Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC

Authors


Abstract

Aberrant activation of the Wnt signaling pathway has been reported in different human tumor types, including malignant melanomas. We investigated 37 malignant melanomas (15 primary tumors and 22 metastases) for alterations of 4 genes encoding members of this pathway, i.e.,CTNNB1 (β-catenin gene, 3p22.1), APC (adenomatous polyposis coli gene, 5q22.2), BTRC (β-transducin repeat–containing protein gene, 10q24.3) and ICAT (inhibitor of β-catenin and Tcf-4, 1p36.2). Mutational analysis of CTNNB1 identified somatic mutations in 1 primary melanoma and 1 melanoma metastasis from 2 different patients (5%). Both mutations affected the N-terminal degradation box of β-catenin, which is important for the regulation of β-catenin homeostasis. Another primary melanoma carried a somatic APC missense mutation within the known mutation cluster region in exon 15. Fourteen tumors (40%) showed LOH at microsatellite markers on 1p36. None of the tumors had lost both copies of the ICAT gene, but 1 melanoma metastasis carried a somatic point mutation altering the translation start codon of ICAT. Real-time RT-PCR showed markedly reduced ICAT transcript levels (≤20% relative to normal skin and benign melanocytic nevi) in 28/36 malignant melanomas (78%), including 13/14 tumors with LOH on 1p36. Allelic loss on 10q was detected in 15 tumors (44%). We found neither mutations nor complete loss of expression of the BTRC gene in our melanoma series. Taken together, our results indicate that the Wnt pathway may be altered in malignant melanomas by different mechanisms, including rare somatic mutations in CTNNB1, APC or ICAT, as well as low or absent expression of ICAT transcripts. © 2002 Wiley-Liss, Inc.

Abbreviations:

ALM, acral lentiginous melanoma; ARF1, ADP-ribosylation factor 1 (gene); APC, adenomatous polyposis coli; B2MG, β2-microglobulin; Btrc, β-transducin repeat–containing (protein); CMM, cutaneous melanoma metastasis; Gsk, glycogen synthase kinase; Icat, inhibitor of β-catenin and Tcf-4; Lef-1, lymphoid-enhancing factor 1; LOH, loss of heterozygosity; MAb, monoclonal antibody; NM, nodular melanoma; SSCP, single-strand conformation polymorphism; SSM, superficial spreading melanoma; Tcf, T-cell factor.

Cutaneous malignant melanomas (CMMs) have shown a marked increase in both incidence and mortality over the past decades.1 The genetic alterations associated with the development and progression of these tumors are still poorly understood. Cytogenetic and molecular genetic studies have implicated a number of chromosomal and genetic changes in melanoma pathogenesis.2, 3 The genes known to be aberrant in variable subsets of malignant melanomas include tumor-suppressor genes, such as CDKN2A and PTEN, as well as protooncogenes, such as CDK4, NRAS and MYCC.2–4 A previous study showed that melanoma cell lines frequently carry mutations in the β-catenin gene (CTNNB1).5 The β-catenin protein is a central component of the Wnt (wingless) signal-transduction pathway, which plays an important role in development and tumorigenesis.6–8 The Wnt signal is transduced through cell membrane–associated receptors of the frizzled family and stabilizes β-catenin, which then enters the cell nucleus and forms a complex with members of the Tcf/Lef-1 family of transcription factors.7, 8 Activation of Tcf/Lef-1 by binding to β-catenin induces the transcription of various target genes, including protooncogenes such as MYCC and CCND1.9, 10 Within the cell nucleus, the activity of the β-catenin–Tcf/Lef-1 complex can be inhibited by the protein Icat (inhibitor of β-catenin and Tcf-4), which blocks the interaction between β-catenin and Tcf-4 and thereby antagonizes Wnt signaling.11 In addition, the level of β-catenin in the cell is tightly regulated by a multiprotein complex composed of the APC tumor-suppressor protein, axin and Gsk-3β.6–8 In the absence of Wnt signal, this complex promotes the phosphorylation of serine and threonine residues in the amino-terminal region of β-catenin by Gsk-3β. Phosphorylated β-catenin can be bound by the F-box and WD40 domain–containing protein Btrc, also known as β-Trcp,12 and is thereby targeted for degradation via the ubiquitin/proteasome pathway.13–15

Oncogenic activation of β-catenin by amino acid substitutions or deletions affecting its N-terminal degradation box has been demonstrated in various human tumors, including melanoma cell lines,5 as well as a small fraction of primary melanomas.16, 17 Individual melanoma cell lines with APC mutations have also been reported.5 The BTRC and ICAT gene loci map to the long arm of chromosome 10 (10q24.3) and the short arm of chromosome 1 (1p36.2), respectively.11, 18 Both regions are frequently affected by LOH in melanomas.2, 19–22 Furthermore, a familial melanoma gene locus has been linked to 1p36.23 Thus, in addition to CTNNB1 and APC, the BTRC and ICAT genes are interesting candidates for melanoma-associated tumor-suppressor genes. To better define the role of alterations in Wnt signaling pathway genes in the pathogenesis of melanomas, we determined the expression of β-catenin in a panel of sporadic melanomas from 37 patients and analyzed the CTNNB1, APC, ICAT and BTRC genes in these tumors for alterations at the gene and transcript levels.

