@

Oncogene (2000) 19, 1002-1009
© 2000 Macmillan Publishers Ltd All rights reserved 0950-9232/00 $15.00

www.nature.com/onc

Use of MMTV-Wnt-1 transgenic mice for studying the genetic basis of

breast cancer

Yi Li*', Wendy P Hively' and Harold E Varmus'

‘Division of Basic Science, National Cancer Institute, 49 Convent Drive, Building 49, Room 4A56, Bethesda, Maryland, MD

20892, USA

Wat-1 was first identified as a protooncogene activated
by viral insertion in mouse mammary tumors. Transgenic
expression of this gene using a mouse mammary tumor
virus LTR enhancer causes extensive ductal hyperplasia
early in life and mammary adenocarcinomas in approxi-
mately 50% of the female transgenic (TG) mice by 6
months of age. Metastasis to the lung and proximal
lymph nodes is rare at the time tumors are detected but
frequent after the removal of the primary neoplasm. The
potent mitogenic effect mediated by Wat-1 expression

does not require estrogen stimulation; tumors form after

an increased latency in estrogen receptor o-null mice.
Several genetic lesions, including inactivation of p53 and
over-expression of Fgf-3, collaborate with Wat-1 in
leading to mammary tumors, but loss of Sky and
inactivation of one allele of Rb do not affect the rate
of tumor formation in Wat-1 TG mice. Oncogene (2000)
19, 1002-1009.

Keywords: Wnt-1; breast cancer;
mammary; transgenic; MMTV

cancer models;

 

Introduction

Infection of most strains of mice, such as C3H, with
mouse mammary tumor virus (MMTV) leads to a high
incidence of mammary tumors (reviewed by Nusse,
1991). The sites of insertions by MMTV proviruses
have been extensively mined in order to identify genes
that are deregulated to cause tumorigenesis. Wnt-] was
the first protooncogene to be cloned following
activation by viral insertion in mouse mammary
tumors (Nusse and Varmus, 1982). (Its initial name,
int-1, was subsequently changed to Wnt-1 because of
its homology to the Drosophila Wingless (Wg) gene
(Nusse et al., 1991)). Insertional activation of Wnt-]
occurs in approximately 70% of C3H mice that are
chronically infected with MMTV (Nusse and Varmus,
1982). Other candidate protooncogenes that are some-
times activated by MMTV proviral insertions include
two additional members of the Wnt family, Wnt-3
(Roelink et al., 1990) and Wnt-10b (Lee et al., 1995);
three members of the fibroblast growth factor family,
Fegf-3/int-2 (Dickson et al., 1984), Fef-4/hst (Peters et
al., 1989), and Fef-8/AIGF (MacArthur et al, 1995);
Notch-4/int-3 (Lee et al., 1995); and int-6 (Asano et al.,
1997), encoding a subunit of the translation initiation
factor eIF3. Some of these genes, such as Fgf-3 and
Wnt-10b, have been validated as oncogenes by

 

*Correspondence: Y Li

transgenic expression (Kwan et al., 1992; Lane and
Leder, 1997; Muller et al., 1990).

The Wnt-1 gene encodes a member of a large
family of secreted proteins that are cysteine-rich,
glycosylated, and poorly soluble (reviewed by Nusse
and Varmus, 1992). Presently, at least 18 distinct Wnt
family members have been identified in mammals.
Having a propensity to associate with the extracellular
matrix, Wnts act on both Wnt-producing and adjacent
cells through cell surface receptors to control cell fate
and patterning (reviewed by Nusse and Varmus,
1992). In mice, Wnt-I is expressed exclusively in the
developing central nervous system (CNS) and aduit
testes (Jakobovits et al., 1986; Shackleford and
Varmus, 1987; Wilkinson et al., 1987), and it is
required for CNS patterning and development of the
midbrain and cerebellum (McMahon and Bradley,
1990; Thomas and Capecchi, 1990). Its Drosophila
ortholog, Wg, controls segment polarity and many
other developmental processes (Wodarz and Nusse,
1998).

A brief overview of the Wnt signaling pathway

One of the major intracellular responses to Wnt-1
signaling is to stabilize and increase the level of
cytosolic f-catenin (Figure 1), a multi-functional
protein that associates with membrane-bound E-
cadherin, as well as several DNA binding proteins,
such as members of the TCF/LEF family (reviewed by
Kinzler and Vogelstein, 1996). Heterodimers of -
catenin and transcriptions factors translocate to the
nucleus and transactivate a number of genes, including
c-myc (He et al., 1998), cyclin D1 (Shtutman ef ai.,
1999; Tetsu and McCormick, 1999), WISPs (Pennica
et al., 1998), and possibly cyclooxygenase-2 (Howe et
al., 1999). Depending upon the cell type, Wnt
signaling activates different genes, affecting various
stages of development and several types of cancer.
The receptors for Wnts have been identified as a
class of seven transmembrane proteins known as
Frizzled (Fz) (Bhanot et al, 1996). The ligand-
receptor interaction is facilitated by extracellular
proteoglycans and, inhibited by Fz-related proteins,
dickkopf, and cerberus. After binding a Wnt ligand,
Fz transmits a signal to cytoplasmic phosphoproteins
in the disheveled (Dvl) family via unknown mechan-
isms. Dvl1 inhibits the constitutively active kinase
activity of glycogen synthase kinase type 3 (GSK3),
which normally phosphorylates f-catenin and targets
it for degradation. Cytosolic levels of B-catenin are
additionally regulated by adenomatous polyosis coli
(APC), which targets B-catenin for proteosome-
mediated degradation, and by another large protein,
Axin, also an inhibitor of Wnt signaling. A complex
containing APC, Axin, B-catenin, GSK3, and GSK-
‘binding protein/Frat-1 has been observed in lysates
prepared from certain cell types (reviewed by Barish
and Williams, 1999). /

Several members of the Wnt family transform
cultured cells, when overexpressed. For example,
overexpression of Wnt-1, Wnt-2, Wnt-3, and Wnht-3a
(but not Wnt-4, Wnt-5a, Wnt-5b, and Wnt-7b) leads to
morphological transformation of mammary epithelial
cells such as CS7MG (Brown et al., 1986; Shimizu et
al., 1997). Continued overexpression is required for the
transformation phenotype induced by Wat-/ (Li et al.,
1999; Mason et al., 1992).

