Molecular analysis of E-cadherin and cadherin-11 in Wilms' tumours

Original Paper

Molecular analysis of E-cadherin and cadherin-11 inWilms' tumours

Stephan Schulz1*, Karl-Friedrich Becker1,2, Evelyn Braungart1, Claudia Reichmuth1, Barbara Klamt3,

Ingrid Becker1, Mike Atkinson2, Manfred Gessler3 and Heinz HoÈ¯er1,2

1 Institut fuÈr Allgemeine Pathologie und Pathologische Anatomie der Technischen UniversitaÈt MuÈnchen, Klinikum rechts der Isar, Ismaninger Strasse 22,D-81675 Munich, Germany

2 GSF-Forschungszentrum fuÈr Umwelt und Gesundheit, Institut fuÈr Pathologie, D-85764 Neuherberg, Germany3 Theodor-Boveri-Institut fuÈr Biowissenschaften (Biozentrum) der UniversitaÈt WuÈrzburg, Physiologische Chemie I, D-97074 WuÈrzburg, Germany

*Correspondence to:Stephan Schulz, Institut fuÈrAllgemeine Pathologie undPathologische Anatomie derTechnischen UniversitaÈt MuÈnchen,Klinikum rechts der Isar,Ismaninger Strasse 22, D-81675MuÈnchen, Germany

Received: 25 May 1999

Revised: 30 August 1999

Accepted: 8 December 1999

Abstract

Different studies of Wilms' tumours have demonstrated a loss of heterozygosity (LOH) of

chromosome 16q ranging from 17 to 25%. In order to search for a potential tumour suppressor

gene on 16q, we chose the calcium-dependent cell adhesion molecules E-cadherin and cadherin-11

as candidate genes, which are both located on the long arm of chromosome 16. E-cadherin is

known to be expressed in epithelial structures, whereas cadherin-11 is supposed to be expressed in

mesenchymal structures and developing epithelium, including renal tubules. For the present study,

fresh frozen tissue from 30 Wilms' tumours and corresponding non-tumour tissues were analysed.

Single nucleotide polymorphisms of the E-cadherin and cadherin-11 genes were chosen and

analysed for allelic inactivation by polymerase chain reaction (PCR) ampli®cation and sequence

analysis. Loss of expression of one E-cadherin allele was seen in 10% (2/20) of the informative

cases. Two out of 11 informative cases (18%) showed loss of expression of one cadherin-11 allele.

No length alterations of either the E-cadherin or the cadherin-11 messenger RNAs were identi®ed

using reverse transcription PCR and agarose gel electrophoresis in tumour tissue. Sequencing of

the entire E-cadherin coding region in seven cases showed the wild-type sequence. These data

imply that E-cadherin and cadherin-11 are not likely to play typical tumour suppressor roles in

Wilms' tumour. Interestingly, the E-cadherin immunohistochemistry showed a deviation from the

normal reaction pattern in 50% of the cases, with 27% (8/30) showing an apical or cytoplasmic

reaction and 23% (7/30) being completely negative. Northern blot analysis revealed that the

overall expression of cadherin-11 is much stronger than that of E-cadherin. In several cases, the

expression levels of the two genes were inversely correlated, suggesting the existence of a

regulatory mechanism. Analysis of differential expression of the various cadherins and their

subsequent signal transduction pathways might contribute to a better understanding of the

complexity of Wilms' tumour formation. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: Wilms' tumour; nephroblastoma; E-cadherin; cadherin-11; expression; LOH; cDNA;

sequence

Introduction

Previous studies of Wilms' tumour (nephroblastoma)have shown loss of heterozygosity (LOH) on chromo-some 11p in up to 50% of cases [1]. The subsequentlyidenti®ed Wilms' tumour suppressor gene, WT1, islocated on 11p13 [2,3]. Mutations of this gene werefound in 10±15% of sporadic Wilms' tumours [4,5].The WT2 gene has been postulated to be located at11p15, based on allelic loss and chromosome transferstudies [6±8]. Besides these alterations on chromosome11p, LOH has also been detected on 16q in frequenciesof up to 25% of Wilms' tumours, suggesting theexistence of additional suppressor genes [9±13]. In oneof these studies, LOH on 16q was correlated with anadverse clinical outcome [12,13]. Using seven differentmarkers located on the long arm of chromosome 16,Klamt et al. could further support these ®ndings bydemonstrating LOH in 13% of cases [14].