MATERIAL AND METHODS

Patients

We investigated sporadic malignant melanomas from 37 patients (12 male and 25 female, mean age 67 years) operated on at either the Department of Dermatology or the Department of Neurosurgery, Heinrich-Heine-University. The tumor series included 15 primary cutaneous melanomas (7 NMs, 5 ALMs and 3 SSMs) and 22 melanoma metastases (15 CMMs, 1 regional lymph node melanoma metastasis, 1 spinal and 5 intracerebral melanoma metastases). The Clark level and tumor thickness of the primary melanomas are listed in Table I. Parts of each tumor were frozen immediately after operation and stored at –80°C. The tumor cell content of each specimen was histologically evaluated (Table I). Peripheral blood samples for the extraction of constitutive DNA were available from 34 patients.

Table I. Summary of Patient Data and Molecular Genetic Results
Tumor no.Tumor type1Clark level/ thickness (mm)Age (years)SexLocalizationTumor cell content (%)LOH on 1p362ICAT mutation3ICAT expression real-time4ICAT expression duplex4LOH on 10q2BTRC mutation3BTRC expression real-time4BTRC expression v1/v25CTNNBI mutation3β-Catenin nuclear staining6β-Catenin cytoplasmic staining6β-Catenin membrane staining6β-Catenin negative cells6APC mutation3
  • 1

    ALM, acral lentiginous melanoma; CMM, cutaneous melanoma metastasis; i.c. MM; intracerebral melanoma metastasis; l.n. MM; melanoma metastasis in a regional lymph node; NM, nodular melanoma; PM, polyploid melanoma; spinal MM; spinal melanoma metastasis; SSM, superficial spreading melanoma.

  • 2

    LOH, loss of heterozygosity; +, LOH at one or more microsatellite markers analyzed from 1p36; −, no LOH detected at the 1p36 microsatellite markers investigated; n.a., not analyzed.

  • 3

    −, no mutation detected; n.a., not analyzed.

  • 4

    Values indicate the expression level in tumor relative to the mean expression level determined for 2 normal skin samples and 4 benign melanocytic nevi.

  • 5

    BTRC transcript variant 1 (v1) to BTRC transcript variant 2 (v2) ratio as determined by reverse transcription-PCR.

  • 6

    Presence of melanoma cells with nuclear, cytoplasmic, cell membrane–associated or absent β-catenin immunoreactivity was estimated separately for each case and scored as follows: +, >10% positive tumor cells; (+) <10% positive tumor cells, −, no positive tumor cells detected.

  • 7

    The percentage in parentheses refers to microdissected specimens.

M5ALMIV/4.068FPlantar700.10.30.91.2(+)++
M13ALMIII/1.467FPlantar600.70.9+1.51.1++
M15ALMIV/5.580FPlantar70 (>90)70.30.6+1.11.0++
M48ALMIV/1.775FPlantar70n.a.n.a.n.a.n.a.n.a.++
M21SSMIV/1.755FLeg801.4n.a.0.90.9+++c.4431G>T(Q1477H)
M37SSMIV/1.187FNeck60 (>90)70.30.3+1.21.2+++
M9NMIV/4.570MLeg700.20.20.61.2(+)+
M12NMIV/3.974FShoulder800.20.21.00.9++
M19NMV/4.675FLeg900.20.10.61.0+++
M24NMIV/4.957FFoot800.70.80.51.1++(+)
M29NMIV/2.929FAbdomen80+0.40.20.61.0+++
M39NMIV/6.068MAbdomen80+0.1<0.1+0.41.1c.48del289(del exon2-3)++
M47NMIV/2.272FNeck800.20.2+0.61.0+
M54NMIV/1.577MShoulder700.60.61.10.9++
M55PMV/3564FAbdomen90+0.20.2+0.91.1++
M1CMM65MChest90+0.1<0.11.60.9++(+)+
M2CMM63FLeg800.1<0.10.81.2+(+)
M3CMM85FScalp80+0.1<0.10.81.1(+)+
M4CMM72FLeg800.20.10.61.1++(+)
M10CMM78FInguinal80+0.20.1+0.90.8(+)++
M14CMM66MArm80+0.1<0.10.80.9(+)+
M17CMM71MSupraclavicular700.20.10.70.9++
M18CMM59FAbdomen900.1<0.10.81.0(+)++
M23CMM86FLabia majora80+<0.1<0.10.71.0+++
M26CMM69FChest80n.a.0.1<0.1n.a.0.31.0+++
M38CMM83FLeg800.20.2+0.60.8++
M45CMM40MLeg80+0.20.2+1.31.0+(+)n.a.
M46CMM63FAbdomen80+c.1A>G (M1-E13del)0.10.1n.a.0.61.1(+)+
M50CMM88FLeg700.91.0+0.90.9(+)+
M64CMM72MAxilla900.1<0.10.81.0(+)(+)+
M11l.n. MM43MAxilla800.1<0.11.01.5++
M32Spinal MM76FSpinal80+0.10.2+0.50.9(+)++
M33i.c. MM69MCerebral80n.a.0.20.1n.a.0.51.1+++
M34i.c. MM53FCerebral90+0.10.1+0.60.8(+)+
M35i.c. MM76FCerebral70+0.10.2+0.80.9c.133delTCT (S45del)++
M36i.c. MM60MCerebral80+0.20.2+0.50.8(+)++
M44i.c. MM72MCerebral900.10.1+1.30.9+++

