Wnt5a Cloning, Expression, and Up-Regulation in Human ... · Vol. 1, 215-222, February 1995 Clinical Cancer Research 215 Wnt5a Cloning, Expression, and Up-Regulation in Human Primary

Vol. 1, 215-222, February 1995 Clinical Cancer Research 215

Wnt5a Cloning, Expression, and Up-Regulation in Human Primary

Breast Cancers’

Sue Lejeune, Emmanuel L. Huguet,

Andrew Hamby, Richard Poulsom,

and Adrian L. Harris2

Imperial Cancer Research Fund, Molecular Oncology Laboratory,

Institute of Molecular Medicine, John Radcliffe Hospital, Headington,

Oxford, 0X3 9DU, United Kingdom

ABSTRACT

Wnt genes are involved in mouse mammary cancer, but

their role in human cancer is unknown. Human Wnt5a was

cloned from a placental cDNA library and used to assessexpression by ribonuclease protection and in situ hybridiza-

tion in human breast cell lines and in normal, benign, and

malignant breast tissues. Human WntSa shows over 99%

homology at amino acid level with mouse Wnt5a, and 90%with Xenopus Wnt5a. It was expressed only at low levels inbreast cell lines and normal breast tissue. Benign prolifera-

tions and invasive cancer respectively showed 10-fold and4-fold higher Wnt5a than normal breast tissues. The greaterup-regulation in benign conditions suggests a robe in aber-

rant differentiation. In situ hybridization localized the signal

to the epithelial component. Wnt5a is the first member of theWnt family to demonstrate overexpression in human breast

cancer. It was not associated with factors known to affect

breast cancer prognosis such as lymph node status or epi-

dermal growth factor receptor status.

INTRODUCTION

In mouse mammary tumor virus-induced breast cancer,

analysis of insertion sites has shown activation of endogenous

genes, mt genes (1). mt-i (now WntJ) was the first gene isolated

and shows strong homology to a Drosophila developmental

gene, wingless (2), involved in pattern development. Two other

members of this family, Wnt3 and Wnt2 (also called mt-related

protein, irp) are also involved in mouse mammary cancer (3, 4).

Fifteen Wnt genes have been isolated in vertebrates. They

are expressed in many adult and embryonic tissues (5) and are

involved in morphological development. The importance of

their role is reflected by the severity of the phenotypic abnor-

malities that result from aberrant Wnt expression. Thus, Wnt

genes have important roles in development and in cancer.

The role of Wnts in mouse mammary carcinogenesis has

been extensively studied and there is evidence that they are

secreted proteins, processed via the Golgi apparatus (6), which

remain tightly associated with the extracellular plasma mem-

Received 9/23/94; accepted 9/28/94.

, This work was funded by the Imperial Cancer Research Fund and

Oxfordshine Health Authority.2 To whom requests for reprints should be addressed.

brane or matrix (7). Writs produce morphological effects on

some mouse mammary cancer cell lines by transfection in

autocnine (8) and paracrine mechanisms (9).

A survey of expression in normal mouse mammary gland

development showed that some Wnts are expressed in virginal

breast, some in pregnancy, and others in lactation (10). How-

ever, Wntl, which is involved in carcinogenesis is not expressed

in normal mouse mammary tissue. This implicates Writ gene

family members in normal breast development and suggests

aberrant expression of other members can contribute to malig-

nancies (1 1).

Evaluation of the normal expression of Wnt genes in hu-

man breast epithelium and cancer would contribute to under-

standing the role of Wnt genes in human cancer. It has been

shown that some of the Wnt genes are expressed in human breast

tissue, and that quantitative differences exist in the Win expres-

sion profile of normal and proliferative lesions (12).

We chose Wnt5a as a candidate human gene to clone and

evaluate because it is expressed in normal mouse breast epithe-

hum to a low extent, and also in mouse breast cancer cell lines

(10). Furthermore it has some different properties from the

human Wnt genes previously cloned in its effects on cell gap

junctions (13) and Xenopus development (14). Interactions be-

tween Wnt genes with different normal functions may contribute

to malignant transformation (1 1).

MATERIALS AND METHODS

Isolation of 384-Base Pairs Fragment of Human WntSa

from Fetal Brain cDNA Library. Two hundred ng of a

human fetal brain library in plasmid pCDM8 (obtained from Dr.

D. Simmons and Dr. J. Fawcett, Institute of Molecular Medi-

cine, Oxford, United Kingdom) was used as a template for PCR.