The cell±cell adhesion molecule E-cadherin isexpressed in most epithelial tissues and is known toplay an important role in the development of epithelialstructures. During kidney development for instance,sequential expression and the formation of adherensjunctions by E-cadherin are considered to be essentialfor the establishment of a polarized epithelium alongthe human nephron [15]. E-cadherin has a proven roleas a tumour suppressor gene, which is mutated in avariety of different carcinomas, such as diffuse-typegastric cancers, lobular breast cancers, and carcinomasof the endometrium and ovary [16±20]. Furthermore,inactivation of the E-cadherin-mediated cell adhesionsystem can also result from reduced expression of E-cadherin in vivo [16]. Cadherin-11 is predominantlyexpressed in mesenchymal cells and in different typesof developing cells, for instance myoblasts and epithe-lial cells of differentiating renal tubules [21±23].

Both cadherin genes are located on chromosome

Journal of PathologyJ Pathol 2000; 191: 162±169.

Copyright # 2000 John Wiley & Sons, Ltd.

16q, a region of great interest in Wilms' tumourbecause of the known allele loss. In the present study,these two genes were tested as possible tumoursuppressor genes in Wilms' tumours. Analysis forallelic loss and chromosomal deletion were performedby cDNA ampli®cation and sequencing. Immunohisto-chemistry and northern blots were used to analyseprotein and mRNA expression.

Materials and methods

Material

The frozen tissue of 30 Wilms' tumours and corre-sponding normal tissues as controls (from blood oradjacent normal kidney tissue) were obtained throughthe German Nephroblastoma Study (SIOP 93±01/GPOH) [14]. Most of the patients, except thoseyounger than 6 months, had received preoperativechemotherapy over 4 weeks, according to SIOP 93±01guidelines. Although some of the tissues focallyrevealed regressive changes and necrosis, RNA andDNA extraction yielded suf®cient amounts of totalRNA and high-molecular-weight DNA, using theguanidinium isothiocyanate and CsCl2 centrifugationmethod in each case [24].

Allelic inactivation analysis

Allele-speci®c expression of E-cadherin and cadherin-11 was determined as previously described [25,26].Brie¯y, after reverse transcription of the total cDNA,two intragenic single nucleotide polymorphisms of theE-cadherin mRNA [26] and one intragenic singlenucleotide polymorphism of the cadherin-11 mRNA[27] were ampli®ed and directly sequenced. ThecDNAs of tumour and corresponding normal tissueswere ampli®ed in 234±278 base pair (bp) longfragments, using a single-step PCR protocol. Wede®ned allelic inactivation in tumour tissues as thedisappearance of an mRNA containing a polymorph-ism expressed in the non-tumour tissues of the samepatient [25].

Analysis of E-cadherin and cadherin-11 mRNAintegrity

After reverse transcription, the entire coding regions ofE-cadherin and cadherin-11 from all 30 Wilms'tumours were PCR-ampli®ed in three overlappingfragments ranging from 675 to 1095 bp in length. E-cadherin cDNA was ampli®ed using a two-step PCRprotocol with 35 cycles each. For ampli®cation of theentire cadherin-11 cDNA, a single-step protocol with40 cycles was used (cycle conditions: 1 min 94uC, 1 min55±57uC, and 1 min 72uC). The primer pairs used havealready been described [22,25±28]. To detect lengthalterations, the PCR products were visualized byagarose gel electrophoresis. In addition, the entire E-cadherin coding region from seven tumours wassequenced, using a cycle sequencing protocol and

internal primers. Sequence analysis was performed onan automated sequencer (ABI prism2 377, PerkinElmer) and the results were compared with the wild-type sequence deposited in the EMBL/GenBank DataLibraries, accession No. Z13009.