DNA and RNA Extraction

DNA and RNA were extracted from frozen tumor tissue by ultracentrifugation as described elsewhere.24 DNA was extracted from leukocytes according to a standard protocol.25 From 2 cases (M15, M37), DNA was additionally extracted from formalin-fixed and paraffin-embedded specimens after microdissection of tumor areas with a high tumor cell content (>90%). In M46, constitutional DNA for LOH analysis on 1p36 was purified from microdissected, microscopically tumor-free epidermal tissue.

LOH Analysis

The details of the method used to assess LOH were reported elsewhere.26 LOH on chromosome band 1p36 was analyzed by evaluating the following 8 microsatellite loci: D1S468 (1p36.32), D1S1608 (1p36.32), D1S2870 (1p36.32), D1S2666 (1p36.31), D1S214 (1p36.31), D1S503 (1p36.23) (all located distal to the ICAT locus), D1S489 (1p36.22) and D1S507 (1p36.21) (which map centromerically to the ICAT locus). Chromosome 10 was studied for LOH at the following loci: D10S249 (10pter), D10S215 (10q23), D10S541 (10q23), D10S209 (10q25), D10S587 (10q25-26) and D10S212 (10q26). LOH data on chromosome 10 were reported before.22

Analysis for Homozygous ICAT Deletion

All tumors were screened for homozygous deletions involving the ICAT gene by duplex-PCR analysis using primers for exon 3 of ICAT together with primers for the APRT gene on 16q24.3. The respective primer sequences are listed in Table II. PCR was performed with 20 ng of genomic DNA as template. An initial denaturation of 5 min at 95°C was followed by 28 cycles of 20 sec at 95°C, 20 sec at 56°C and 20 sec at 72°C. The final extension reaction was performed for 5 min at 72°C. PCR products were separated on 3% agarose gels, and ethidium bromide–stained bands were recorded with the Gel-Doc 1000 system (Bio-Rad, Hercules, CA). Quantitative analysis of the signal intensities obtained for the target gene and the reference gene was performed with the Molecular Analyst software (version 2.1, Bio-Rad). Only reductions in the target/reference gene ratio of <0.3 relative to the target/reference ratio obtained for the constitutional DNA were considered as indicative of a homozygous ICAT deletion.