Amplification of cDNA was carried out using 500 ng of each of

the degenerate forward (5’-GGGGAATTCCA’�’/GGA”/GTGT/

�AA”/TGT/CCAT3’) and reverse (5’-AAAATCTAGA’�’/

GCA/GCACCA/GTG/GAA3) oligonucleotide primers pre-viously described by Gavin et a!. (5). PCR products were

separated on a 2% agarose gel. Products of the correct size

(predicted from known Writ sequences) were recovered, then

further amplified using the above primers and ligated into the

plasmid pBluescript KS+ (Stratagene). JM1O9 cells were trans-

formed with the reaction products, and the nucleotide sequences

of clones containing inserts of correct size were determined by

dideoxy chain termination sequencing. Clones with significant

homology to known Wnts were identified using the FASTA

program (GCG) and included one clone containing a 384-base

pair fragment of human Wnt5a.

Isolation of Human Wnt5a from Placental cDNA Li-

brary. Replica colony lifts of approximately i0� recombinant

clones of a human placental library in the plasmid pCDM8 were

prepared using Hybond-N membranes (Amersham). The mem-

branes were hybridized to a Wnt5a probe synthesized using the

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216 Wnt5a Up-Regulation in Human Breast Cancer

384-base pair fragment isolated as described above. The probe

was generated by incorporating [a-32PJdCTP (Amersham) in a

PCR amplification of the 384-base pair fragment using the

above degenerate primers. The resultant species were end filled

and separated from unincorporated nucleotides by passage

through a Sephadex G-50 spin column (Boehninger Mannheim).

After hybridization of the probe to the membranes, positive

clones were identified by autoradiognaphy, and subjected to

further rounds of screening. After four rounds of screening,

positive clones were selected and sequenced. Clones with sig-

nificant homology to known W,its were identified and included

one containing 798 base pairs of the 3’ end of the human Wnt5a

cDNA as well as some 3’ untranslated sequences.

Further sequence 5’ to the 798-base pair 3’ terminus was

obtained using a nested PCR strategy. A primary PCR reaction

was carried out using 200 ng of a placental cDNA library in

pCDM8 as template. The primers used were a mouse Wnt5a-

specific forward primer FPI (5’-ATGAAGAAGCCCATTG-

GAATA-3’) corresponding to the 21 extreme 5’ nucleotides of

mouse Wnt5a, and a human Wnt5a-specific reverse primer RPI

(5 ‘-GCACGCCCGGCTCATGGCGTF-3 ‘) corresponding to a

known sequence in the 798-base pair partial clone. Fragments of

correct size (approximately 462 base pairs) were recovered and

used as template for nested PCR. In the nested PCR reaction the

forward and reverse primers (FP2 and RP2) were 3’ and 5’ of

the primers FP1 and RP1, respectively. Primer FP2 (5’-

AANTCNTGGTGGTCNCTNGG-3’) was a fully degenerate

primer, and primer RP2 (5’-GTGTI’ATCCACAGTGCT-3’)

was a human Wnt5a-specific primer corresponding to the ex-

treme 5’ region of the 798-base pair partial clone. Fragments of

correct size (approximately 234 base pairs) were recovered,

subcboned into plasmid pBluescnipt 5K, and sequenced. Clones

with significant homology to Wnt5a were identified and in-

cluded one containing 234 base pairs of human Wnt5a sequence

5’ to the 798-base pair partial clone.

A nested PCR strategy was also used in order to clone the

extreme 5’ region of Wnt5a. In a primary PCR reaction, 200 ng

of human placental cDNA library in pCDM8 was used as

template. The primers used were a pCDM8-specific primer and

the human Wnt5a-specific internal primer RP1. Products of a

large enough size to contain the 5’ end of Wnt5a were recovered

and used as template in the nested PCR. In the nested PCR

reaction, primers FP1 and RP2 were used. Fragments of correct

size (approximately 366 base pairs) were recovered, cloned into

TA cloning vector (Invitrogen), and sequenced. A clone con-

taming the 5’ end of Wnt5a was identified. Thus the sequences

of the original 798-base pain partial clone and the two nested

PCR products combined to give the full-length sequence.

Chromosomal Localization of Human Wnt5a. Twen-

ty-jig DNA samples from a panel of human-rodent hybrid cell

lines, and control human, mouse, and hamster DNA (obtained

from Dr. N. Spurn, Imperial Cancer Research Fund, London,

United Kingdom) were digested with EcoRI, fractionated on a

0.7% agarose gel, and transferred to Hybond-N membranes

according to Southern’s protocol (15). The membranes were

then hybridized to a random primer generated [a-32P]dCTP-

labeled probe, using a 1.4-kilobase XbaI fragment of the original

(placental) partial human Wnt5a clone as template. Signals were

detected by autoradiography.