Wnt-I is not normally expressed in the mammary
gland, nor has it been directly implicated in human
breast cancer. However, several other Wnt family
members are expressed in breast tissue, and some are
overexpressed in breast tumors (reviewed by Bergstein
and Brown, 1999). In addition, genes encoding several
components and targets of the Wnt signaling pathway,
including f-catenin, APC, E-cadherin, cyclin D1, c-
myc, and WISPs, have been found to be mutated or
deregulated in several types of human tumors, such as
breast cancer (Bieche et a/., 1999), colon cancer (He et
al., 1998), melanoma (Rimm et al., 1999; Rubinfeld et
al., 1997), hepatocellular carcinoma (de La Coste et al.,
1998), and pilomatricoma (Chan et al., 1999).

The Wnt-1 transgenic (TG) mouse model

Wnt-1 TG mice were initially made to test the
oncogenicity of Wnt-1 (Tsukamoto et al., 1988). The
transgene (Figure 2), is controlled by the Wht-1
promoter and an MMTV LTR inserted upstream of
the gene in the opposite transcriptional orientation, in
a fashion reminiscent of a typical viral insertion into
the Wnt-1 locus in MMTV-induced tumors. Ectopic
Wnt-I expression exerts a potent mitogenic effect on
mammary epithelium; ductal hyperplasia is noticeable
in the mammary end-buds by 18 days of gestation
(Cunha and Hom, 1996) and very apparent 2 weeks
after birth in the TG females (Lin et al., 1992).
Because of the extensive ductal hyperplasia, female
TG mice can not deliver milk to their young.

About 50% of virgin female Wnt-1 TG mice in the
SJL strain develop adenocarcinomas by 6 months of

 

 

Nuclear Membrane v

Target
Genes

Figure 1 Illustration of the Wnt signal transduction pathway
(kindly provided by J-M Li). See text for explanation

MMTV-Wnt-1 transgenic model
Y Li et al

 

age; the rest succumb to tumors by 1 year. Breeding
females develop tumors slightly earlier than virgin mice
(Shackleford e¢ al., 1993; Tsukamoto et al., 1988). This
acceleration may be caused by either hormonal
influence on cell growth or the. increased mass of the
mammary epithelium attributed to pregnancies and
lactation. Hyperplasia is also extensive in the primary
mammary glands of adult male TG mice; about 15%
of them develop palpable mammary tumors by | year
of age (Kwan et al., 1992; Tsukamoto et al., 1988).
Although metastasis does not seem to occur frequently
at the time mammary tumors are detected (Tsukamoto
et al., 1988), the majority of female Wnt-1 TG mice
develop lymph node and/or jung metastasis after
removal of the primary tumor (L Godley and WP
Hively, unpublished observation).

Tumors found in Wnt-l TG mice are usually
moderately differentiated and comprised of ducts with
multiple layers of epithelial cells, that show significantly
higher than normal nucleus-to-cytoplasm ratio and
occasionally pleomorphic nuclei and mitotic figures.
The lumens usually contain pyknotic cells suggestive of
apoptosis. Widespread necrosis and hemorrhage are
sometimes noticeable in these tumors. In addition,
extensive fibrosis is present in neoplasms induced by the
Wnt-1 transgene. Hyperplastic glands of Wnt-1 TG mice
also display a prominent fibrotic response, which may
start as early as 7 days postnatally in TG females (G
Cunha, personal communication).

Variations in genetic backgrounds usually do not
influence the time course of tumor development
mediated by the Wnt-/] transgene (Bocchinfuso et al.,
1999; Donehower et al., 1995; Shackleford et al., 1993;
Tsukamoto et al., 1988). The original TG line was made in
C57BL/6 X SJL F1 mice. Subsequently, interbreedings
with other strains (FVB/N, BALB/c, 129/J, CS58BL/6)
have been found to be similar to SLJ in tumor latency
(Tabie 1). But a much longer latency has been observed in
some mixed backgrounds (C Alexander, personal
communication).

CEE] LR es
wiv LTR Wnt-1 > Wnet

M SV40 poly A+
promoter signal

Figure 2. Wnt-/ transgene construct. The 7 kb transgene contains
the MMTV-LTR approximately 1 kb upstream of the mouse
Wnt-] gene. The MMTV-LTR was placed in the opposite
transcriptional orientation and is used as an enhancer. The
Wnt-1 coding sequences are shown as filled boxes. A fragment
containing the SV-40 splice and polyadenylation sites (850 bp)
was placed downstream of the last exon of Wnt-1

 

Table 1 Genetic lesions crossed to the MMTV-Wat-/ transgene

 

Genetic

Genotype background

MMTV-Fef-3 TG FVB/N

References

Muller e¢ al., 1990;
Kwan et al., 1992

 

Sky —/- 129/Sv x CS7TBL/6 Lu et al., 1999;
WP Hively (unpublished)
ps3 —/- 129/Sv Donehower e7 al., 1992, 1995
ERa—/{- C57BL/6 Lubahn et al., 1993;
Bocchinfuso et al., 1999
MMTV-TGF CS57BL/6 A Chytil, Y-L Chen and

HL Moses (unpublished)

 

1003

 

Oncogene
MMTV-Wnt-1 transgenic model
Y Li et af

 

1004

 

Figure 3 Histology of mammary glands from an 18-week-old
MMTV-Wnt-1 TG virgin female. (a) Hyperplastic mammary
gland (25 x). (b) Mammary adenocarcinoma (25 x )

Many genetic lesions and epigenetic changes, such as
levels of mammogenic hormones, have been implicated
in breast carcinogenesis. However, the complex
molecular interplay leading to breast cancer is very
poorly understood. All mammary epithelial ceils
expressing the transgene in the Wnt-1 TG line are at
risk for tumor development. Indeed, ductal hyperplasia
occurs throughout the mammary tissue early in
development, yet tumors appear stochastically after
several months. Therefore, other cooperative events
must have accompanied expression of the Wht-1
transgene in the few cells that expanded into tumors.
A number of methods have been applied to uncover
these synergistic events. Among them are hormonal
manipulations, insertional activation of protoonco-
genes using retroviral infection, and breeding with
TG and knockout mice carrying other genetic lesions
implicated in breast cancer.

Hormonal and stromal influences on Wnt-1-induced
hyperplasia and tumors

Estrogen is essential in mammary development and
plays a very important role in carcinogenesis of the
breast (reviewed by Pike ef al., 1993). It stimulates
ductal morphogenesis and branching through nuclear
receptors—ERa and possibly the recently identified
ER (Kuiper et ai., 1996; Mosselman et al., 1996). ERa
is expressed in both mammary epithelium and the
stroma (Daniel ef al., 1987). ERB is detectable in
mammary tissues at low levels, but its role in
mammary proliferation remains elusive (Krege et al.,
1998).

