1 UMR 144 CNRS-Institut Curie, Institut Curie, Section de Recherche, 26 rue
d'Ulm, 75248, Paris, Cedex 05, France
2 Department of Molecular Cell Biology, The Weizmann Institute of Science,
Rehovot, 76100, Israel
* Author for correspondence (e-mail: marina.glukhova{at}curie.fr)
Accepted 12 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ß-catenin, Progenitor cell, Mammary gland, Basal epithelial cell, Hyperplasia, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The origin of the two major mammary epithelial cell lineages remains poorly
understood. Serial transplantation experiments have suggested that the adult
mouse mammary epithelium harbors long-lived, bipotent and lineage-restricted
progenitors with a high proliferative potential
(Smalley and Ashworth, 2003;
Smith and Boulanger, 2003
).
Moreover, a technique that allows the maintenance of early mammary progenitors
in vitro was recently developed (Dontu et
al., 2003
). However, the location of the progenitor cell
population in the mammary epithelium and its phenotypic characteristics remain
to be established. Ultrastructural studies revealed a candidate cell type -
undifferentiated `pale' or `light' cells resting on the basement membrane or
the suprabasal surface of myoepithelial cells
(Smith and Boulanger, 2003
;
Smith and Medina, 1988
). These
cells are rare, but they are present at all stages of mammary development and
are distributed throughout the mammary tree. Welm et al. have shown that the
cell population expressing Sca1, a presumable progenitor cell marker, is
located in the luminal layer of mammary gland
(Welm et al., 2002
).
Similarly, the experiments performed in vitro with separated luminal and
myoepithelial cells have suggested that bipotent mammary precursor cells
belong to the luminal compartment (Pechoux
et al., 1999
; Smalley et al.,
1999
). However, other studies provided evidence that luminal cells
in the human and mouse mammary glands originated from progenitors with basal
characteristics (Bocker et al.,
2002
; Jonkers et al.,
2001
). We recently described a mouse mammary epithelial cell line
with progenitor properties (Deugnier et
al., 2002a
). In vitro, these cells exclusively express basal cell
markers, such as P-cadherin and K5/14, whereas in vivo, when injected into the
cleared mammary fat pad, they are able to self-renew and to produce
differentiated progeny restricted to the luminal secretory lineage. On the
contrary, a cell line displaying progenitor properties isolated by Gudjonsson
et al. from human breast presents essentially luminal characteristics. In
clonal cultures or when grafted subcutaneously in nude mice, these cells are
able to differentiate into myoepithelium
(Gudjonsson et al., 2002
).
These apparently contradictory data suggest that phenotypically diverse
progenitor populations may exist in the mammary epithelium, either permanently
or at specific developmental stages.
Numerous studies have shown that the Wnt/ß-catenin signaling pathway
is involved in the maintenance of the progenitor cell population in the skin,
intestine and other tissues (Alonso and
Fuchs, 2003; Sancho et al.,
2003
). In epithelial cells, ß-catenin is engaged in the
formation of cadherin-containing cell-cell junctions, whereas non-junctional
ß-catenin is rapidly degraded by the ubiquitin-proteasome system.
Activation of Wnt signaling results in stabilization of ß-catenin, its
translocation to the nucleus, binding to Lef/Tcf transcription factors and the
transactivation of target genes, including those that encode important
regulators of growth, survival and differentiation (see Wnt gene homepage at
http://www.stanford.edu/~rnusse/pathways/targets.html).
Wnt/ß-catenin signaling is involved in the regulation of cell fate during
development, and its aberrant activation due to ß-catenin stabilization
contributes to tumorigenesis (Nelson and
Nusse, 2004
; Polakis,
2000
).
Several members of the Wnt family are differentially expressed during
mammary development (Buhler et al.,
1993; Gavin and McMahon,
1992
; Lane and Leder,
1997
; Weber-Hall et al.,
1994
). However, with the exception of Wnt4, their functions have
not been studied yet. Overexpression of Wnt4 in mouse mammary epithelial cells
grafted into cleared mammary fat pads of virgin mice results in the formation
of ramified outgrowths, mimicking the branching pattern typically seen during
early pregnancy (Bradbury et al.,
1995
). Furthermore, Brisken et al. have reported that Wnt4 acts
downstream of the progesterone receptor to induce ductal side branching during
pregnancy (Brisken et al.,
2000
).