Ribonuclease Protection Assays. Ribonuclease protec-

tion assays were carried as described in Ref. 16. Briefly, a

384-base pair fragment of human Wnt5a was cloned into pBlue-

script KS. The EcoRV linearized plasmid was used to generate

antisense [a-32P]CTP-labeled probes with T7 RNA polymerase

(Gibco). Probes were hybridized to 10-jig samples of total RNA

extracted from tissues and cell lines by a single-step extraction

method (17). Hybridization was carried out at 45#{176}Cfor 16 h.

Each hybridization also contained an antisense probe for

GAPDH3 as a loading control. GAPDH probes were prepared

from a 120-base pain b fragment of GAPDH cloned into pBlue-

script 5K (18). Unhybridized probe was digested with RNase A

and Ti, and protected fragments were electrophoresed on poly-

acrylamide gels. Dried gels were autoradiographed.

Image Analysis. Autoradiographs of nibonuclease pro-

tection assays were scanned using a Bio-image analyser (Milli-

gan Bioresearch) to determine RNA abundance. Wnt5a values

were normalized to GAPDH to allow for loading. MCF1O RNA

was included in all assays as a positive control. This was

assigned a unit level of expression and all other values were

standardized to this.

Cell Lines. The following breast cell lines were obtained

from ATCC (Bethesda, MD): T47D (ATCC HTB 133),

MDA231 (ATCC HTB26), MCFJO (ATCC CRL1O3I7),

MDA415 (ATCC HTB128), MDA453 (ATCC HTBI31),

MDA157 (ATCC HTB24), BT2O (ATCC HTB19), SKBR3

(ATCC HTB3O). ZR9B1 1 (ZR-75-1), ZR4, and ZR1 1 were

obtained from Dr. E. Valvenius (Department of Pathology, Uni-

versity Hospital, Uppsala, Sweden). MCF7s were obtained from

Dr. B. Durkacz (Cancer Research Unit, University of Newcastle

upon Tyne). Adniamycin-resistant MCF7s were obtained from

Dr. K. Cowen (National Institutes of Health). Lines 2-5-2a,

3-4-1, 5-3-1, 6-1-1, MTSV1-7, and MTSV4-1 were obtained

from Dr. J. Taylor (Imperial Cancer Research Fund, London,

United Kingdom).

Cell Culture. T47D, MDA23I, MCF1O, MDA453,

SKBR3, MCF7, Adniamycin-resistant MCF7, ZR9B1 1, ZR4,

and ZR1 1 were maintained in DMEM with 10% FCS. BT2O was

maintained in Eagle’s MEM supplemented with 15% FCS and 2

mM glutamine. MDA4I5 was maintained in DMEM, iS% FCS,

1 jiM hydrocortisone, 10 jig/mI insulin, and 10 jig/mb glutathi-

one. MDA1S7 was maintained in RPMI 1640 and 10% FCS.

MTSV1-7, MTSV4-l, 2-5-2a, 3-4-1, 5-3-1, and 6-1-1 were

maintained in DMEM: Ham’s F12 (i:1), 10% FCS, 10 jig/mb

insulin, and 5 jig/ml hydrocortisone. All cultures were grown on

plastic dishes in 5% CO,-95% air in humidified incubators. All

cultures were free of Mvcoplastna.

Handling of Clinical Samples. Protocols for handling of

clinical samples and assays for hormone and growth factor

receptors were followed as detailed in Leieune et a!. (19).

Briefly, Tumors were considered to be ER positive if they

contained at least 10 fmol of specific binding sites per mg of

cytosolic protein, and EGFR positive if they contained at least

3 The abbreviations used are: GAPDH, glycenaldehyde-3-phosphate de-hydnogenase; ER, estrogen receptor; EGFR, epidenmal growth factor

receptor; ATCC, American Type Culture Collection.



FPI

ataaaaaaacccattaaaatattaagcccaggagt’ gctttggggatggctggaagtgca

H K K P I G I L S P 1 V /, L G M A (�. S A

atgtcttccaagttcttcctagtggctttggccatatttt tctccttcgcc-aggt tqta61 � , , +.------, 121)

M S S K F F L V A L A 1 F F S F S Q 2 V

FP2

attgaagccaattcttaataatcactaacitatgaataaccctgttcagatgtcagaaqt a

121 + , ,

I K A N S W W S L G H N N P V Q M S F

tatattataggagcacagcct ctctgcagccaactggcaggactttctcaaggacaqaag

181 ‘ --‘ + ‘ + ‘ 245

Y I I S A Q 1’ L C S Q L A G L S 0 5 Q F

241 - ‘ - i.