Immunohistochemistry

Frozen sections were ®xed with acetone and incubatedwith two E-cadherin-speci®c monoclonal antibodies:HECD-1 (Takara, Genevilliers, France; 1 : 500) andAEC (anti-E-cadherin, clone 36, Transduction Labora-tories, Lexington, KY, USA; 1 : 1000). For detection, acommercial peroxidase system was used (FAST DABperoxidase substrate table set, Sigma GmbH, Deisen-hofen, Germany). Frozen tissue from a normal child'skidney and a normal adult kidney were used as positivecontrols. Negative controls included the omission ofprimary and/or secondary antibodies. Staining inten-sity was assessed semiquantitatively by two patholo-gists and speci®ed as the percentage of positive cells(Table 3).

Northern blot analysis

Standard hybridization of northern blots was per-formed using the following cDNA probes: an E-cadherin probe, consisting of 1372 base pairs spanningexons 6±13, cloned into an expression vector after PCRampli®cation (a gift from Dr G. Handschuh, Munich)[29], and a cadherin-11 probe, consisting of 1867 bases,comprising bp 1794 to bp 3660 of human cadherin-11(a gift from Dr A. Markus) [22]. For the northernblots, 10 mg of total RNA from each case were used,blotted and hybridized with [a-32P]-dATP-labelled E-cadherin probe, washed under stringent conditions,and ®nally exposed. After radioactivity had faded, theblots were stripped [24] once and rehybridized with thecadherin-11 probe.

Results

Allelic inactivation analysis

Analysis of intragenic E-cadherin polymorphismsshowed loss of expression of one allele in 10% (2/20)of informative cases (Figures 1a±1d and Table 1).Allele-speci®c loss of expression of cadherin-11 wasdetected in 18% (2/11) of the informative cases (Figures1e±1h and Table 1). One tumour (case 1, Figure 1a and1b) showed allele-speci®c loss of expression of E-cadherin and cadherin-11. Histologically, case 5, whichalso showed LOH, had an anaplastic component.However, none of the LOH cases had a signi®cantlypoorer clinical outcome, or any of the classicalsyndromes often associated with Wilms' tumours,such as the WAGR or the Beckwith±Wiedemannsyndrome.

Cadherins in Wilms' tumours 163

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 162±169.

Analysis of E-cadherin and cadherin-11 mRNAintegrity

To analyse the integrity of the E-cadherin mRNA,

cDNA of all 30 cases was ampli®ed in three over-

lapping fragments using a nested PCR protocol; 24

cases showed reproducible PCR products of apparent

wild-type length (Figure 2a). In six cases, no ampli®ca-

tion products could be seen, even after repeated

application of nested PCR. In order to exclude small

mutations in the ampli®ed E-cadherin fragments, seven

cases were selected for sequencing of the entire coding

region: three cases with allele-speci®c expression loss

and four cases with an abnormal staining pattern in

immunohistochemistry for E-cadherin (apical staining

of tubules, see below). The E-cadherin sequences in all

seven cases were found to be identical to the wild-type

cDNA sequence (EMBL/GenBank Data Libraries,

accession No. Z13009).In contrast to the E-cadherin results, cadherin-11

mRNA was present in all tumours. The cadherin-11

ampli®cation products showed no gross structural

alterations and only PCR fragments of the wild-type

length were seen (Figure 2b). Interestingly, a single-

step PCR protocol was suf®cient to amplify cadherin-

11 mRNA in all 30 cases; in contrast, a nested PCR

protocol had to be used for ampli®cation of E-

cadherin mRNA.