 

Oncogene

Hyperplastic ductal growth in the Wnt-1 TG animals
persists despite estradiol deprivation due to ovariect-
omy (Bocchinfuso et al., 1999; Lin et al., 1992). Albeit
delayed, tumors continue to form in ovariectomized
mice (Bocchinfuso et al., 1999), suggesting that Wnt-1
does not require estrogen signaling for stimulating
proliferation and inducing tumors. These results were
confirmed and strengthened by experiments in which
the MMTV-Wnzt-/ transgene was crossed into the ERa
knockout (ERKO) mice. In homozygous ERKO mice,
mammary glands are underdeveloped, with rudimen-
tary ducts confined to the nipple area (Lubahn et ai.,
1993). The presence of the Wnt-J transgene stimulated
hyperplastic ductal growth in ERKO mice, and the
females developed mammary tumors at twice the age of
Wnt-1 TG females with one or two intact ERa genes
(Bocchinfuso et al., 1999). It remains to be determined
if the increased latency to tumor development observed
in ovariectomized or ERKO mice is the result of
reduced mass of mammary epithelium, the loss of
cooperative functions of ER signaling in Wht-I-
induced oncogenesis, or both.

The majority of human breast tumors are ERa-
positive and respond to anti-hormone therapy;
however, most malignant tumors are ERa-negative
(McGuire and Clark, 1985). Together with the fact that
only a small percentage of mammary epithelial cells
express ERa (Petersen et al., 1987; Ricketts et al,
1991), it has been suggested that breast cancer may
initiate from ER-positive cells but become ER-negative
and estrogen-independent in its growth at later stages
(Moolgavkar et al., 1980). The observation that
mammary tumors arise in both ERKO and ovariecto-
mized mice supports an alternative model that a
fraction of breast cancers may directly evolve from
ERa-negative cells (Nandi et al., 1995), an idea that
needs to be tested with other oncogenic transgenes.

The potent mitogenic effect of Wnt-1 on mammary
epithelial cells may not depend upon other mammo-
genic hormones either. For example, a similar degree
of abnormal side branching was observed in mammary
epithelial transplants derived from Wat-1 TG mice that
were either wild-type or nullizygous for progesterone
receptor « (PRa, C Brisken and R Weinberg, personal
communication). Likewise, in ovariectomized and/or
adrenalectomized mice, Wnt-1 continued to stimulate
hyperplastic growth in transplanted and reconstituted
glands (Edwards et al., 1992; Lin et al., 1992).

The reciprocal interactions between parenchyma and
stroma are important in mammary development,
remodeling, and carcinogenesis (reviewed by Cunha
and Hom, 1996). For example, signaling through the
epidermal growth factor receptor (EGFR) in the
mesenchyme is required for ductal growth and
branching morphogenesis, since epithelium trans-
planted from wild-type mice fails to proliferate in the
fat-pad from EGFR-null mice (Wiesen et al., 1999).
Interestingly, this requirement also seems to be
diminished in Wnt-1 TG mice. Transplantation of
epithelium from Wnt-] TG animals into the fat-pad of
EGFR nullizygous mice only modestly impaired
hyperplastic growth (G Cunha, personal communica-
tion). Epithelial-stromal interactions in tumor forma-
tion have also been studied by experiments in which
mammary epithelial cells from Wnt-1 TG animals were
transplanted into rat mammary fat-pad. Transplanta-
tion led to fibrotic proliferation in rat mesenchyme (G
Cunha, personal communication), suggesting that the
alteration of stromal differentiation is mediated by the
Wnt-1-expressing epithelial cells. Wnt-mediated epithe-

lial-mesenchymal interactions have also been reported .

in other tissues. For example, Wnt-induced mesench-
ymal reactions may regulate axonal growth and
guidance in developing limbs. Several members of the
Wnt family expressed in limb ectoderm induce
production of neurotrophin-3 in the underlying
mesenchyme (Patapoutian et al., 1999).

Collaboration between Wnt-1 and other genes in
oncogenesis

‘Mammary tumors induced by MMTV occasionally
show transcriptional activation of both Wat-1 and
fibroblast growth factor3 (Fgf3, Peters et al., 1986),
suggesting that these two genes collaborate in
oncogenesis. Fgf3 belongs to a family of heparin-
binding proteins that are both mitogenic and angio-
genic. Signaling by FGFs is mediated by transmem-
brane receptors (FGFRs) that phosphorylate and
activate several substrates, leading to the activation
of mitogen-activated protein kinases (reviewed by
Faham et al., 1998). Although Wnts and FGFs act
through very different pathways, they are both required
for development of primary body axis, neural axis,
limbs, and other structures, suggesting that these two
families of growth factors may collaborate in develop-
ment in ways that resemble synergistic roles in tumor
formation.

Transgenic female mice expressing MMTV-Fe/f3
show extensive mammary hyperplasia but rarely
develop tumors (Muller et al., 1990). When this
transgene was bred into Wni-1 TG mice, tumors
developed faster in bi-transgenic females than in
females bearing either transgene alone, providing direct
evidence of cooperation between these two growth
factors (Kwan et al., 1992). The acceleration is even
more dramatic in the bi-transgenic males. Additional
evidence of synergistic interactions between Wnt-/ and
members of the Fgf family comes from infection of Wnt-
1 TG animals with MMTV (Shackleford ef al., 1993).
Infection accelerates tumor formation, and up to ten
tumors per mouse were observed in infected animals.
Approximately 40% of the mammary tumors showed
insertional activation of Fgf3, a small percentage of
them had insertional activation of both Fgf3 and Fgf-4
or Fgf4 alone. Another member of the Fgf family, Fgf-8,
was also found to be insertionally-activated and/or
overexpressed in some of these tumors (Kapoun and
Shackleford, 1997; MacArthur et al., 1995). Collabora-
tion between members of the Wnt and FGF families has
also been observed in experiments in which infection of
MMTV-Fef3 TG mice with MMTV led to frequent viral
insertions in Wnt-1 or Wnt-10b loci (Lee et al., 1995).

Tumor growth factor £ (TGF) stimulates cell
growth under some conditions, but, more commonly,
inhibits celi proliferation, especially in the mammary
gland (reviewed by Massague, 1998). For example,
transgenic expression of TGF inhibits tumor forma-
tion in mice expressing an MMTV-7GF«e transgene
(Pierce et al., 1995). But in a recent cross between our
MMTV-Wni-I TG mice and MMTV-TGFB TG
animals, no éffects were observed on the rate of

MMTV-Wnt-1 transgenic model
Y Li et al

 

tumor appearance, histology, or the size of the tumor
induced by the Wnt-] transgene (A Chytil, Y-L Chen
and HL Moses, personal communication). Assuming
adequate levels of expression, it appears TGF cannot
inhibit proliferation of mammary epithelia stimulated
by the Wnt-/ transgene.

