the fresh

tumors. 60, 40, and 40% of the promoter CpGs in

ENC1, RARRES1, and TP53INP1 respectively

were methylated in 6, 13, and 19% of the mela-

noma tumors (Supp. Info. Figure S3).

Methylation of Candidate Gene Promoters in

Other Cancer Types

To assess the possible specificity of methyla-

tion of the four candidate genes to melanoma, we

repeated the same Epityper assays on a limited

number cell lines from cancers of the colon,

esophagus, and brain (glioma). These different

cancer types were assessed to determine whether

the candidate genes might play a more general

role in tumor suppression. Two cell lines from

each tumor type were assessed for their methyla-

tion status for PPP1R3C, ENC1, RARRES1, andTP53INP1. Using the same cut-off as for the mel-

anoma cell lines and tumors, none of these cancer

cell lines were methylated for ENC1 and

RARRES1, but the two colon cancer cell lines

(Col15 and LIM2405) showed >50% methylation

for PPP1R3C, and the two esophageal cancer cell

lines (OE19 and OE33) were methylated (>40%)

for TP53INP1 (Table 2).

From scanning the CpGs of PPP1R3C and

ENC1 amplicon 1, it was apparent that the first

CpG reflected the global methylation pattern.

However, it should be noted that the first CpG in

amplicon 1 does not reflect the first CpG site of

the gene, it only refers to the first CpG site in

the amplicons studied here. As for PPP1R3C (see

above), CpG1 methylation of ENC1 was highly

correlated with global methylation of this region

(Pearson correlation ¼ 0.87), and which in turn

was inversely correlated with transcript levels in

melanoma cell lines (Fig. 6). In contrast, in the

other cancer cell lines CpG1 methylation showed

no correlation with the global methylation pattern

(Figs. 3B–6B). This may indicate that the associa-

tion between methylation at this site and gene

silencing could potentially be pigment cell line-

age specific. The methylation status of each

potential CpG methylation site for the four candi-

date genes in all melanoma cell lines, melano-

cytes, melanoma tumor samples, and the other

cancer types is summarized in Figure 2 and

Supp. Info. Figure S1.

DISCUSSION

The objective of this study was to identify

novel TSGs inactivated by promoter methylation

in melanoma. We used a microarray-based strat-

egy in a panel of melanoma cell lines treated

with 5AzadC and TSA as an initial screening

approach. Selected candidate genes were fol-

lowed up using the Epityper assay in a much

larger panel of melanoma cell lines, as well as a

panel of fresh melanoma samples, melanocyte

cultures, and cell lines from other cancer types.

We identified four genes that were not previously

known to be silenced by DNA methylation in

melanoma.

PPP1R3C encodes a protein phosphatase 1

(PP1) regulatory subunit which forms complexes

with glycogen phosphorylase, glycogen synthase,

and phosphorylase kinase necessary for its regula-

tion of PP1 activity. Little is known about its

function and its potential deregulation in human

cancer. In the present study, the PPP1R3C CpG

island presented a high proportion of methylation

(>50%) in 57% of the melanoma cell lines tested.

This was well correlated with concomitant down-

regulated expression of this gene. PPP1R3C was

also found to be methylated in 25% of melanoma

tumors. The difference in CpG1 methylation

TABLE 2. Analysis of Methylation Status of ENC1, PPP1R3C, RARRES1, and TP53INP1 in Other Kind of Cancers,such as Colon Cancer, Esophageal Cancer, and Glioma

Cancer types Cell lines ENC1 PPP1R3C RARRES1 TP53INP1

Colon cancer Col15 U M U ULIM2405 U M U U

Esophageal cancer OE19 U U U MOE33 U U U M

Glioma T46 U U U UT50 U U U U

We used the same cutoff determined from melanoma cell lines and tumors in Table 1, 60% for ENC1, 50% for PPP1R3C, and 40% for RARRES1 and

TP53INP1.



between samples of the melanocyte lineage and

other cancer types may reflect differential tran-

scription factor occupancy of this site. Interest-

ingly, CpG1 is part of an Sp1 transcription factor

binding site, raising the possibility that Sp1 may

have different effects on regulating transcription

of PPP1R3C in various cell types.

ENC1 belongs to the family of p53-induced

genes (it is also known as PIG10) and encodes an

actin-binding protein involved in differentiation

of neural crest, colon and far cells (Zhao et al.,

2000). ENC1 is highly expressed in adult brain

and spinal cord, and in numerous cell lines

derived from nervous system tumors low mRNA

levels were detectable (Hernandez et al., 1998).

Although initially described as a TSG in neuro-

blastoma, high levels of ENC1 expression have

been described in medulloblastoma (Yokota et al.,

2004), parathyroid adenomas (Forsberg et al.,

2005), hairy cell leukemia (Hammarsund

et al., 2004), glioblastomas and astrocytomas (Kim

et al., 2000), and colon cancer (Fujita et al.,

2001), indicating that it may more commonly

function in an oncogenic manner if inappropri-

ately expressed. In our study, ENC1 was highly

expressed in melanocytes while its expression

was decreased in melanoma cell lines in parallel

with a high methylation percentage. The ENC150UTR was highly methylated (>60%) in 37% of

the melanoma cell lines and in 6% of the fresh

melanomas. We have identified a CpG site with

which the methylation status of the entire ENC150UTR region is highly correlated. Further confir-

mation is required as is the need to correlate

mRNA levels with protein expression. Functional

experiments are also needed to confirm ENC1 as

a tumor suppressor gene in melanoma.