Expression of Wnt1 and Wnt10b in luminal epithelial cells, driven by the
mouse mammary tumor virus (MMTV)-promoter, affects mammary development, and
induces hyperplasia and mammary tumors
(Lane and Leder, 1997;
Tsukamoto et al., 1988
).
Similarly, activation of ß-catenin signaling by stabilization of
ß-catenin in luminal epithelial cells results in the development of
mammary tumors, including adenocarcinoma and squamous metaplasia
(Imbert et al., 2001
;
Michaelson and Leder, 2001
;
Miyoshi et al., 2002a
;
Miyoshi et al., 2002b
;
Rowlands et al., 2003
).
Conversely, overexpression of axin, which favors ß-catenin degradation in
luminal cells, impairs lobuloalveolar development
(Hsu et al., 2001
). The
expression of a dominant-negative ß-catenin/engrailed chimera in luminal
cells induces apoptosis, showing that the transactivation capacity of
ß-catenin is essential for mammary epithelial cell survival
(Tepera et al., 2003
).
We used the K5 promoter to target the expression of stabilized N-terminally truncated ß-catenin to the basal epithelial cell layer of the mammary gland. The resulting activation of ß-catenin signaling was accompanied by precocious lobuloalveolar development in pregnancy, persistent luminal cell proliferation in lactation and accelerated involution, and led to basal-type mammary hyperplasia and invasive carcinomas.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Whole-mount mammary gland staining, histology, BrdU-incorporation and TUNEL assays
In all cases, only mice with litters of seven or eight pups were used for
analysis. For whole-mount staining, the fourth mammary glands were spread on
microscope slides, fixed overnight in methacarn (60% methanol, 30% chloroform,
10% acetic acid), rehydrated and stained with carmine alum overnight. After
dehydration, samples were cleared in xylene and digital images were acquired
with a JVC KYF50 color video camera in a Leica MZ8 binocular using the Scion
Image software.
Prior to embedding in paraffin, mammary gland specimens were fixed in methacarn or 4% paraformaldehyde in PBS and dehydrated. For histological analysis, 6 µm sections were cut and stained with Hematoxylin and Eosin.
To assess cell proliferation, mice were injected intraperitoneally with 0.25 mg 5-Bromo-2'-deoxyUridine (BrdU)/g body weight 2 hours prior to sacrifice. BrdU incorporation was detected on sections by immunohistochemistry. After light counterstaining with Hematoxylin, 1000-1500 nuclei per sample were counted.
To detect apoptotic nuclei, paraformaldehyde-fixed paraffin sections were analyzed by TdT digoxygenin nick-end labeling with Apoptag kit (Serologicals) following manufacturer's instructions.
Digital images were acquired with a JVC KYF50 color video camera in a Nikon Optiphot-2 microscope using the Scion Image software.
Immunohistochemistry
For immunohistochemistry, the sections were incubated in 1%
H2O2 to block endogenous peroxidase activity. To
retrieve nuclear antigens on paraffin-embedded skin sections, slides with
sections were incubated for 20 minutes in 10 mM sodium citrate buffer, pH 6.0
at 90°C. Furthermore, the sections were incubated for 60 minutes in 5%
FCS, overnight with primary antibodies and for 2 hours at room temperature
with appropriate secondary antibodies. Nuclei were stained with 1 µg/ml
DAPI (Sigma) for immunofluorescence studies, or counterstained with
Hematoxylin for immunohistochemistry. An epifluorescence Leica DMRBE
microscope and a CCD Hamamatsu C5985 camera were used for image
acquisition.
Antibodies
The following primary antibodies were used for immunostaining: rabbit
polyclonal anti-ß-catenin, anti-laminin (Sigma-Aldrich, 1/100),
anti-keratin 5 (Covance, 1/2000) and anti ß-casein (a gift from Dr D.