Collaboration between Wnt-1 and loss of a tumor
suppressor gene

Several tumor suppressor genes are mutated or
downregulated in human breast cancer. Inherited
mutations of some of them predispose to breast
neoplasm. For example, mutations of BRCA-1 and
BRCA-2 are found in approximately 50% and 30%,
respectively, of families predisposed to breast cancer

‘(Ford et al., 1998). Somatic p53 mutations are found

in about 35% of sporadic and 85% of familial breast
cancers (Crook et al., 1998), and germline alterations
of p53 are associated with a predisposition to several
cancers, including breast cancer (the Li-Fraumeni
syndrome). RB, a cell cycle regulator, is also mutated
in a small percentage of sporadic human breast
cancers (Berns et al., 1995). In addition, p21/WAFI/
CIPI, a_ cyclin-dependent kinase inhibitor that
regulates Gi-S cell cycle progression, is downregu-
lated in some breast tumors, especially those with
poor prognosis (Jiang et al., 1997; Wakasugi et al.,
1997).

The impact of the loss of a tumor suppressor gene on
tumorigenesis has been documented in animal modeis
using targeted gene disruption, loss of heterozygosity
(LOH) assays, and transgenic overexpression of a
dominant-negative version of a tumor suppressor
gene. Mice deficient for the p53 tumor suppressor gene
(p53+/— and p53—/—) develop tumors of non-
epithelial origin (Donehower et al., 1992). To analyse
the effect of p53 inactivation on mammary oncogenesis,
p53 knockout mice were bred with Wnt-1 TG mice
(Donehower et al., 1995). p53 nullizygotes (both females
and males) expressing the Wnt-] transgene develop
mammary tumors much earlier than mice containing at
least one wild-type allele, suggesting that inactivation of
p53 plays an important role and collaborates with Wnt-
J in mammary oncogenesis. In addition, p53-null
tumors are more anaplastic and less fibrotic than
tumors that carry at least one copy of the p53 gene
(Donehower et al., 1995).

Although the absence of one copy of p53 did not
significantly alter the time at which MMTV-Wnt-/
transgene induced tumors appeared, approximately
50% of the tumors in p53-heterozygous, Wnt-1 TG
mice displayed loss of the wild-type locus. This
frequent occurrence of LOH contrasts with the very
rare loss of the wild-type p53 allele in mammary
tumors from p53 heterozygotes carrying an MMTV-c-
myc transgene (Elson et al., 1995; McCormack et al.,
1998). It is notable that inactivation of p53 collaborates
with MMTV-c-myc, MMTV-H-ras, and MMTV-neu
transgenes to produce lymphomas and salivary tumors,
but rarely mammary tumors (C-X Deng, personal
communication, Elson et al., 1995; Hundley ef al.
1997).

Mutations in. BRCA-I and BRCA-2 are often
associated with loss of p53 in breast carcinogenesis in
humans (Crook et al., 1998). Induction of mammary

1005

 

Oncogene
MMTV-Wnt-1 transgenic model
Y Li et al

 

1006

tumors in the mouse by mammary-specific Brcal
inactivation is dramatically accelerated by inactivation
of p53 (Xu et al., 1999). However, loss of one allele of
Brca-1 (T Wynshaw-Boris, personal communication) or
Brca-2. (XS Cui and LA _ Donehower, personal
communication) does not seem to influence the
kinetics of tumor formation induced by the Wnt-1
transgene. It remains to be determined whether tumors
that are heterozygous for Brcal or Brca2 show LOH
or alteration in karyotype. The availability of mice that
carry loxP-flanked (floxed) alleles of Brcal and Brca2
will permit better tests for synergy between the loss of
Brcal or Brca2 and inheritance of a Wnt-] transgene in
tumor formation.

Germline mutations in one copy of the Rb gene
predispose humans to retinoblastomas and osteosarco-
mas. Mice nullizygous for Rb die during embryogen-
esis; heterozygotes develop tumors primarily in the
pituitary and thyroid glands but rarely in mammary
glands (Jacks et al., 1992). To determine if the loss of
Rb affects the development of tumors in Wnat-1 TG
animals, we have crossed Wnt-/ TG animals with mice
heterozygous for Rb. Absence of one allele of Rb did
not affect the age at which the tumor was detected, and
none of 25 tumors examined by restriction mapping
showed loss of the wild-type locus (WP Hively,
unpublished). The lack of acceleration may be due to
the complementary expression of one or both of the
other two members of the Rb gene family (p/07 and
p130) in the mouse mammary gland. In fact, the
presence of normal p/07 aileles has been shown to
inhibit Rb deficiency-mediated tumor formation in the
mouse retina (Robanus-Maandag ef al., 1998). Elim-
ination of all three members of the Rb gene family in
Wnt-I1 TG mice would further clarify the role of their
inactivation in mammary oncogenesis. One way to
eliminate their functions is to generate transgenic mice
expressing the gene encoding the amino terminal
domain (T,,;) of the Simian virus 40 T antigen, which
inactivates all three members of the Rb family (Saenz
Robles et al., 1994; Symonds ez al., 1994).

Inactivation of one or both alleles of p2/ did not
accelerate tumor formation in Wnt-1 TG mice (Jones et
al., 1999). But, interestingly, tumors from p2/+/—
mice grew significantly faster, with a higher mitotic
index and increased cyclin D1-associated phosphoryla-
tion of Rb, than those from either p2/+/+ or
p21—/— mice (Jones et al., 1999).

Additional tumor suppressor genes that collaborate
with the Wnt-] transgene to induce tumor formation
may be identified by scanning the whole genome for
LOH. Application of this approach has led to the
identification of two regions on mouse chromosomes 9
and 16 that are frequently deleted in insulinomas and
carcinoid tumors in TG mice expressing the Simian
virus 40 large T antigen (Dietrich et al., 1994).
Furthermore, using this technology in Fl hybrid mice
between FVB/N and Mus musculus castaneus, Radany
and colleagues (1997) have found that a marker on
chromosome 4 from Mus musculus castaneus was
frequently lost in MMTV-H-Ras transgene-induced
mammary tumors. In contrast, no single chromosome
was preferentially lost in tumors occurring in Fl
progeny of a similar cross between Wnt-1 TG SJL
mice and Mus musculus castaneus (unpublished data of
K Hong et al., cited in Radany et al., 1997).