K L C H L V Q 1) H N Q S : C E C A K V -

SF2

atcaaagaatgccagtatcadt tccgacatcgaaggtggaactgca�cacLaLauata�c

301 � --� ,--- �------ ‘

I F F C 0 5 0 F H H P Is W N C S T .‘ II 1

�(ctgtttttggcagggtqatg(aqataggcagccgcgagacggccttcacara� I

361 --- --- - -,-------, -- -4VT 2 V F G S �.-‘ H� (I � � 5 5 F S A F 2 5 1

gtuaqcqcagcaggggtggt9aacQCCat0aQCCUQUCatUCCgCgagggCq4gCt� 1

421 --- -----‘ ---: :- A A G ‘.‘ 2 1. 5 ‘� 5 15 A C H F C 51:.��

20 fmol of specific binding sites per mg of membrane protein.

Human tissue samples were selected to represent normal breast

tissue, benign breast disease, and breast cancer.

In Situ Hybridization. A single-stranded antisense RNA

probe to Wnt5a was transcribed from EcoRI-linearized Wnt5a

DNA using T3 DNA polymerase (Promega), and 35S-UTP

(-800 Ci/mmol; Amersham International) as the sole source of

UTP. Histological sections of human tissues that had been fixed

in neutral-buffered formalin and embedded in paraffin wax were

treated in the manner described by Senior et a!. (20) with minor

modifications. In summary, 1 X 10#{176}�cpm of unhydrolyzed probe

in 10 ml buffer was hybridized overnight at 55#{176}Cto sections

permeabilized with proteinase K. Posthybnidization steps in-

cluded several large volume washes in a 50% formamide buffer

at 55#{176}Cto remove unhybrized probe, RNase A treatment to

digest single-stranded and imperfectly hybridized domains, and

extensive washing to remove these cleaved fragments were

performed. The final washes were in 0.SX SSC (1X SSC is

0.15 M NaC1 and 0.015 M sodium citrate) at 65#{176}Cfor 30 mm twice.

Slides were dehydrated and processed for autoradiography (Ilford

K2) at 4#{176}Cfor 7 to 10 days. Latent images were developed with

Kodak D-19, and sections were Giemsa counterstained.

The �3-actin mRNA was used as a positive control for the

presence of mRNA species, as described previously by Wright

et a!. (21). Labeled Wnt5a sense transcripts were used as con-

trols for nonspecific niboprobe binding.

RESULTS

Isolation of WntSa cDNA. A partial length cDNA was

initially isolated by using PCR primers to homologous domains

in Wnt genes. This was used to isolate a cDNA from a human

placental library, which, in combination with nested PCR prod-

ucts from the same library provided the full human Wnt5a

cDNA coding sequence shown in Fig. 1 with the corresponding

amino acid sequence. The sequence comparison of human

Wnt5a with mouse and Xenopus Wnt5a shows extensive con-

servation with amino acid level homology of 99% and 90%,

respectively.

Chromosomal Localization of Wnt5a. The chromo-

some location of the human Wnt5a was determined by screening

hamster-human and mouse-human hybrid cell lines which had

known human chromosome karyotypes. Southern blotting

showed a Wnt5a signal located on human chromosome 3 (Fig.

2), which was absent in a mouse-human hybrid cell line carrying

a p1 1 to p terminus deleted chromosome 3 (data not shown).

This suggests that Wnt5a is on chromosome 3p.

Expression of Wnt5a in Human Breast Cell Lines. To

assess expression in human breast cancer, a panel of human

breast cell lines was initially analyzed, since they represent a

pure epithelial population. Cell lines from normal breast duct

luminal epithelium, benign epithelial proliferation, and in situ or

invasive components of breast epithelium were studied (Table 1

and Fig. 3). Levels of expression measured by nuclease protec-

tion assays were low, requiring 7 days of development. One line

from luminal epithelium had higher levels (MTSV1-7) than the

others.