Figure 1. Allele-speci®c expression analysis of intragenic single nucleotide polymorphisms of the E-cadherin and cadherin-11 cDNA.(a±d) Analysis of E-cadherin polymorphism of two cases showing expression of both alleles in the `normal' tissue (a, c) and loss ofexpression of one allele in the tumour tissue (b, d). (e±h) Analysis of cadherin-11 polymorphism of two cases showing expression ofboth alleles in the `normal' tissue (e, g) and loss of expression of one allele in the tumour tissue (f, h)

Table 1. Allele-speci®c expression analysis of E-cadherinand cadherin-11 using intragenic single nucleotide poly-morphisms

Polymorphisms

(nucleotide position) n

Informative

cases

Allelic

inactivation

E-cadherin (nt 2076and nt 2797)

30 20/30 2/20 (10%)

Cadherin-11 (nt 1100) 30 11/30 2/11 (18%)

164 S. Schulz et al.


Immunohistochemistry

E-cadherin immunohistochemistry on fresh frozen

material showed a speci®c reaction in 23 of the 30

(77%) cases. Six (20%) of the cases had a strong, 17

(57%) had a moderate to weak, and seven (23%) had

no immunoreactivity in the epithelial components

(Tables 2 and 3). The seven cases negative for E-

cadherin were composed of three tumours with a

dominant blastemal component, two stroma-rich

tumours, and two tumours with rhabdoid differentia-

tion without epithelial components. The normal E-

cadherin staining pattern with lateral, intercellular,

staining of tubule cells (Figure 3a) was found in 14 of

the 22 E-cadherin-positive tumours. Interestingly, eight

tumours showed a deviation from this pattern, six had

apical staining of tubular structures (Figure 3b) and

two cytoplasmic staining (Figure 3c and Tables 2 and

3). In two cases, E-cadherin-positive and -negative

tubular structures were adjacent to one another

(Figure 4); inspection of the corresponding haematox-

ylin and eosin (H&E)-stained slide eliminated necrosisas an explanation of this result (Figure 4, inset).

Northern blot analysis

Northern blot analysis was used to compare theexpression levels of E-cadherin and cadherin-11. Inter-estingly, there were marked variations in signalintensities among the 30 cases. In the E-cadherinnorthern blots, cases 4 and 6 were negative, case 10demonstrated a weak signal, and case 9 showed astrong signal (Figure 5a). In contrast, cadherin-11showed inverse results; here cases 4 and 6 bothdemonstrated a clear signal, case 10 had a strongsignal, whereas the signal of case 9 was weak(Figure 5b), suggesting an inverse correlation of theexpression of E-cadherin and cadherin-11 in thesecases.

Discussion

Allelic inactivation of a gene is functionally similar toloss of heterozygosity (LOH) [24]. The allele-speci®closs of E-cadherin expression in 10% of our cases,determined with intragenic polymorphisms, is compar-able to the published results of Klamt et al. [14], whofound LOH in 13% of their cases. These frequenciesare lower than those from other reports, which showedLOH frequencies on 16q ranging from 17 to 25%[9±13]. On the other hand, the 18% allele-speci®c loss

Figure 2. Ampli®cation of the E-cadherin and cadherin-11 cDNA. (a) E-cadherin PCR fragments from the 3k-end (1095 bp); (b)cadherin-11 PCR fragments from the 3k-end (972 bp). M=molecular weight marker (Boehringer, Mannheim). Ten different Wilms'tumour samples are indicated by the numbers. In samples 4, 6, and 16, no E-cadherin cDNA was seen. Cadherin-11 cDNA could beampli®ed in all cases. +=positive control; x=negative control

Table 2. Pattern of E-cadherin immunoreactivity

No. of tumours

E-cadherin immunoreactivity

Lateral Apical Cytoplasmic Negative

30 15 (50%) 6 (20%) 2 (7%) 7 (23%)



of the cadherin-11 gene described in the present study

is within this range. Interestingly, one of the two cases

showed allelic inactivation of E-cadherin and cadherin-

11, suggesting that a large part of the recently

described cadherin gene cluster might have been

affected [30]. This result also supports the ®ndings of

Klamt et al. [14], who found that in all cases with LOH

of 16q, the complete long arm of chromosome 16 was

lost. Such large losses greatly complicate the search for

potential suppressor genes on 16q which might be

involved in Wilms' tumour initiation or progression.In addition to their ®ndings on 16q, Klamt et al. [14]