 

Oncogene

Molecular characterization of tumors from Wnt-1 TG
animals

Chromosomal rearrangements including aneuploidy,
chromosomal translocations and duplications, and
amplification of selected genes are common in tumor
cells (reviewed by Wright, 1999). Loss of p53 function
frequently leads to deregulated ceil cycle control and
chromosomal instability, which favors tumor growth
(reviewed by Prives and Hall, 1999). Mammary tumors
from Wnt-1 TG mice with one or two functional copies
of p53 display occasional chromosomal abnormalities
as shown by comparative genome hybridization (CGH,
Kallioniemi et al., 1992), which detects regions of
expansion and deletion in all chromosomes. As
expected, Wnt-1 induced tumors without any p53
function usually have more than one chromosomal
abnormality. Tumors that arose in p53 heterozygotes
and experienced LOH at the p53 locus displayed even
more extensive alterations (at least three regions of
DNA gain or loss) (Donehower et al., 1995).

In general, it is difficult to anticipate what specific
genes in an amplified chromosomal region may have
synergized with an oncogenic transgene to induce
neoplasm. But the distal region of chromosome 7,
which was amplified in a Whnt-I-induced p53—/—
tumor, is the site of Fgf3 (Donechower et al., 1995).
Molecular hybridization using an Fgf3-specific probe
confirmed that Ffg3 was amplified and abundantly
expressed in this tumor (Donehower et al., 1995). This
is different from human breast cancer, in which the
syntenic region of chromosome 8q is frequently
amplified (Brison, 1993; Lammie et al., 1991; Theiliet
et al., 1989), but Fgf3 mRNA is not detected (Penault-
Llorca et al., 1995); however, a linked gene, PRAD-1/
CycinD1, is usually overexpressed in such tumors
(Motokura et al., 1991).

Spectral karyotyping (SKY) labels each chromosome
with a different color, allowing detection of chromo-
somal translocations and duplications (Liyanage et al.,
1996). We have analysed some tumors from Wnt-1 TG
mice that were p53+/— or p53—/—. Translocations,
trisomy, and aneuploidy have been detected in cells
cultured from some of these tumors (Z Weaver and
WP Hively, unpublished). Karyotype instability in
mammary tumors has been reported in mammary-
specific Brcal knockout mice (Xu et al., 1999) and
other transgenic models including MMTV-c-myc
(McCormack et al., 1998; Weaver et al., 1999).

As a physiologic response to genotoxins, p53 is
rapidly induced to cause cell cycle arrest and/or
apoptosis. Inactivation of p53 is often accompanied by
accelerated cell growth and attenuated apoptosis
(reviewed by Ko and Prives, 1996). p53 deficiency
(p53+/—, p53—/—) enhances cell proliferation in the
Wnt-1 transgene-derived tumors, but the modestly
ongoing apoptosis that accompanies Wnt-1 overexpres-
sion does not seem to be attenuated (Jones e¢ al., 1997).
Similarly, absence of one allele of p53 does not affect the
apoptotic index in mammary tumors induced by an
MMTV-c-myc transgene (McCormack et al., 1998).

Normal telomeres are essential to cell survival.
Telomerase is usually activated in human cancer cells,
presumably to overcome shortened telomeres due to
excessive cell replication (reviewed by de Lange and
DePinho, 1999). Normal telomeres (20-50 kb) are
present in both hyperplastic glands and carcinomas
from Wnt-1 TG mice, regardless of the p53 status
(Broccoli et al., 1996). Interestingly, despite the
presence of long telomeres, telomerase activity and
the RNA component of the enzyme were consistently
upregulated in these tumors compared with normal
and hyperplastic glands (Broccoli et al., 1996),
suggesting that activation of the telomerase machinery
in at least some mammary tumors does not depend
upon telomeric shortenings. Breeding Wnt-1 TG
animals with mice that carry a mutated gene for
telomerase or a component of the telomeric complex
(Blasco et al., 1997; Rudolph et al., 1999) will help
address whether the telomere activation is required for
mammary oncogenesis induced by the Wnt-/ transgene.

Another approach to uncovering the molecular
basis of tumorigenesis is to identify differentially-
expressed genes during various stages of tumor
formation. Several methods including subtractive
hybridization, differential display, serial analysis of
gene expression (SAGE), and cDNA expression array
technology have been used. A number of genes have
been found to be deregulated in Whnt-1-induced
tumors and Wnt-I-transformed cells by these and
other methods. For example, using PCR to screen for
differentially expressed tyrosine kinases, we found that
Sky, which encodes a member of the Axl/Ufo family
of receptor tyrosine kinases, is barely detectable in the
mammary glands from virgin animals and in
preneoplastic mammary glands, but is abundantly
expressed in the mammary tumors of Wnat-1 TG
mice (Taylor et al., 1995). Recently, using a modified
subtractive hybridization approach, Pennica et al.
(1998) have found that two novel genes, WISP] and
2, are overexpressed in Wnt-J-transformed mammary
cells and that they are transcriptionally regulated by
Wnt-I expression and aberrantly expressed in colon
cancer.

Screening for upregulated genes in tumors might
also help identify collaborating factors in tumor
formation. But alteration of the transcriptional
apparatus during neoplastic conversion may dereg-
ulate many non-collaborating genes. An example of
such a non-synergistic element is Sky. Tumor
development in Whot-] transgenic mice was not
affected by breeding Sky knockout mice (Lu et al.,
1999) with Wnt-1 TG animals (WP Hively, unpub-
lished), suggesting that overexpression of Sky is not
necessary for Wnt-1 mediated oncogenesis.

Involvement of other components of the Wnt signaling
pathway in mammary carcinogenesis

Many other components of the Wnt signaling path-
way have been implicated in mammary tumorigenesis.
Mutations in APC, a negative regulator of the Wnt
signaling pathway, have been reported to confer an
increased risk for development of breast cancer in
Ashkenzi Jews (Redston et al., 1998; Woodage et al.,
1998). Min mice, which carry a nonsense mutation at
one APC locus, also have increased risk for mammary
carcinomas after carcinogen treatment (Moser et al.,
1993, 1995). With the use of additional TG mice, it
will be interesting to determine if deregulated
expression of other components of the Wont-1
signaling pathway, such as over-expression of f-

MMTV-Wnit-1 transgenic model
Y Li et af

 

catenin and inactivation of &-cadherin, also induce
mammary tumors.