Cell lines established from malignant pleural effusions and

metastasis had lower levels than the luminal cell line MTSV1-7

481 . -. 54)

S C V C S H A A H P K 1) 1. 1 5 1) W 1 H

ggctgcggcgacaacatcqaCtatggctacCgCtttgCCaaqgagttCgtggaCgCc:3

541 + --‘-- ‘ -------‘ + 15_la

G C G D N I 1) Y (1 5 5 F A K F F V D A F

gagcgggagcgcat ccacgccaacqqct cIt acgagagt gct cgcat cct cat qaa as

601 -- ------‘ _-_-- � ,E P E F I H A K S V F S A P 1 F H N

ca�aacaa cgaggccggccgcaggaCggtgtaCaacctggctgatgtggcc�gcsa4t4C

661 - -----‘-- -‘ ---‘ --- 7,)))

H N N K A C) P P T V V N L A D V A C K C

catqgggt gt ccggctcatgt agcctgaagaCatgctggctgcagctqgCagacttCcq(

721 ----� ----- --� 7817

H (7 � S S S C S S K T C N L Q 1 A S F F

aaggtgggtgatgccctgaaggagaagtacgacagcqcggcggcCatgCggctcaacagc

781 �----------‘------ +- , 845

K V C D A L K E K Y D S A A A M S L N 77

cggggcaagt tggtacaggtcaacagccgct tcaactcgcccaccacacaagacctggtc841 �-- #{149}- - -, ca:

S G K L V Q V N S S F N S P T T Q S 1. V

tacat cgaccccagcCCtgaCtaCtgCgtgcgCaatgagagCaccggct cgctq5gcacg

901 ---- ----‘ ‘ ‘--- ---- . 1131)

V I [3 F S P D S C V P N F S I 77 77 1. 77 1

in all but one case (BT2O). Thus, in vitro cell lines rarely

expressed Wnt5a.

Expression in Human Breast Tissue. Expression was

then studied in normal breast tissue, tissue from benign breast

diseases, and primary breast cancers. The clinical details of the

patients (e.g., age, tumor receptor status) are given in Table 2.

Clinical Cancer Research 217

cagggccqcctgtgCaaCaag8CgtCggagggCatggatggCtqC�3dqCt catgtgctgc

961 ‘ ‘ -‘--- ----‘ a::Q G S L C N K T S K C M D G C F: :. 0 C C

ggccgtggctaC9dCCagttC8agaCCgtgCag8CggagCgCtqCC.�Ct 3caaqt tccac

1021 -‘ ‘

G R G V 0 0 F K T V Q T 0 5 C H C K F H

tggtgccgctaCgtCaagtgCaagaagtgCaCggagatCgtggaCC�gL’JtgtgtgCdag

1051 + + . 141)

w C C Y V K C K F C T E I V D �) F � C F

tag1141 ---1143

Fig. I Nucleotide sequence of human Wnt5a. The location of PCR

primers FP1, FP2, RP1, and RP2 are underlined. Predicted amino acid

sequence shown below nucleotide sequence. Conserved cysteine resi-

dues are shown in bold. �, stop codon.



Table I Comparison of Wnt5a expression in breast cell lines derived

from ductal epithelium, in situ and invasive components of breast

carcinoma, and breast cancer metastases

Cell line

Expression

standardizedType to MCF1O

MCFIOZR9B1I

ZR1IZR4T47D

MCF7MDA231MDA415

MDA453

MDAI57

BT2O

SKBR3Adniamycin-resistant

MCF7

2-5-2A

3-4-I

5-3-I

6-1-I

MTSV1-7

MTSV4-1

Immortal nonmalignant

ER+ breast cancer

ER+ breast cancerER + breast cancer

ER+ breast cancerER+ breast cancerER- breast cancerER- breast cancerER- breast cancer

ER- breast cancerER- amplified EGFnER- amplified erbB-2Drug-resistant MCF7

Derived from benign

component of breastDerived from in situ component

Derived from in situ component

Derived from in situ component

Luminal normal cells, SV4O

immortalized

Luminal normal cells, SV4O

immortalized

Normal tissues (n = 15) were obtained either from reduction

mammoplasties (n = 7), or from normal tissue adjacent to

tumors (n = 8). Benign breast disease samples consisted of

fibroadenomas (n = 5), fibrocystic disease (n = 3), and benign


ahcdefghijklmnopqrl23

..otr . I

Fig. 2 Southern blot of human-rodent hybrid cell lines with human Wnt5a probe. Lane 1, human genomic DNA; Lane 2, mouse genomic DNA; Lane

3, hamster genomic DNA. Lanes a-r, DNA from hybrid human-rodent cell lines, each beaning human chromosome DNA. Lanes: a, chromosome 1;

b, chromosome 2; c, chromosome 3; d, chromosome 5; e, partial chromosome 6;f, chromosome 7; g, chromosome 8; h, chromosome 9; i, chromosome

I 1 ; j, chromosome 12, k, chromosome 13; 1, chromosome 14; m, chromosome 15; n, chromosome 16; o, chromosome 17; p, chromosome 19; q,chromosome 20 and partial 4; r, chromosome 21. Arrows in Lanes I and c, signal for human genomic DNA (Lane 1) corresponds to that in Lane