found that many cases with 16q LOH also showed

allelic loss of 11p, or multiple allelic losses which

included 11p. These results suggest that allelic loss at

16q alone might not be suf®cient for tumour initiation,

which might require additional allelic loss at 11p. It is

also possible that allelic loss on 16q occurs during

Wilms' tumour progression and has no in¯uence on

tumour initiation.The results of the E-cadherin cDNA ampli®cation

and sequencing, in which no length or sequence

alterations were detected, indicate that E-cadherin is

not likely to play a role as a typical suppressor gene in

Wilms' tumour. Interestingly, the cases in which no E-

cadherin PCR product was seen, despite repeated

nested ampli®cation, showed a positive signal in the

single-step cadherin-11 PCR. This suggests that the

cadherin-11 mRNA is more strongly expressed in

Wilms' tumours than the E-cadherin mRNA. As in

the E-cadherin analysis, ampli®cation of the entire

cadherin-11 mRNA exclusively showed the wild-type

length, implying that gross structural alterations are

rare in this gene. However, small deletions or inser-

tions and point mutations could still have been present

that would not be detected using our approach.

Although the cadherin-11 PCR results do not allow a

®nal conclusion, they suggest that cadherin-11 is also

not likely to act as a typical suppressor gene in Wilms'

tumour.The deviation from the normal lateral E-cadherin

expression pattern seen by immunohistochemistry in

eight cases has also been seen in other cancers [31±33].

A genetic defect in one of the intracellular ligands of E-

cadherin, alpha- and beta-catenin, which link the

cadherin cell adhesion apparatus to the actin cyto-

skeleton, may account for this result [34±36]. Although

one case (case 9, Table 3) with an abnormal E-cadherin

Table 3. Summary of the results

CaseNo.

Histologicalsubtype

E-cadherin IHC

E-Cdhnorth

Cdh-11north

Allelic analysis

E-CdhPCR

Cdh-11PCR ClinicIntensity Localization E-Cdh Cdh-11

1 Epithelial 3+ + + + LOH LOH & &2 Blastemal + + + + LOH N & &3 Triphasic 2+ + + + N het & &4 Stromal x x x + N N % &5 Triphasic ± An 2+ Apical + + N LOH & &6 Rhabdoid x x x + het N % & Met. (lung)7 Triphasic 2+ Apical + + het het & &8 Epithelial 3+ Apical + + N het & &9 Triphasic 2+ Apical + + het het & & Bilat., Rec.

10 Triphasic + + + + N N & &11 Stromal + Cytopl + + het het & &12 Triphasic 3+ + + + het N & & WAGR, Met. (lung)

13 Stromal 2+ + + + het N & &14 Triphasic 2+ Apical + + het het & &15 Blastemal x x x + het het & &16 Blastemal x x x + N N % & Met. (lung)

17 Blastemal + + + + het N & &18 Rhabdoid x x x + N N % &19 Triphasic 3+ + + + het N & & Met. (lung)

20 Blastemal x x x + N N % &21 Stromal 2+ Cytopl + + het het & &22 Epithelial 3+ + + + het N & & Met.

23 Triphasic 2+ + + + het N & &24 Triphasic 3+ + + + N N & &25 Stromal x x x + N N % & Met., Rec., #

26 Triphasic 2+ Apical + + het N & &27 Epithelial 2+ + + + het N & &28 Triphasic 2+ + + + het het & & BWS29 Triphasic ± An + + + + het N & &30 Epithelial 2+ + + + het N & &

An=anaplasia. IHC=immunohistochemistry; intensity (percentage of stained cells): x=no cells stained; +=up to 30%; 2+=30±60%; 3+=more than60%. Localization: cytopl=cytoplasmic reaction; +=reaction on intercellular boundaries; x=no immunoreactivity. Cdh=cadherin; north=northern blot;

+=signal detectable; x=no signal. &=positive signal in PCR; %=negative signal in PCR. LOH=loss of heterozygosity; het=heterozygous; N=non-

informative. WAGR=WAGR syndrome; BWS=Beckwith±Wiedemann syndrome; Bilat.=bilateral tumour; Met.=metastasis; Rec.=recurrent; #=death.