RARRES1 (retinoic acid receptor responder ¼tazarotene induced gene 1, TIG1) was identified

as a retinoic acid receptor-responsive gene (Nag-

pal et al., 1996). A tumor suppressor role for

RARRES1 has been reported in prostate cancer

(Jing et al., 2002) as its expression decreased in

vitro invasiveness and in vivo tumorigenicity. A

recent study has associated RARRES1 promoter

hypermethylation with gastric carcinoma (Shutoh

et al., 2005) and Youssef et al., have shown its

down-regulation in different cancer cell lines is

related to levels of >35% methylation of the

promoter (Youssef et al., 2004). The authors

Figure 6. Correlation between ENC1 global methylation and CpG1 in (a) melanoma cell lines andmelanocytes (av. mel) (Pearson correlation: r ¼ 0.87) and (b) melanoma fresh tumors and cell lines fromother tumor types.

18 BONAZZI ET AL.


concluded that silencing of RARRES1 by pro-

moter hypermethylation is common in human

cancers and may contribute to the loss of retinoic

acid responsiveness in some neoplastic cells. In

the present study, we have shown for the first

time that RARRES1 methylation and expression

is associated with malignant melanoma. The

RARRES1 promoter was highly methylated

(>40%) in 59% of melanoma cell lines, and this

was associated with a down-regulation of its

mRNA expression. The gene was also methyl-

ated in 13% of the fresh tumors tested.

The TP53INP1 gene (tumor protein 53-

induced nuclear protein 1), cloned by three dif-

ferent teams (Carrier et al., 1999; Okamura et al.,

2001; Tomasini et al., 2001), encodes a p53-in-

ducible protein which promotes apoptosis and

cell cycle arrest in G1 phase (Tomasini et al.,

2003). Decreased TP53INP1 expression has been

described in breast carcinoma (Ito et al., 2006b),

anaplastic carcinoma of the thyroid (Ito et al.,

2006a) and gastric cancer, where its loss was

inversely correlated with tumor size, positive

lymph node metastasis and aberrant p53 expres-

sion (Jiang et al., 2006). In our study, >40%

methylation of the TP53INP1 50UTR region was

correlated with a decrease of its mRNA expres-

sion in 35% of our panel of melanoma cell lines

and in 18% of the uncultured melanomas.

It should be noted that similarly to the samples

reported by Furuta et al., (Furuta et al., 2004),

none of our cell lines showed PTEN promoter

hypermethylation, which has been shown or sug-

gested in some melanomas (Zhou et al., 2000;

Mirmohammadsadegh et al., 2006). Similarly,

using the criterion of a minimum 2-fold change

after drug treatment, we have no evidence that

the p14ARF promoter is methylated in any of the

7 cell lines tested in which the gene is not homo-

zygously deleted. This observation contrasts with

a recent study (Freedberg et al., 2008) describing

methylation of the p14ARF promoter in some

melanoma cell lines and metastases.

To date, three different studies have reported

microarray-based screening following treatment of

uveal or cutaneous melanoma cell lines with the

DNA methylation inhibitor 5-aza-20-deoxycyti-dine (5AzadC) (van der Velden et al., 2003;

Gallagher et al., 2005; Muthusamy et al., 2006) to

find new TSGs silenced by methylation. Van der

Velden et al., (van der Velden et al., 2003) identi-

fied 19 genes, including TIMP3 (tissue inhibitor

of metalloproteinase 3) and TYRP1 (tyrosinase

related protein 1), that were differentially ex-

pressed between a demethylated derivative clone

of a primary uveal melanoma cell line and its

untreated control. Gallagher et al., (2005) com-

pared different highly tumorigenic derivative

melanoma cell lines to their poorly tumorigenic

parental cell lines (established from cutaneous su-

perficial spreading melanomas). This group

focused on TSPY, a Y chromosome specific gene

normally expressed in the germ cells of the testis,

which was hypermethylated in the derivative cell

lines. The third study, from Muthusamy et al.,

(2006) showed the tumor suppressor properties of

HOXB13 and SYK, which were inhibited by

methylation in 89% of the melanoma cell lines

analyzed. There are some significant limitations

to each of these studies. Firstly, they each ana-

lyzed only a small number of samples (n ¼ 1, van

der Velden et al., n ¼ 3, Gallagher et al., n ¼ 6,

Muthusamy et al.). Secondly, they did not com-

bine 5AzadC treatment with that of the histone

deacetylase inhibitor Trichostatin A (TSA) - for

which a synergy between demethylation and his-

tone deacetylase inhibition has been showed for

the re-expression of genes silenced in cancer

(Cameron et al., 1999; Baylin et al., 2001).

Thirdly, they chose to confirm the methylation

status of the candidate TSGs by bisulfite

sequencing, which is time consuming and

requires a large number of clones to be

sequenced for accurate representation of the

DNA methylation profile. In contrast, we have

used Illumina genome-wide expression arrays

which are rapid, cost effective, straightforward,

and can be applied to large number of samples

using a range of drug treatments. However, this

screening cannot be done using uncultured sam-

ples. The Epityper assay we have used is a sensi-

tive and high-throughput method of DNA

methylation analysis which is much more quanti-

tative than other methods, less subjective to the

limitation in the number of clones analyzed and

favorable to a high-throughput experiment as it is

less time consuming and more cost-effective.

(Ehrich et al., 2005; Coolen et al., 2007). A num-

ber of CpG rich regions can be interrogated, large

numbers of samples can be screened cost-effec-

tively for a number of gene promoters simultane-

ously. Nevertheless, there are some limitations to

the approach, for example, the methylation status

of different alleles cannot be easily determined

and sometimes not all CpGs within an amplicon

are able to be detected because of overlapping

fragment masses. However, this study demon-

strates the advantages of using such a global



approach to identify novel melanoma TSGs.

These require validation in larger panels of fresh

tumors as well as functional characterization of

their potential roles in melanoma development.

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