Medina); rat monoclonal anti-HA tag (Roche, 1/100); and mouse monoclonal
anti-ß-catenin (Transduction Laboratories, 1/100), anti-BrdU (Pharmingen,
1/100), anti keratin 8 (Progen, 1/100), anti--SM-actin (Sigma-Aldrich,
1/100), anti-cyclin D1, anti-p63 (Pharmingen, 1/100), anti-keratin 10 (Dako,
1/100) and anti-hair keratin AE13 (a gift from Dr T. T. Sun, New York, USA;
1/20).
Alexafluor-conjugated secondary antibodies (1/1000, Molecular Probes) were used for immunofluorescence labeling. The Dako Envision + System HRP kit (Dako) was used for immunohistochemistry.
RT-PCR
Total RNA was isolated from fourth (abdominal) and third (thoracic) frozen
mammary glands with RNA-plus reagent (Bioprobe Systems, Montreuil-Sous-Bois,
France). Two µg of total RNA was treated with RNase-free DNase (10 U;
Roche) for 10 minutes at 37°C to remove any contaminating DNA, and reverse
transcribed with Mo-MuLV reverse transcriptase (200 U, Promega) primed with
random hexamers (1 µg, Roche). Control reactions in which reverse
transcriptase was omitted were included for each sample.
PCRs included 0.5 µl of cDNA from the reverse transcriptase reaction, 5 U Hotgoldstar (Eurogentec), 1.5 mM MgCl2, 0.4 µM primers, 200 µM deoxy-NTP, in a total volume of 25 µl. PCR conditions were as follows: 95°C for 10 minutes (one cycle); 94°C for 30 seconds; Tm, 30 seconds; 72°C for 1 minute. PCR products were detected by electrophoresis in agarose gels and photographed using a Vilber Lourmat TFX20 system.
Quantitative PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). The thermal cycling conditions included an initial denaturation step at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds followed by 1 minute at either 65°C for cyclin D1, or 60°C for the rest of the transcripts. The target gene transcript levels relative to the glyceraldehyde phosphate dehydrogenase (Gapd) transcript levels were calculated as 2(Ct Gapd-Ct target gene). Two independent experiments were performed in duplicate in each case.
Gene-specific primers (Eurogentec) were designed using the Oligo 4.O software and are listed in Table 1.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
K5-N57-ßcat mice were viable and fertile. As expected, they
presented skin lesions similar to those observed previously in mice expressing
N-terminally truncated ß-catenin in basal epithelial cells
(Celso et al., 2004
;
Gat et al., 1998
;
Van Mater et al., 2003
).
Homozygous and heterozygous transgenic animals were used for further
analysis.
Expression of N57-ß-catenin in basal mammary cells induces precocious side branching and secretory cell differentiation in pregnancy
K5-N57-ßcat females were able to feed normal-sized litters,
suggesting that, overall, mammary differentiation was complete. However,
K5-
N57-ßcat mouse mammary glands presented several developmental
abnormalities. In particular, at 7.5, 10.5 and 13.5 days of pregnancy,
transgenic mouse glands contained significantly more lateral buds and short
side branches than did those from wild-type animals
(Fig. 2A). A BrdU incorporation
assay revealed considerably more proliferating luminal epithelial cells in
K5-
N57-ßcat glands than in wild-type mammary glands on day 7.5 of
pregnancy (20.5±0.2% versus 11.4±1.9%, respectively). An
anti-ß-casein antibody stained wild-type glands only weakly at 7.5 days
of pregnancy, whereas numerous alveoli from K5-
N57-ßcat mice were
positive for ß-casein at this developmental stage
(Fig. 2B). Semi-quantitative
analysis of milk gene expression by RT-PCR confirmed that at 7.5 days of
pregnancy transgenic glands contained higher levels of ß-casein
transcripts than their wild-type littermates
(Fig. 3). Furthermore, at 13.5
days of pregnancy, WAP and
-lactalbumin mRNA levels were higher in
K5-
N57-ßcat glands than in those from the wild-type mice
(Fig. 3).
|
|
Expression of N57-ß-catenin in basal mammary cells leads to persistent proliferation in lactation and premature mammary involution
Whole-mount staining did not reveal any significant difference between
lactating wild-type and transgenic glands. However, at peak lactation (six
days postpartum), when cell division had stopped in wild-type glands,
BrdU-incorporating luminal epithelial cells were still present in
K5-N57-ßcat glands, revealing that cells were still proliferating
(Fig. 2C, upper panel). Similar
to wild-type animals, apoptotic nuclei were extremely rare in mammary glands
from 6-day-lactating K5-
N57-ßcat mice
(Fig. 2C, lower panel).