Table II. Summary of Oligonucleotide Primers Used for SSCP/Heteroduplex Analyses of BTRC and ICAT (Application 1), Screening for Homozygous ICAT Deletion (Application 2), Expression Analysis of BTRC and ICAT by Real-Time RT-PCR (Application 3), Expression Analysis of ICAT by Duplex RT-PCR (Application 4) and Relative Expression of BTRC Transcript Variants 1 and 2 (Application 5)
Primer namePrimer sequenceFragment length (bp)Application
BTRC-RT/F5′-gtg gcc tcg gcg att atg gac-3′321 (variant 1)1, 5
BTRC-RT/R5′-cag tct tca tag cag tgc ttg c-3′213 (variant 2) 
BTRC-1/F5′-aca gta tgt tta gca agc act gc-3′2281
BTRC-1/R5′-gtg ccc atg ttg gta atg aca c-3′  
BTRC-2/F5′-gca gtg gtc aga gtc aga tc-3′2031
BTRC-2/R5′-gtt cag cag cac ata gtg att tg-3′  
BTRC-3/F5′-gag aac att ctg tca tac ctg g-3′2071
BTRC-3/R5′-agc att ccc gtc agg agg -3′  
BTRC-4/F5′-ggc aga acg aag agg atg-3′2351
BTRC-4/R5′-aag gcc gct tac tat ttt ctg-3′  
BTRC-5/F5′-gga gtt tac tgt tta cag tat g-3′2331
BTRC-5/R5′-ggt gaa tca acg tgt tta gc-3′  
BTRC-6/F5′-cgg tca gag tgt ggg atg-3′2271
BTRC-6/R5′-aat gta ctt gtc atc aaa gtc tac-3′  
BTRC-7/F5′-acc gag ctg ctg tca atg-3′2451
BTRC-7/R5′-ctc atg gcc ttc taa cac tc-3′  
BTRC-8/F5′-gac ata gaa tgt ggt gca tg-3′1981
BTRC-8/R5′-tct tcc gga atg ctc cac-3′  
BTRC-9/F5′-cag gga cac tct gtc tac-3′2151
BTRC-9/R5′-agt atg agg tca gtg tat gg-3′  
BTRC-TAQ-F35′-ctg cag gga cac tct gtc tac-3′1223
BTRC-TAQ-R35′-gaa gtc cca gat gag gat tgt g-3′  
ICAT-1/F5′-cag ctc tca ggc aga gca ag-3′2011
ICAT-1/R5′-gga gga tga gtg gct tct gc-3′  
ICAT-2/F5′-ggt tgt gca tcc tca gca tgg-3′2381
ICAT-2/R5′-ccc aca cta ctg tat ggc cac-3′  
ICAT-3.1/F5′-cgg ctc ctg gga gga gtg ag-3′1501
ICAT-3.1/R5′-ccc atc ttc cgc agc atg ag-3′  
ICAT-3.2/F5′-gga aga gtc cgg agg aga tg-3′1621, 2
ICAT-3.2/R5′-gtg gct cca ccc tcc aat ag-3′  
ICAT-4/F5′-cag gga aca ggt gca tgc tg-3′2051
ICAT-4/R5′-ggg aac cac aag tcg gtg cc-3′  
ICAT-5/F5′-ctc aga gag tgc cgc gat cc-3′2421
ICAT-5/R5′-cca gga gcc aca cag atc tc-3′  
ICAT-TAQ/F5′-gaa gag tcc gga gga gat gt-3′653
ICAT-TAQ/R5′-cca atc ttc cgc agc atg ag-3′  
ICAT-RT/F5′-gag cac ctg ttt gcc tga ag-3′3474
ICAT-RT/R5′-gcc ctt caa cag cat cca gg-3′  
APRT-1/F5′-tgg gaa agc tgt tta ctg cg-3′1362
APRT-2/R5′-cag gga aca cat tcc ttt gc-3′  
ARF1-TAQ/F15′-gac cac gat cct cta caa gc -3′1113
ARF1-TAQ/R35′-tcc cac aca gtg aag ctga tg-3′  
B2MG-RT/F25′-ctc gct ccg tgg cct tag-3′3804
B2MG-RT/R5′-atc ttc aaa cct cca tga tg-3′  

SSCP/heteroduplex Analysis and DNA Sequencing

The mutation cluster region within exon 15 (codons 1255–1641) of the APC gene was amplified by PCR in 6 overlapping fragments, using the oligonucleotide primers and PCR conditions reported before.27 PCR products were screened for mutations by SSCP/heteroduplex analysis as described.27 Each fragment was evaluated under at least 2 different gel or temperature conditions. Exon 3 of CTNNB1 was amplified by PCR as described,27 followed by SSCP/heteroduplex analysis. For the detection of intragenic CTNNB1 deletions affecting exon 3, cDNA or genomic DNA from tumor specimens was amplified using primer pairs flanking exon 3 or exons 2–4.27 The entire BTRC coding region was amplified by RT-PCR from each tumor and then subjected to SSCP/heteroduplex analysis. For mutation analysis of ICAT, exons 1–4 and the 5′ coding part of exon 5 were amplified by PCR from genomic DNA. The respective primer sequences are listed in Table II. PCR products with aberrant SSCP/heteroduplex patterns were sequenced in both directions. The somatic origin of the detected mutations was confirmed by sequencing of the respective patient's constitutive (leukocyte) DNA.

Expression Analyses at the mRNA Level

Expression of BTRC and ICAT transcripts was determined by real-time RT-PCR using the ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA), which allows continuous measurement of the PCR product amount by means of SybrGreen fluorescent dye. BTRC and ICAT mRNA levels were normalized to the transcript levels of the housekeeping gene ARF1. Primer sequences used for real-time RT-PCR assays are listed in Table II. As nonneoplastic reference tissues, normal skin samples from 2 adults were used for expression analyses. As a further reference, we determined the BTRC and ICAT mRNA expression levels in 4 benign nevi from 4 different individuals. In addition to real-time PCR experiments, we evaluated ICAT mRNA expression by duplex RT-PCR, amplifying a 347 bp fragment spanning the entire ICAT coding sequence together with a 380 bp fragment from the B2MG transcript (for primer sequences, see Table II). The GenBank database contains sequences for 2 distinct transcript variants of BTRC, i.e, transcript variant 1 (accession NM_033637) and transcript variant 2 (accession NM_003939). Variant 1 carries an additional fragment of 108 nucleotides within the coding region, which is absent in variant 2, and encodes an in-frame 36–amino acid longer isoform of Btrc than variant 2. Relative expression of these 2 transcript variants was assessed by simultaneous RT-PCR amplification of both mRNAs, using primers located proximally and distally to the insertion in variant 1 (for primer sequences, see Table II).