Ali three members of the Dv/ family (Dv/1, Dvi2 and
Dvi3) are expressed in mammary glands, with Dvll
being most abundant (Tsang et al., 1996). Dvi proteins
transmit signals from the Fz receptor for Wnt-1 to B-
catenin via unknown mechanisms (see Introduction).
Mice nullizygous for Dvil are normal except for
abnormalities in social behavior and sensorimotor
gating (Lijam et ai., 1997). The mammary epithelia
lacking the dominant member of this family might be
expected to respond poorly to Wnt-1 induced cell
proliferation and tumor formation. But, Dv// nullizyg-
osity did not affect the rate of tumor formation in Wnt-
I TG mice (N Lijam, WP Hively, HE Varmus and T
Wynshaw-Boris, unpublished). Since Dv/2 and Dvi/3 are
also expressed in the mammary gland, they might have
substituted for Dv// in mediating the Wnt-1 signal.

Syndecan-1 is a member of the transmembrane
proteoglycan family that regulates ceil morphology
and growth (Leppa et al., 1992). Proteoglycans
facilitate the binding of Wnt ligands to Fz receptors
(Lin and Perrimon, 1999; Wodarz and Nusse, 1998).
Consistent with this finding, Wnt-1 TG mice that carry
two null alleles of syndecan-] very rarely develop tumors
(C Alexander, personal communication), suggesting
that syndecan-1 may be an important factor in
mediating Wnt-1 signaling in the mammary gland.

Prospects

Although initially developed to document the onco-
genic potential of Wnt-1, our line of MMTV-Wnt-1
TG mice has been useful in studying many aspects of
mammary tumorigenesis: the cooperation between
cancer genes, the influence of estrogen receptors and
growth hormones, and the concomitant changes in
genomic instability and gene expression.

Different stages of tumor progression can be
discerned in Wnt-1 TG mice, and some of the
collaborative lesions accompanying Wnt-1 overexpres-
sion in tumor formation have been defined. There-
fore, this line may be a convenient source of
hyperplastic glands and invasive and metastatic
tumors for various approaches designed to identify
molecular signatures of tumor progression. Compar-
ing the expression profile of the Wnt-J-derived
tumors with those of tumors derived from Wat-1
TG mice crossed with other genetically modified lines
may offer additional insights into the complex nature
of mammary oncogenesis. Additional benefits in the
characterization of these tumors include identification
of transcriptional targets of the Wnt-1 signaling
pathway.

Recently, a novel method has been used to transduce
oncogenes into somatic cells of a specific tissue
(reviewed by Fisher et al., 1999) using sub-group A
avian leukosis virus (ALV-A) as a vector. Transgenic
expression of tv-a, encoding the receptor for ALV-A,
from a cell type-specific promoter, permits tissue-
specific infection with ALV-A, which does not
produce infectious virus in mammalian hosts. Conse-
quently, combinatorial effects of genetic lesions can be
examined in a single TG line by infecting with mixtures
of ALV-A viruses expressing different oncogenes. In
addition, ALV-A expressing the gene encoding Cre

1007

 

Oncogene
MMTV-Wnt-1 transgenic model
Y Li et al

 

1008

recombinase can be used to inactivate tumor suppres-
sor genes flanked with loxP recombination sites. Since
ALV infection requires mitotic cells which are widely
available only during late pregnancy, breeding the
Wnt-1 transgene into mice expressing tv-a from a
mammary-specific promoter may provide both replicat-
ing epithelial cells (eliminating the requirement for
pregnancies) and a cancer predisposing factor allowing
more rapid formation of tumors. The TVA technology

References

Asano K, Merrick WC and Hershey JW. (1997). J. Biol.
Chem., 272, 23477 —23480.

Barish GD and Williams BO. (1999). Signal Networks and
Cell Cycle Control. Gutkind JS (ed.). Human Press.

Bergstein I and Brown AMC. (1999). Breast Cancer:
Molecular Genetics, Pathogenesis and Therapeutics. Bow-
cock AM (ed.). Human Press: Totowa, New Jersey, pp.
181-198.

Berns EM, de Klein A, van Putten WL, van Staveren IL,
Bootsma A, Klijn JG and Foekens JA. (1995). Int. J.
Cancer, 64, 140-145.

Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, Macke
JP, Andrew D, Nathans J and Nusse R. (1996). Nature,
382, 225-230.

Bieche I, Laurendeau I, Tozlu 8, Olivi M, Vidaud D,
Lidereau R and Vidaud M. (1999). Cancer Res., 59,
2759-2765.

Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM,
DePinho RA and Greider CW. (1997). Cell, 91, 25-34.
Bocchinfuso WP, Hively WP, Couse JF, Varmus HE and

Korach KS. (1999). Cancer Res., 59, 1869 — 1876.

Brison O. (1993). Biochim Biophys Acta, 1155, 25-41.

Broccoli D, Godley LA, Donehower LA, Varmus HE and de
Lange T. (1996). Mol. Cell Biol., 16, 3765-3772.

Brown AM, Wildin RS, Prendergast TJ and Varmus HE.
(1986). Cell, 46, 1001-1009.

Chan EF, Gat U, McNiff JM and Fuchs E. (1999). Nat.
Genet., 21, 410-413.

Crook T, Brooks LA, Crossland S, Osin P, Barker KT,
Waller J, Philp E, Smith PD, Yulug I, Peto J, Parker G,
Allday MJ, Crompton MR and Gusterson BA. (1998).
Oncogene, 17, 1681 — 1689.

Cunha GR and Hom YK. (1996). J. Mammary Gland Biol.
Neoplasia, 1, 21—37.

Daniel CW, Silberstein GB and Strickland P. (1987). Cancer
Res., 47, 6052-6057.

de La Coste A, Romagnolo B, Billuart P, Renard CA,
Buendia MA, Soubrane O, Fabre M, Chelly J, Beldjord C,
Kahn A and Perret C. (1998). Proc. Natl. Acad. Sci. USA,
95, 8847-8851.

de Lange T and DePinho RA. (1999). Science, 283, 947-949.

Dickson C, Smith R, Brookes S.and Peters G. (1984). Cell,
37, 529-536.

Dietrich WF, Radany EH, Smith JS, Bishop JM, Hanahan D
and Lander ES. (1994). Proc. Natl. Acad. Sci. USA, 91,
9451-9455.

Donehower LA, Godley LA, Aldaz CM, Pyle R, Shi YP,
Pinkel D, Gray J, Bradley A, Medina D and Varmus HE.
(1995). Genes Dev., 9, 882-895.

Donehower LA, Harvey M, Slagle BL, McArthur MJ,
Montgomery CA, Butel JS and Bradley A. (1992). Nature
(London), 356, 215-221.

Edwards PA, Hiby SE, Papkoff J and Bradbury JM. (1992).
Oncogene, 7, 2041-2051.

Elson A, Deng C, Campos-Torres J, Donehower LA and
Leder P. (1995). Oncogene, 11, 181-190.