F’, i.e. , chromosome 3. The absence of Wnt5a signal in a human-mouse hybrid cell line carrying a p terminus deleted chromosome 3 (data not shown)

suggests that Wnt5a is on human chromosome 3p.

phyllode tumors (n = 2). Tumors (n = 28) were selected to

represent different subgroups according to known prognostic

factors (node, ER, and EGFR status). In contrast to the cancer

cell lines, Wnt5a was commonly expressed in primary breast

cancer (Fig. 4). Comparing normal breast tissue to tumor tissue

I showed levels that were 4-fold higher on average in the latter0 (Table 2). Ten of 28 tumors had levels higher than the highest

expression in the cell lines from normal breast tissue. Thus, the

<1 cell lines reflected normal breast expression, but not tumor0 levels. Tumor levels were significantly greater than normal

� tissue levels (P 0.0004, Mann-Whitney U test).

0 Benign breast tissue also showed much higher expression

< I than normal breast tissues, similar to and often greater than0 levels in many of the tumors. Two different types of benign

?8 breast disease were studied: fibroadenomas and fibrocystic dis-<1 ease. The former is a benign lesion involving epithelial and

< 1 stromal elements. The latter is a nontumorous collection of cysts

and ducts with some epithelial hyperplasia. In both types of0 benign breast disease there was high expression of Wnt5a (Fig.

1 � nuclease protection). Benign levels were significantly greater1 than those of normals (P = 0.0012 Mann-Whitney U test) and

<1 also than those of tumors, although to a smaller extent (P =

5 0.03).

< 1 In situ hybridization using the Wnt5a probe was carried out

to assess localization of expression. This showed that levels

were low and difficult to detect in normal breast but were

detectable in a few ducts and lobules. In some larger ducts the

mRNA appeared to be expressed in luminal cells but not the

myoepithelial population. In fibroadenomas there was high ex-

pression in the epithelial component uniformly throughout the

tumor (Fig. 5, a and b). In fibrocystic disease it was again



wnt5a

gapdh


ab c de f gh i j k 1 mnop q r s

v- �

-�-

LT:____a.____A

-�

A

--_�w-�--�

�.� � A �-A..

Fig. 3 RNase protection assay on cell lines. Wnt5a and GAPDH signals are shown. Lanes a-f, human mammary epithelial cell lines; Lanes g-s, human

breast cancer cell lines. a, 2-5-2a; b, 3-4-1; c, 5-3-1; d, 6-1-1; e, MThV1-7;f, MRSV4-1; g, MCF1O; h, ZR1 1; i, ZR4;j, MDA41S; k, MDA4S3; I, MDA4S7;m, BT2O; n, SKBR3; o, Adniamycin-resistant; p, MCF7; q, AR9B11; r, T47D; s, MDA231.

Table 2 Wnt5a expression assayed by RNase protection in humanbreast cancer

Sample Type

Normal

Benign Tissue PrimaryTotal (a = 10) (a 15) (n - 28)

Age (yr)Mean 49 39 42 56SE 2.13 3.1 4.8 2.3

Median 49 38 46 57Min,a max 18, 86 25, 52 18, 78 32, 86

Wnt5a

(RNA units)Mean 7.1 18.7 1.5 5.9SE 10.4 17.2 1.5 6.4Median 3.0 11.5 1.0 4.0Mm, max 0, 45 0, 45 0, 5 0.5, 31

ER (fmol/mg)cytosol

protein

Mean 128SE 168

Median 50.5Mm, max 3, 695

EGFR (fmol/mg)membraneprotein

Mean 29SE 27Median 25Mm, max 0, 127

Node statusPositive 11

Negative 15

a Mm, minimu m; max, maximum.

expressed in the epithelial component (Fig. 5, c and d). Simi-

larly, in the malignant tumors (Fig. 5, e and f) Wnt5a was

expressed within the epithelial element of the tumor and in

clumps of invading cells throughout the stroma. There was no

increased localization at the invading edge. The sense Wnt5a

probes used as controls for nonspecific hybridization showed a

uniform and low background signal (data not shown).

Thus, in many cases of benign breast proliferation and

breast cancers. Wnt5a is overexpressed in the epithelium. This

was not due to gene amplification since Southern blots of the

high-expressing cases showed no evidence of this (data not

shown).