Immunohistochemistry

Expression of β-catenin was immunohistochemically determined on formalin-fixed, paraffin-embedded tumor sections using the anti-β-catenin MAb 14 (Transduction Laboratories, Lexington, KY) and an established immunohistochemic protocol.27 Antibody binding was visualized with the avidin-biotin-peroxidase complex method and 3′3′-diaminobenzidine-tetrahydrochloride as chromogen. All sections were counterstained with hematoxylin.

RESULTS

Immunohistochemistry for β-catenin revealed the typical membrane-associated expression pattern in the nonneoplastic epidermis adjacent to the investigated primary malignant melanomas and the cutaneous melanoma metastases (Fig. 1a,c,f). The melanoma cells themselves showed heterogeneous β-catenin staining patterns (Table I, Fig. 1a–f). Membrane-associated immunoreactivity was retained in variable fractions of tumor cells in the majority of cases, with individual tumors demonstrating predominantly membranous labeling (Fig. 1e). Most tumors, however, showed additional or predominant cytoplasmic immunoreactivity (Fig. 1a,b). Seven malignant melanomas contained tumor cells with nuclear β-catenin accumulation (Fig. 1b–d). In addition, variable fractions of immunonegative melanoma cells, ranging from <10% to 80% of the entire tumor cell pool represented in the investigated tissue sections, were seen in 16 tumors (Table I, Fig. 1f).

Figure 1.

Representative results of immunohistochemic stainings for β-catenin in melanomas. (a) Strong cytoplasmic immunoreactivity in tumor M39, a primary melanoma that carried a deletion of exons 2 and 3 of CTNNB1 (see Fig. 2c,d). Note the membranous staining in the adjacent epidermis on the right. (b) Cytoplasmic and partially nuclear expression of β-catenin in primary melanoma M21, which carried an APC missense mutation (see Fig. 2e,f). (c,d) Nuclear β-catenin accumulation in tumor M19, an NM (c) and tumor M4, a CMM (d). Both tumors lacked detectable CTNNB1 or APC mutations. (e) Predominantly cell membrane–associated staining pattern in a CMM (M10) without a demonstrated CTNNB1 or APC mutation. (f) A tumor area with β-catenin-negative melanoma cells in a CMM (M50). Membranous staining in the adjacent epidermis serves as internal positive control for the immunostaining procedure. All sections were counterstained with hematoxylin.

SSCP/heteroduplex analysis followed by DNA sequencing identified 2 melanomas with mutations in the CTNNB1 gene, both of which demonstrated strong cytoplasmic but no nuclear β-catenin staining (Table I, Fig. 1a). Intracerebral melanoma metastasis M35 carried a somatic CTNNB1 mutation resulting in deletion of codon 45 (c.133delTCT:S45del) (Fig. 2a,b). Tumor M39, an NM, expressed an aberrant transcript with deletion of exons 2 and 3 (c.-48del289; Table I, Fig. 2c,d). The identical mutation was identified in a regional lymph node melanoma metastasis (M49) obtained from the same patient (Fig. 2c). One primary melanoma (M21) carried an APC missense mutation affecting codon 1471 (c.4411G>T:A1471S) (Table I, Fig. 2e,f). Immunohistochemically, this particular tumor showed focal nuclear β-catenin accumulation (Fig. 1b).

Figure 2.

Demonstration of the mutations detected in CTNNB1 (a–d), APC (e,f) and ICAT (g,h) in malignant melanomas. (a) SSCP/heteroduplex analysis showed aberrant bands (arrows) in melanoma M35 (lane 4) compared to 3 other melanomas that demonstrated the wild-type pattern (lanes 1–3). (b) DNA sequencing revealed an in-frame deletion of 3 bp (c.133delTCT:S45del) in 1 CTNNB1 allele of M35. Arrow indicates the first deleted nucleotide. (c) RT-PCR analysis of tumor M39 (lane 1) detected a shorter PCR fragment of 531 bp (arrow) in addition to the normal PCR fragment of 817 bp. The same fragment was also detected in a cutaneous metastasis of M39 (M49, lane 2) but not in normal skin (lane 3). (d) DNA sequencing revealed that the aberrant band was due to deletion of exons 2 and 3 from the CTNNB1 transcript. (e) SSCP/heteroduplex analysis of APC demonstrated aberrant bands (arrows) in tumor M21 (lane 1) compared to the same patient's constitutional DNA (lane 2) and 2 wild-type controls (lanes 3, 4). (f) DNA sequencing identified a point mutation (c.4431G>T) that translates into an amino acid exchange from glutamine to histidine at codon 1477. The sequence shown is derived from reverse sequencing of the coding strand. (g)ICAT mutation analysis revealed an aberrant SSCP band (arrow) in melanoma metastasis M46 (lane 3), which was absent in this patient's constitutive DNA (lane 4), as well as in 2 unrelated melanomas (lanes 1, 2). (h) M46 carried an ICAT point mutation, affecting the first nucleotide in the start codon (c.1A>G:M1-E13del).