Faham S, Linhardt RJ and Rees DC. (1998). Curr. Opin.
Struct. Biol., 8, 578 —586.

 

Oncogene

may help test candidate collaborative events in context
of the Wnt-1 transgene.

Acknowledgments

We thank S Orsulic, S Ortiz, G Fisher and M Chamorro
for critical review of the manuscript. Y Li is supported by
the Department of Defense Breast Cancer Research
Program.

Fisher GH, Orsulic S, Holland EC, Hively WP, Li Y, Lewis
BC, Williams BO and Varmus HE. (1999). Oncogene, 18,
5253-5260.

Ford D, Easton DF, Stratton M, Narod S, Goldgar D,
Devilee P, Bishop DT, Weber B, Lenoir G, Chang-Claude
J, Sobol H, Teare MD, Struewing J, Arason A, Scherneck
S, Peto J, Rebbeck TR, Tonin P, Neuhausen S,
Barkardottir R, Eyfjord J, Lynch H, Ponder BA, Gayther
SA, Zelada-Hedman M et al. (1998). Am. J. Hum. Genet.,
62, 676 —689.

He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da
Costa LT, Morin PJ, Vogelstein B and Kinzler KW.
(1998). Science, 281, 1509-1512.

Howe LR, Subbaramaiah K, Chung WJ, Dannenberg AJ
and Brown AM. (1999). Cancer Res., 59, 1572 —1577.

Hundley JE, Koester SK, Troyer DA, Hilsenbeck SG, Subler
MA and Windle JJ. (1997). Mol. Cell Biol., 17, 723-731.

Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA
and Weinberg RA. (1992). Nature, 359, 295-300. .

Jakobovits A, Shackleford GM, Varmus HE and Martin
GR. (1986). Proc. Natl. Acad. Sci. USA, 83, 7806-7810.

Jiang M, Shao ZM, Wu J, Lu JS, Yu LM, Yuan JD, Han QX,
Shen ZZ and Fontana JA. (1997). Int. J. Cancer, 74, 529 —
$34.

Jones JM, Attardi L, Godley LA, Laucirica R, Medina D,
Jacks T, Varmus HE and Donehower LA. (1997). Cell
Growth Differ., 8, 829-838.

Jones JM, Cui XS, Medina D and Donehower LA. (1999).
Cell Growth Differ, 10, 213-222.

Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray
JW, Waldman F and Pinkel D. (1992). Science, 258, 818 -
821.

Kapoun AM and Shackleford GM. (1997). Oncogene, 14,
2985-2989.

Kinzler KW and Vogelstein B. (1996). Cell, 87, 159-170.

Ko LJ and Prives C. (1996). Genes Dev., 10, 1054-1072.

Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M,
Mahler JF, Sar M, Korach KS, Gustafsson JA and
Smithies O. (1998). Proc. Natl. Acad. Sci. USA, 95,
15677 — 15682.

Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S and
Gustafsson JA. (1996). Proc. Natl. Acad. Sci. USA, 93,
5925-5930.

Kwan H, Pecenka V, Tsukamoto A, Parslow TG, Guzman
R, Lin TP, Muller WJ, Lee FS, Leder P and Varmus HE.
(1992). Mol. Cell Biol., 12, 147-154.

Lammie GA, Fantl V, Smith R, Schuuring E, Brookes S,
Michalides R, Dickson C, Arnold A and Peters G. (1991).
Oncogene, 6, 439-444.

Lane TF and Leder P. (1997). Oncogene, 15, 2133-2144.

Lee FS, Lane TF, Kuo A, Shackleford GM and Leder P.
(1995). Proc. Natl. Acad. Sci. USA, 92, 2268 —2272.

Leppa S, Mali M, Miettinen HM and Jalkanen M. (1992).
Proc. Natl. Acad. Sci. USA, 89, 932-936.

Li YX, Papkoff J and Sarkar NH. (1999). Virology, 255,
138-149.
Lijam N, Paylor R, McDonald, MP, Crawley JN, Deng CX,
Herrup K, Stevens KE, Maccaferri G, McBain CJ,
Sussman DJ and Wynshaw-Boris A. (1997). Cell, 90,
895-905.

Lin TP, Guzman RC, Osborn RC, Thordarson G and Nandi
S. (1992). Cancer Res., 52, 4413-4419.

Lin X and Perrimon N. (1999). Nature, 400, 281-284.

Liyanage M, Coleman A, du Manoir S, Veldman T,
McCormack S, Dickson RB, Barlow C, Wynshaw-Boris
A, Janz S, Wienberg J, Ferguson-Smith MA, Schrock E
and Ried T. (1996). Nat. Genet., 14, 312-315.

Lu Q, Gore M, Zhang Q, Camenisch T, Boast S, Casagranda
F, Lai C, Skinner MK, Klein R, Matsushima GK, Earp
HS, Goff SP and Lemke G. (1999). Nature, 398, 723-728.

Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS
and Smithies O. (1993). Proc. Natl. Acad. Sci. USA, 90,
11162-11166.

MacArthur CA, Shankar DB and Shackleford GM. (1995).
J. Virol., 69, 2501 —2507.

Mason JO, Kitajewski J and Varmus HE. (1992). Mol. Biol.
Cell, 3, 521-533.

Massague J. (1998). Annu. Rev. Biochem., 67, 753-791.

McCormack SJ, Weaver Z, Deming S, Natarajan G, Torri J,
Johnson MD, Liyanage M, Reid T and Dickson RB.
(1998). Oncogene, 16, 2755 —~2766.

McGuire WL and Clark GM. (1985). Semin. Oncol., 12, 12-
16.

McMahon AP and Bradley A. (1990). Cell, 62, 1073-1085.

Moolgavkar SH, Day NE and Stevens RG. (1980). J. Natl.
Cancer Inst., 65, 559 —569.

Moser AR, Luongo C, Gould KA, McNeley MK, Shoe-
maker AR and Dove WF. (1995). Eur. J. Cancer, 31A,
1061-1064.

Moser AR, Mattes EM, Dove WF, Lindstrom MJ, Haag JD
and Gould MN. (1993). Proc. Natl. Acad. Sci. USA, 90,
8977-8981.

Mosselman S, Polman J and Dijkema R. (1996). FEBS Lett.,
392, 49-53. .

Motokura T, Bloom T, Kim HG, Juppner H, Ruderman JV,
Kronenberg HM and Arnold A. (1991). Nature, 350, 512-—
515.

Muller WJ, Lee FS, Dickson C, Peters G, Pattengale P and
Leder P. (1990). EMBO J., 9, 907-913.