Semi-quantitative RT-PCR showed that levels of ß-casein, WAP and
-lactalbumin mRNA were not altered in lactating transgenic mouse glands
(Fig. 3).
To induce involution, pups were removed from their mothers after 6 days of lactation. Three days later, alveoli from wild-type mice were still full of residual milk. Transgenic mouse glands regressed faster, and in 3-day-old involuting glands, most alveoli were collapsed and contained multiple apoptotic cells, as revealed by TUNEL assay (Fig. 2D). Accordingly, at post-weaning day 3, the amount of milk gene transcripts was significantly lower in transgenic glands than in wild-type glands (Fig. 3).
Altered expression of Mmp and Timp in developing K5-N57-ßcat mouse mammary glands
ECM-degrading metalloproteinases (Mmp) and their inhibitors are essential
for the control of mammary development
(Wiseman and Werb, 2002). We
therefore compared Mmp and Timp expression in mammary glands from wild-type
and transgenic mouse by semi-quantitative RT-PCR. On day 7.5 of pregnancy,
MT1-Mmp transcript levels were higher in transgenic glands than in wild-type
glands (Fig. 4). Although Timp1
transcript levels were also elevated in transgenic mouse glands at 7.5 days of
pregnancy, they were significantly lower than in wild-type animals at later
time points (10.5 and 13.5 days) (Fig.
4). Mmp2, Mmp3, Timp2 and Timp3 transcript levels were similar in
pregnant transgenic and wild-type mouse glands (not shown). No changes in
Mmp/Timp expression were detected in lactating transgenic mouse glands
(Fig. 4; data not shown).
MT1-Mmp levels were slightly higher in 3-day-old involuting
K5-
N57-ßcat glands than in their wild-type counterparts, whereas
Timp1 levels were lower, and Mmp2, Mmp3 and Timp3 transcript levels were
considerably higher (Fig. 4).
These data show that activation of ß-catenin signaling in basal mammary
epithelial cells affects the expression of Mmps and their inhibitors during
pregnancy, lactation and involution.
|
|
Thus, in pregnant mice, activation of ß-catenin signaling in basal
mammary epithelial cells leads to the upregulation of Myc and cyclin D1 gene
transcription, and a shift in p63 variant expression towards the Np63
form.
N57-ß-catenin expression in basal mammary epithelial cells induces basal-type hyperplasia in nulliparous females and invasive carcinomas in multiparous mice
Targeted activation of ß-catenin signaling in luminal epithelial cells
using the MMTV-promoter has been reported to induce mammary tumors
(Imbert et al., 2001;
Michaelson and Leder, 2001
;
Miyoshi et al., 2002a
;
Miyoshi et al., 2002b
). To
determine whether expression of
N57-ß-catenin in basal mammary
cells could lead to tumor formation, we analyzed mammary glands from
nulliparous and multiparous K5-
N57-ßcat mice aged between 12 and
18 months. In 10 out of 16 transgenic virgin females, mammary gland wholemount
staining and histological analysis revealed large hyperplastic areas
comprising polyp-like structures and multiple, focal and diffuse thickenings
of the ductal epithelium (Fig.
6A,B). Cells in the hyperplastic areas presented a homogeneous
phenotype; they expressed basal cell-type keratins K5 and K14, and were
negative for K8, a luminal cell marker, and for
-SM-actin, a
myoepithelial cell marker (Fig.