Microsatellite analysis demonstrated LOH at all informative markers from 1p36 in 14 melanomas (40%, Table I). In all instances, the deleted region spanned the ICAT gene locus at 1p36.2. SSCP/heteroduplex analysis of ICAT identified an aberrant band pattern in 1 CMM (M46) with LOH on 1p36. This tumor carried a somatic mutation altering the translation start codon of ICAT (c.1A>G:M1-E13del) (Fig. 2g,h). In addition, an intronic single base pair insertion polymorphism (IVS3+58insG) was detected in one of our patients. None of the tumors showed evidence of homozygous ICAT deletion by duplex-PCR analysis. Real-time RT-PCR revealed expression of ICAT transcripts at approximately equal levels in normal skin and each of the 4 benign melanocytic nevi analyzed. However, ICAT transcript levels were reduced to ≤20% of the mean transcript level determined for the 4 benign melanocytic nevi and normal skin in 28/36 investigated melanomas (78%), including 13 of the 14 tumors with demonstrated LOH at microsatellite markers from 1p36 (Table I, Fig. 3a). Three additional melanomas showed ICAT transcript levels reduced to 30–50% of reference tissue values (Table I). Duplex RT-PCR analysis using a 347 bp fragment spanning the entire ICAT coding sequence together with a 380 bp B2MG fragment revealed identical or very similar results as determined by real-time RT-PCR (Table I, Fig. 3b). The differences in the relative expression levels seen in individual tumors (M5, M15 and M29; Table I) may be explained by the use of 2 distinct reference transcripts (ARF1 and B2MG) in the respective assays. There was no apparent relation between the ICAT transcript level and nuclear immunoreactivity for β-catenin (Table I).

Figure 3.

Analysis of melanomas for expression of ICAT transcripts. (a) Demonstration of markedly reduced ICAT mRNA expression in the CMM M64 relative to the benign melanocytic nevus N7. Abscissa, cycle number; ordinate, amount of PCR product. While the curves for the reference mRNA (ARF1, lower panel) pass the threshold (Ct) value at an approximately equal cycle number in both tumors, the ICAT mRNA curve (upper panel) obtained for M64 is shifted to the right relative to that obtained for N7. The calculated ICAT mRNA expression level in M64 was 0.1 relative to N7 (whose expression value represented the mean value determined for normal skin and 4 melanocytic nevi). (b) Example of ICAT mRNA expression analysis by duplex RT-PCR using B2MG mRNA expression as reference. Lanes: 1 and 2, normal skin samples from 2 different individuals; 3, M3; 4, M34; 5, M46; 6, M64; 7, M13; 8, M50; 9–11, benign melanocytic nevi (N1, N7, N10) from 3 different individuals; 12, no template control. The length of individual PCR products is indicated on the right. Note that ICAT mRNA is expressed at approximately equal levels in normal skin, all benign melanocytic nevi and 2 melanomas (M13, M50). In contrast, 4 melanomas (M3, M34, M46, M64) show markedly reduced signals for ICAT mRNA. Quantitative densitometric analysis of ICAT mRNA signal intensity relative to B2MG mRNA signal intensity revealed that each of these 4 melanomas had an ICAT transcript level of ≤0.1 relative to the level determined for normal skin and benign melanocytic nevi.

As reported before,22 we detected allelic losses at all informative loci analyzed on 10q in the tumors from 15 patients, including 6 primary melanomas and 9 melanoma metastases (Table I). SSCP/heteroduplex analysis of the BTRC gene revealed 5 single-nucleotide sequence polymorphisms (c.684C>T, c.953A>C, c.1276A>G, c.1350T>G, c.1588G>T) but no tumor-associated mutations. We also screened for the intragenic BTRC deletions reported in prostate cancer by combining primers BTRC-RT/F with BTRC-1/R (spanning the 168 bp deletion reported in the TSU cell line) and BTRC-2/F with BTRC-5/R (spanning the 96 bp deletion reported in a prostate cancer xenograft).28 None of our melanomas expressed BTRC transcripts with the 2 reported deletions or any other detectable deletions. Real-time RT-PCR analysis showed that BTRC transcripts were expressed at similar levels in each of the 4 benign melanocytic nevi and the 2 normal skin samples. The majority of melanomas (30/36 tumors) demonstrated BTRC transcript levels of 60% or more relative to the mean transcript level determined for the benign melanocytic nevi and normal skin (Table I, Fig. 4a). Six tumors (2 NMs and 4 CMM) had transcript levels of 30–50%, but none of the tumors demonstated expression values of ≤20% relative to normal skin and benign melanocytic nevi (Table I). RT-PCR analysis revealed that both BTRC transcript variants were expressed in normal skin, benign melanocytic nevi and all investigated malignant melanomas (Table I, Fig. 4b). Densitometric analysis of the signal intensities for BTRC transcript variant 1 relative to BTRC transcript variant 2 showed approximately equal expression of both variants in melanomas.