Nandi 8, Guzman RC and Yang J. (1995). Proc. Natl. Acad.
Sci. USA, 92, 3650-3657.

Nusse R. (1991). Curr. Top. Microbiol. Immunol., 171, 43 -
65.

Nusse R, Brown A, Papkoff J, Scambler P, Shackleford G,
McMahon A, Moon R and Varmus H. (1991). Cell, 64,
231.

Nusse R and Varmus HE. (1982). Cell, 31, 99-109.

Nusse R and Varmus HE. (1992). Cell, 69, 1073 — 1087.

Patapoutian A, Backus C, Kispert A and Reichardt LF.
(1999). Science, 283, 1180-1183.

Penault-Llorca F, Bertucci F, Adelaide J, Parc P, Coulier F,
Jacquemier J, Birnbaum D and deLapeyriere O. (1995).
Int. J. Cancer, 61, 170-176.

Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence
DA, Lee J, Brush J, Taneyhill LA, Deuel B, Lew M,
Watanabe C, Cohen RL, Melhem MF, Finley GG, Quirke
P, Goddard AD, Hillan KJ, Gurney AL, Botstein D and
Levine AJ. (1998). Proc. Natl. Acad. Sci. USA, 95, 14717—
14722.

Peters G, Brookes S, Smith R, Placzek M and Dickson C.
(1989). Proc. Natl. Acad. Sci. USA, 86, 5678 — 5682.

Peters G, Lee AE and Dickson C. (1986). Nature, 320, 628 —
631.

Petersen OW, Hoyer PE and van Deurs B. (1987). Cancer
Res., 47, 5748-5751.

Pierce Jr DF, Gorska AE, Chytil A, Meise KS, Page DL,
Coffey Jr RJ and Moses HL. (1995). Proc. Natl. Acad. Sci.
USA, 92, 4254-4258.

MMTV-Wot-i transgenic model
Y Li et af

@

 

Pike MC, Spicer DV, Dahmoush L and Press MF. (1993).
Epidemiol Rev., 15, 17-35.

Prives C and Hall PA. (1999). J. Pathol., 187, 112-126.

Radany EH, Hong K, Kesharvarzi S, Lander ES and Bishop
JM. (1997). Proc. Natl. Acad. Sci. USA, 94, 8664-8669.

Redston M, Nathanson KL, Yuan ZQ, Neuhausen SL,
Satagopan J, Wong N, Yang D, Nafa D, Abrahamson J,
Ozcelik H, Antin-Ozerkis D, Andrulis I, Daly M, Pinsky
L, Schrag D, Gallinger S, Kaback M, King MC, Woodage
T, Brody LC, Godwin A, Warner E, Weber B, Foulkes W
and Offit K. (1998). Nat. Genet., 20, 13-14.

Ricketts D, Turnbull L, Ryall G, Bakhshi R, Rawson NS,
Gazet JC, Nolan C and Coombes RC. (1991). Cancer Res.,
§1, 1817-1822.

Rimm DL, Caca K, Hu G, Harrison FB and Fearon ER.
(1999). Am. J. Pathol., 154, 325-329.

Robanus-Maandag E, Dekker M, van der Valk M, Carrozza
ML, Jeanny JC, Dannenberg JH, Berns A and te Riele H.
(1998). Genes Dev., 12, 1599-1609.

Roelink H, Wagenaar E, Lopes da Silva S, and Nusse R.
(1990). Proc. Natl. Acad. Sci. USA, 87, 4519-4523.

Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porfiri E and
Polakis P. (1997). Science, 275, 1790-1792.

Rudolph KL, Chang S, Lee HW, Blasco M, Gottlieb GJ,
Greider C and DePinho, RA. (1999). Cell, 96, 701 —712.
Saenz Robles MT, Symonds H, Chen J and Van Dyke T.

(1994). Mol. Cell Biol., 14, 2686-2698.

Shackleford GM, MacArthur CA, Kwan HC and Varmus
HE. (1993). Proc. Natl. Acad. Sci. USA, 90, 740-744.
Shackleford GM and Varmus HE. (1987). Cell, 50, 89-95.
Shimizu H, Julius MA, Giarre M, Zheng Z, Brown AM and
Kitajewski J. (1997). Cell Growth Differ, 8, 1349-1358.
Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico
M, Pestell R and Ben-Ze’ev A. (1999). Proc. Natl. Acad.

Sci. USA, 96, 5522-5527.

Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe
S, Jacks T and Van Dyke T. (1994). Cell, 78, 703-711.
Taylor IC, Roy S, Yaswen P, Stampfer MR and Varmus HE.

(1995). J. Biol. Chem., 270, 6872 — 6880.

Tetsu O and McCormick F. (1999). Nature, 398, 422 —426.

Theillet C, Le Roy X, De Lapeyriere O, Grosgeorges J,
Adnane J, Raynaud SD, Simony-Lafontaine J, Goldfarb
M, Escot C and Birnbaum D. (1989). Oncogene, 4, 915-
922.

Thomas KR and Capecchi MR. (1990). Nature, 346, 847-
850.

Tsang M, Lijam N, Yang Y, Beier DR, Wynshaw-Boris A
and Sussman DJ. (1996). Dev. Dyn., 207, 253 —262.

Tsukamoto AS, Grosschedi R, Guzman RC, Parslow T and
Varmus HE. (1988). Cell, 55, 619-625.

Wakasugi E, Kobayashi T, Tamaki Y, Ito Y, Miyashiro I,
Komoike Y, Takeda T, Shin E, Takatsuka Y, Kikkawa N,
Monden T and Monden M. (1997). Am. J. Clin. Pathol.,
107, 684-691.

Weaver ZA, McCormack SJ, Liyanage M, du Manoir S,
Coleman A, Schrock E, Dickson RB and Ried T. (1999).
Genes Chromos. Cancer, 25, 251-260.

Wiesen JF, Young P, Werb Z and Cunha GR. (1999).
Development, 126, 335-344.

Wilkinson DG, Bailes JA and McMahon AP. (1987). Cell,
50, 79-88.

Wodarz A and Nusse R. (1998). Ann. Rev. Cell Dey. Biol., 14,
59-88.

Woodage T, King SM, Wacholder S, Hartge P, Struewing
JP, McAdams M, Laken SJ, Tucker MA and Brody LC.
(1998). Nat. Genet., 20, 62-65.

Wright EG. (1999). J. Pathol., 187, 19-27.

Xu X, Wagner KU, Larson D, Weaver Z, Li C, Ried T,
Hennighausen L, Wynshaw-Boris A and Deng CX. (1999).
Nat. Genet., 22, 37-43.

009

 

Oncogene