Features of carcinomas known to relate to tumor phenotype

were compared with Wnt5a expression. There was no correba-

tion of either ER or EGFR with Wnt5a expression (P = 0.3 and

0.5, respectively, Spearman’s rank correlation coefficient; Table

2), nor was there a correlation with node status (P = 0.1). There

was no association with menopausal status, as indicated by age

>50 years or <50 years old (Table 2). ER and EGFR were

inversely related to each other, as previously reported (Ref. 22;

P = 0.005, Spearman’s rank correlation coefficient). ER was

related to age and EGFR inversely as has been described before

(Ref. 22; P = 0.0007 and 0.036, respectively, Spearman’s rank

correlation coefficient).

In relation to patient age, there was no significant differ-

ence between normal and benign samples but the primary cancer

patients were significantly older than the normal (P = 0.02) and

benign (P = 0.0013) samples. However the differences in

Wnt5a expression between the samples cannot be a function of

the different age distributions, as Spearman’s rank correlation of

Wnt5a with age as a continuous variable shows no correlation

(P = 0. 1 1). Furthermore the group of tumors which overlap the

normal and benign samples in age (n = 9, age <52 years) have

no different a level of Wnt5a than those which are older (n = 19,

age >52 years, P = 0.86). Also, the differences in the level of

Wnt5a between normal samples and cancers are maintained

whether the cancer patients considered are over or under 52

years of age. Thus, the significant differences in Wnt5a expres-

sion seen between the groups are not related to age distribution

differences.

DISCUSSION

The human Wnt5a gene shows marked homology to other

species including mouse (23) and Xenopus (24). This is char-

acteristic of all of the Wnt family members which are highly

conserved (25). They show greater homology to the family

member in different species than to other family members

expressed in the same species. Wnt5a is located on the terminal

region of chromosome 3 beyond the 3pl 1 band, whereas mouse

Wnt5a is on chromosome 14. However, human chromosome 3 is

syntenic with mouse chromosome 9, suggesting chromosomal

rearrangements at this locus during evolution. The region

3p2l-25 is known to be involved in loss of heterozygosity in

human cancer (26), including 30% of breast cancers (27). How-



I.

.- . 5.. ‘ %_�� . a,

. -:

. . - .

� - . ,�;a.. �


NOFfl11II

Benign

gap

gap - �--

.4

Fig. 4 RNase protection assay

on human breast tissues. Wnt5a

and GAPDH signals are shown

for 15 normal breast tissue sam-

ples, 10 samples of benign dis-

ease, and 21 of the 28 tumors

examined (data for remaining 7tumors not shown in this figure

but included in Table 2).

�sIaIignant5 a -�-�-� � _.

gapw

-. --

_,�_� ‘..� -� � �

� ___Fig. 5 In situ hybridization of Wnt5a in human breast tissues, a, fibroadenoma, light field; b, fibroadenoma, dark field; c, fibrocystic disease, light

field; d, fibnocystic disease, dank field; e, carcinoma, light field; f, carcinoma, dark field. e, epithelial component; s, stroma; c, carcinoma.

ever, the work of Clark et a!. (28) suggests that Wnt5a is outside

this region and therefore not the gene involved.

During the course of this work, the isolation of overlapping

clones was reported giving the total sequence of the human

Wnt5a cDNA isolated from a human fetal fibroblast library. We

have similarly cloned overlapping clones from a human placen-

tab library. Our results independently confirm the sequence

published by Clark et a!. (28), as well as the chromosomal

localization of the gene. Our sequence differs from that of Clark

ci a!. (28) at a few nucleotides but these do not give rise to

amino acid differences.

Expression was studied in a range of human breast cancer

cell lines representing ER-positive and -negative types as well

as those having amplification of EGFR or erbB-2. With the

exception of BT2O, expression in the cell lines was very low

with levels similar to or lower than those found in normal

tissues. The breast cancer cell line BT2O showed high wnt5a

expression, similar to the elevated levels found in benign pro-

liferative lesions. In the mouse, Wntl expression in mammary

epithelium produces abnormal morphology in a hormone-inde-

pendent fashion (29). In relation to this, the level of expression

of Wnt5a in breast cancer cell lines appears unrelated to hor-

mone receptor status.