Figure 4.

Analysis of melanomas for expression of BTRC transcripts. (a) Demonstration of approximately equal BTRC mRNA expression levels in the CMM M64 and the benign melanocytic nevus N7. Abscissa, cycle number; ordinate, amount of PCR product. Note that in both tumors the curves obtained for BTRC mRNA (upper panel) and the reference mRNA (ARF1, lower panel) pass the threshold (Ct) at an approximately equal cycle number. The calculated BTRC mRNA expression level in M64 was 0.8 relative to N7. (b) Expression analysis of BTRC transcript variants in malignant melanomas by RT-PCR. Lanes: 1 and 2, normal skin samples from 2 different individuals; 3, M3; 4, M34; 5, M46; 6, M64; 7, M13; 8, M50; 9–11, benign melanocytic nevi (N1, N7, N10) from 3 different individuals; 12, no template (negative control). Note that BTRC transcript variants 1 (BTRCv1) and 2 (BTRCv2) are expressed at approximately equal levels in normal skin, benign melanocytic nevi and malignant melanomas.

DISCUSSION

Mutations of genes encoding members of the Wnt signaling cascade, in particular CTNNB1 and APC, are frequent in various types of human cancer, including among others colorectal carcinoma, hepatocellular carcinoma and hepatoblastoma, as well as primitive neuroectodermal tumors.6–8, 27, 29 Robbins et al.30 identified a melanoma-associated mutant β-catenin molecule that served as a tumor-specific antigen recognized by tumor-infiltrating lymphocytes. Others have reported CTNNB1 and APC mutations in a significant percentage (23%) of melanoma cell lines.5 Rimm et al.16 studied a series of 65 primary and metastatic melanomas for CTNNB1 mutation and β-catenin expression but identified only 1 melanoma with CTNNB1 mutation (S45P) while 28% of the cases (18/65) showed focal nuclear β-catenin immunoreactivity. Omholt et al.17 reported 1 missense CTNNB1 mutation (S45P) in a metastatic melanoma among a panel of 68 primary and metastatic melanomas analyzed. The mutation was already present in the corresponding primary melanoma. In the series of Omholt et al.,17 30% of the primary melanomas showed at least focal cytoplasmatic and/or nuclear immunoreactivity for β-catenin. Our findings confirm and extend these data. We detected CTNNB1 mutations in melanomas from 2/38 patients (5%) investigated. The mutation identified in M35 (S45del) has been reported before in other tumor types, such as colorectal carcinoma and Wilms' tumor.31–33 This mutation affects a serine residue, which is involved in the downregulation of β-catenin through phosphorylation and proteasomal degradation. The second tumor (M39) expressed an aberrant CTNNB1 transcript lacking exons 2 and 3, which encode for the N-terminal destruction box that is indispensable for β-catenin degradation. Both M35 and M39 showed strong cytoplasmic β-catenin immunostaining but no nuclear β-catenin accumulation. The reason for the absence of nuclear β-catenin staining in these 2 cases is not known, but studies on other tumor types have noted that CTNNB1 mutation is not invariably associated with nuclear β-catenin immunoreactivity.34, 35 One melanoma of our series (M21) carried an APC missense mutation (A1471S). This mutation was associated with cytoplasmic and nuclear β-catenin accumulation, possibly caused by decreased phosphorylation and enhanced stability of β-catenin due to functional impairment of the complex formed by APC, axin and Gsk-3β.6–8 In addition, wild-type APC has a nuclear export function, loss of which is assumed to contribute to nuclear β-catenin accumulation in APC mutant cells.36

The finding that more melanomas have nuclear and/or cytoplasmic β-catenin accumulation than carry detectable mutations in CTNNB1 or APC suggests that the pathway may be activated in these tumors through aberrations in other genes. Persad et al.37 showed that the Pten tumor-suppressor protein is involved in the regulation of nuclear β-catenin accumulation and Tcf-mediated transcriptional activation in an APC-independent manner. Pten-null cells have elevated nuclear β-catenin levels, which could be reduced upon re-expression of wild-type Pten.37 In a previous analysis, we have found PTEN mutations in 4 of the melanomas investigated here (M15, M24, M36 and M54).22 None of these tumors carried CTNNB1 or APC mutations, all had cytoplasmic β-catenin immunostaining and 1 tumor (M24) demonstrated focal nuclear β-catenin accumulation. Our data and results from other groups indicate that PTEN mutations are restricted to a small fraction of melanomas in vivo (approx. 10%),22, 38, 39 though one study found evidence that Pten function is impaired in a higher fraction of melanomas due to epigenetic PTEN silencing.40 However, it remains likely that aberrations of yet other genes contribute to aberrant β-catenin/Tcf activation in these tumors. We therefore investigated the ICAT and BTRC genes for mutation and expression. Both genes encode members of the Wnt signaling pathway and map to chromosomal regions that are frequently affected by allelic loss in melanomas. In line with previous studies,2, 19 we found allelic loss at microsatellite markers from 1p36 in a significant fraction (40%) of the investigated melanomas. LOH on 1p36 was more frequent in melanoma metastasis (55%) than in primary melanomas (20%), confirming the previous notion that 1p losses are a late event in melanoma progression.19 The frequency of LOH on 10q in the melanomas in our study (43%) also corresponds well to the reports by other groups, which showed allelic losses on this chromosome arm in 30–50% of the melanomas investigated.20, 21, 41