pattern by immunohistochemistry did have a poor

clinical course (bilateral tumours and tumour recur-

rence), abnormal E-cadherin immunoreactivity and a

poor clinical outcome were not signi®cantly related to

one another. Interesting, though, was the observation

of metastasis in three cases (Nos. 6, 16, and 25,

Table 3), which showed a negative reaction in the E-

cadherin immunohistochemistry. The lack of E-

cadherin expression might have been responsible for

tumour progression in these cases, a phenomenon

A B

C D

Figure 3. E-cadherin immunoreactivity on fresh frozen material, using monoclonal antibody HECD-1. (a) Normal fetal kidney withtypical membrane-bound (intercellular) reaction on tubular epithelium; (b) Wilms' tumour with intercellular reaction (case 23); (c)Wilms' tumour with apical labelling of tubular epithelium (case 9); (d) Wilms' tumour with cytoplasmic and nuclear staining of singlecells (case 11)

Figure 4. E-cadherin immunohistochemistry on Wilms' tumour (case 10) with negative and positive tubular structures next to eaachother. The inset shows the same spot on a consective H&E-stained slide: both tubular structures were composed of vital cells



described earlier by Perl et al. [37]. Cadherin-11

immunoreactivity could not be analysed, since no

speci®c monoclonal antibodies had been available.The fact that E-cadherin-positive and -negative

tubules could be identi®ed next to each other in two

cases may have resulted from uncoordinated develop-

ment due to defects in transcription factors leading to

different cadherin expression patterns in neighbouring

tubules. Alternatively, some pre-existing tubules may

have been surrounded by epithelial tumour cells during

tumour progression. If these pre-existing tubules

belonged to the proximal tubule system, they should

be E-cadherin-negative, but positive for cadherin-6

[23,38].Overall, the expression of cadherin-11 was much

stronger than that of E-cadherin, as demonstrated by

our northern blot analysis. This statement is supported

by the observation of distinct cadherin-11 signals in all

cases analysed, even those that were negative for E-

cadherin. Furthermore, the autoradiography time to

get a signal was 12 times longer for the E-cadherin

than for the cadherin-11 blots. Since the cDNA probes

used were both much longer than 400 bp (E-cadherin

probe 1372 bp, cadherin-11 probe 1867 bp), it is

unlikely that differences in af®nity to the correspond-

ing mRNA are responsible for this observation. It is

interesting that the expression of E-cadherin and

cadherin-11 was apparently inversely correlated in

four cases. A coordinated regulation of genes in the

cadherin cluster, i.e. up-regulation of cadherin-11 when

E-cadherin is down-regulated and vice versa, may at

least in part explain this phenomenon. It is possible

that cadherin-11 may compensate for the calcium-

dependent homophilic adhesive function of E-cadherin

when the latter is down-regulated. A similar regulatory

phenomenon has been described for E-cadherin and N-

cadherin [39]. Generally, E-cadherin expression is

characteristic for epithelial differentiation, whereas

cadherin-11 expression is predominantly seen in

mesenchymal cells, which is con®rmed by the tumour

tissues analysed in the present study. This phenomenon

might in part explain the inversely correlated expres-

sion pattern described above. Expression analysis of

other cadherins and of relevant signal transduction

pathways might help to provide important insights

into the complex mechanism of Wilms' tumour forma-

tion. Further study will be needed to demonstrate

whether other genes on chromosome 16q could play

important roles as tumour suppressor genes in Wilms'

tumour.

Acknowledgements

We thank Dr G. Handschuh, Dr A. Markus, Dr J. Mueller, Dr

S. Candidus, C. Schott, and U. Mueller for advice and technical

support and all the patients of the SIOP93 Study and their

clinicians and pathologists for providing samples for molecular

analysis. The work of MG was supported by SFB 172.

Figure 5. Northern blot analysis with E-cadherin and cadherin-11 probes. (a) E-cadherin blot (12 days exposed); (b) cadherin-11blot (1 day exposed); (c) agarose gel prior to blotting. The same blot was hybridized with the E-cadherin probe, stripped, and thenhybridized with the cadherin-11 probe



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Molecular analysis of E-cadherin and cadherin-11 in Wilms' tumours

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