In tissues, Wnt5a expression was generally higher than that

in cell lines. Benign and malignant proliferative lesions of the

breast respectively showed levels of Wnt5a 10-fold and 4-fold




4 T. Bui, E. Huguet, and A. L. Harris, unpublished data.

higher than those in normal tissue. In the mouse Wnt5a is

expressed in normal breast tissue and its regulation has been

studied over a short period of reproductive history (10). Wnt5a

is present in early pregnancy but is undetectable by day 17.5 of

pregnancy. It is clearly difficult to reproduce such studies in

humans, but our results suggest that human breast tissue gener-

ally resembles that of the mouse in its expression of Wnt5a,

although no major endocrine effects involving steroid hormones

were demonstrated in that pre- and postmenopausal breast levels

were no different in any of the patient groups. This does not,

however, exclude pregnancy-specific regulation events. Wnt5a

was highest in the benign proliferative lesions, and analysis in

human breast cancer showed up-regulation of Wnt5a in 10 of 28

carcinomas above the highest level in normal breast tissues. The

up-regulation was not due to gene amplification in the breast

cancers and may therefore be related to transcriptional activa-

tion or RNA stabilization. The elevated level of Wnt5a is inde-

pendent of several other factors known to affect prognosis or

biology of human breast cancer such as EGFR and estrogen

receptor. In order to understand further the basis of up-regula-

tion of Wnt5a in human breast disease, human breast cell lines

transfected with a panel of oncogenes and cultured in different

extracellular matrices have been studied for regulation of

Wnt5a. There is evidence that Wnt5a is up-regulated severalfold

in these circumstances.4 Thus, Wnt5a may be secondarily reg-

ulated in response to a range of other genetic changes. Since

Wnts have recently been shown to modulate cell adhesion by

regulation of cadherins, it is possible that Wnt5a may also

contribute to this mechanism of regulation of growth and dif-

ferentiation.

In the mouse, Wnts have a role in the development of

normal mammary tissue, and aberrant Wnt expression can con-

tribute to mammary hyperplasia and mammary carcinomas.

Drawing a parallel between these observations and human

Wnt5a, the expression of the gene in normal breast suggests that

it has a role in this tissue, and its aberrant expression is associ-

ated with proliferative lesions or aberrant differentiation as in

fibroadenomas and fibrocystic disease. Olson and Papkoff (30)

have reported that the level of Wnt5a expression is inversely

related to the proliferative rate of the mouse mammary epithelial

cell line C57MG in vitro. Although this munine result cannot

necessarily be extrapolated to the human situation, it argues

against Wnt5a having a simple growth promoting role in the

benign and malignant lesions we analyzed. Thus, Wnt5a is

expressed at its highest level in benign disease, although it is

also elevated in cancer. It is possible that high Wnt5a expression

occurs at certain stage of normal differentiation, and that cells

blocked in this phase show the observed regulation. Olson and

Papkoff (30) have shown that in the mouse mammary epithelial

cell line C57MG, transfection of Wntl and Wnt2 causes a

30-fold reduction in the expression of endogenous Wnt4 and a

smaller decrease in the expression of Wnt5a. Thus, it is possible

that Wnt5a itself may interfere with the function of other Wnts

in human breast tissue where it is overexpressed.

To assess localization of Wnt5a, in situ hybridization was

carried out on normal tissues, fibrocystic disease, fibroadeno-

mas, and carcinomas. In normal tissues, the level of expression

was generally too low to detect above background, but could be

seen in a few ducts. In fibrocystic disease, fibroadenomas, and

carcinomas Wnt5a was expressed in epithelium. In some cases

it was possible to clearly resolve the basal myoepithelial layer of

the breast from the luminal epithelium and Wnt5a was expressed

in the luminal cell layer.

In fibroadenomas stromal cells are present in a much

greater proportion than in normal epithelium. Dilution of RNA

from the epithelium by matrix and stromal cells shows how high

the level of RNA must be in the fibroadenoma epithebium

compared to normal tissue epithelium. It is possible that the

stromal cells are involved in regulation of Wnt5a expression in

the epithelium or conversely that Wnt5a expression effects

stromal growth. Coculture of luminal cells with stromal cells to

measure Wnt5a regulation would be helpful to assess the role of

such stromal epithelial interactions.

The up-regulation of Wnt5a in both benign and malignant

proliferative disease of the breast suggests an important role of

Wnt genes in breast pathology. Wnt5a is the first member of the

family to demonstrate up-regulation in human breast cancer.

Cell lines established from benign or normal tissues may be

suitable models for further evaluation of the role of Wnt5a to

assess its regulation under variable growth and differentiation

conditions.

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1995;1:215-222. Clin Cancer Res S Lejeune, E L Huguet, A Hamby, et al. breast cancers.Wnt5a cloning, expression, and up-regulation in human primary

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Wnt5a Cloning, Expression, and Up-Regulation in Human ... · Vol. 1, 215-222, February 1995 Clinical Cancer Research 215 Wnt5a Cloning, Expression, and Up-Regulation in Human Primary