The ICAT gene has been mapped to 1p36.2 and shown to encode a negative regulator of the Wnt-signaling pathway.11 Icat protein inhibits the association of β-catenin with Tcf-4 in the cell nucleus and represses transactivation of β-catenin/Tcf-4 target genes in a dose-dependent manner.11 Therefore, Icat may function as a tumor suppressor and its inactivation may lead to tumorigenesis.11 However, to our knowledge, no studies investigating human tumors for ICAT gene mutations or loss of expression are available. Mutational analysis of ICAT in our series of malignant melanomas revealed a single melanoma metastasis (M46) with a somatic point mutation. This particular mutation affected the start codon and predicted expression of a truncated protein lacking the first 13 amino acids of the N terminus. Regarding the function of this truncated Icat protein, it has been shown that Δ13N-Icat is still able to interact with β-catenin and shows no dominant-negative activity.11 The functional significance of the N-terminally deleted Icat in tumor M46 therefore remains to be established.

Our mRNA expression analyses revealed ICAT transcript levels reduced to 20% or less relative to normal skin and benign nevi in more than two-thirds of the melanomas investigated, including the majority of cases with LOH on 1p36. It is possible that the tumor cells in these melanomas have completely lost ICAT expression. The weak residual signals detected in these cases may be derived from contaminating nonneoplastic cells in the biopsy specimens. Relative ICAT mRNA levels of ≤20% were found in all but 1 of the melanoma metastases (95%) and half of the primary melanomas. This suggests that loss of Icat expression may contribute to melanoma progression and metastasis. The mechanism underlying the markedly reduced ICAT mRNA levels in melanomas is unclear at present. Our data indicate that ICAT was not homozygously deleted in any of the cases from our series. It remains to be investigated whether transcriptional silencing by mutation, hypermethylation of ICAT promotor sequences or yet other alterations, e.g., reduced activity of transcriptional activators, increased activity of transcriptional repressors or mutations in noncoding sequences that reduce mRNA stability, are responsible for the markedly lower ICAT mRNA levels in malignant melanomas compared to normal skin and benign melanocytic nevi.

Analysis of the BTRC gene, which encodes a cytoplasmic negative regulator of the Wnt pathway that is important for the ubiquitin-mediated degradation of β-catenin by the proteasome, revealed neither tumor-associated mutations nor complete loss of mRNA expression in our melanoma series. Our negative mutation analysis is in line with the data reported by Chiaur et al.,42 who did not find any deletions or gross alterations of BTRC in 42 human cancer cell lines and 48 tumor samples investigated. In contrast, Gerstein et al.28 reported expression of aberrant BTRC transcripts in 1 prostate cancer cell line (TSU) and 1 prostate cancer xenograft without APC or CTNNB1 mutation. The aberrant BTRC transcripts carried deletions within the coding sequence of 96 or 168 nucleotides, respectively. The TSU cell line demonstrated nuclear expression of β-catenin, suggesting that the BTRC mutation could be the cause for β-catenin activation in this cell line.28 However, in our series of melanomas, we found neither the deletions reported in prostate carcinoma nor any other mutations in the BTRC gene. Nevertheless, a small subset of melanomas demonstrated BTRC transcript levels of 30–50% relative to those detected in normal skin and benign melanocytic nevi, respectively. Small changes in the transcript level of tumor-suppressor genes, such as a constitutional 50% decrease in the expression of one APC allele, may result in a predisposition to tumorigenesis.43 It therefore remains to be investigated whether a 30–50% relative decrease in the BTRC transcript level, as determined for individual melanomas of our series, may facilitate tumor growth. With respect to β-catenin expression, however, there was no apparent association between the BTRC mRNA level and nuclear β-catenin accumulation.

In conclusion, our findings corroborate that mutations in the CTNNB1 and APC genes are present in a small fraction of sporadic malignant melanomas. In addition, we show that expression of ICAT transcripts is commonly reduced or absent in malignant melanomas, an aberration that may be important for melanoma progression by virtue of altered β-catenin/Tcf-4 regulation in the cell nucleus. The BTRC gene, however, showed neither mutations nor complete loss of expression in melanomas; therefore, it is unlikely to be an important target for the frequent 10q deletions in these tumors.

Acknowledgements

J.R. was supported by the Lise-Meitner-Program of the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein-Westfalen.

Ancillary