6D; data not shown). The anti-HA antibody detected the transgene
product,
N57-ß-catenin, in the nuclei of the basal-type
(K5/K14-positive) cells in the hyperplastic ducts
(Fig. 6C). The
BrdU-incorporation assay detected numerous proliferating cells in the basal
layer of the observed multilayered structures (not shown). Although an
anti-laminin antibody detected the basement membrane around all these
hyperplastic structures, the staining was irregular and discontinuous,
suggesting degradation or impaired deposition of the ECM
(Fig. 6F). In accordance with
the basal (K5/K14) keratin expression, most of the cells found in the
hyperplastic structures stained positive with an anti-p63-antibody recognizing
all variants of the protein.
|
|
|
Semi-quantitative RT-PCR revealed no significant differences in the MT1-Mmp and Timp1 transcripts levels between wild-type and transgenic glands or between hyperplastic and apparently normal tissue from the transgenic glands (not shown).
Thus, mammary hyperplasia developing in K5-N57-ßcat mice was
characterized by elevated Myc transcript levels, decreased Timp3 levels and a
shift in p63 variant expression towards the
N-form.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transgenic mice were able to feed normal-sized litters, proving that milk
production and expulsion were not impaired. Thus, constitutive activation of
ß-catenin signaling did not perturb the contractile activity of the
myoepithelium, and the myoepithelial cells in K5-N57-ßcat mice
underwent complete functional differentiation. Mammary hyperplasia comprising
undifferentiated basal cells developed focally and rather late in life.
Activation of ß-catenin signaling in basal cells affected the proliferation, survival and differentiation of luminal cells, suggesting crosstalk between the two mammary epithelium cell layers. Desmosomes, adherens and gap junctions may provide a means of communication between the myoepithelial and luminal compartments. However, the mechanisms underlying signal transduction from basal to luminal cells remain to be elucidated. The results of this study suggest that myoepithelial cells may influence luminal cell growth and differentiation status by modifying the composition and organization of the ECM and by affecting epithelial-stromal relationships.
Activation of ß-catenin signaling in myoepithelial cells affects Mmp and Timp expression
Balanced expression of Mmp and Timp proteins is an important factor in the
control of mammary development and tumorigenesis
(Wiseman and Werb, 2002).
Ectopic expression of Mmp3 in luminal epithelial cells results in precocious
lobuloalveolar differentiation, accelerated involution and development of
mammary tumors, a phenotype reminiscent of that described in this study
(Sternlicht et al., 1999
;
Sympson et al., 1994
;
Witty et al., 1995
).
Similarly, over-expression of MT1-Mmp in luminal cells driven by the
MMTV-promoter leads to hyperplasia in virgin mice and adenocarcinoma in
multiparous animals (Ha et al.,
2001
). The expression of Timp1 in the transgenic mouse mammary
glands overexpressing Mmp3 prevents excessive ECM degradation and rescues
premature apoptosis in involuting glands
(Alexander et al., 1996
).
We found that the expression of Mmps and their inhibitors was altered in
K5-N57-ßcat mice. An increase in MT1-Mmp expression early in
pregnancy was followed by a decrease in Timp1 transcript levels in transgenic
mouse glands. MT1-Mmp, Mmp2 and Mmp3 were expressed at higher levels in
3-day-old involuting K5-
N57-ßcat glands than in their wild-type
littermates, whereas Timp1 and Timp3 were expressed at lower levels. These
perturbations in the Mmp/Timp expression balance suggest changes in ECM
turnover. Resulting alterations in ECM composition and organization can affect
morphogenesis, proliferation and survival of mammary epithelial cells,
contributing to the abnormal mammary phenotype observed in
K5-
N57-ßcat mouse glands. Thus, myoepithelial cells playing an
important role in the control of ECM turnover, and cell-ECM interactions may
actively participate in morphogenesis and tissue remodeling during mammary
development.
Role of myoepithelial cells in the crosstalk between the mammary epithelium and the stroma in normal glands and during tumorigenesis
Differentiated myoepithelial cells from normal breast or benign
myoepithelial lesions were suggested to be `natural tumor suppressors'
(Deugnier et al., 2002b;
Lakhani and O'Hare, 2001
;
Sternlicht et al., 1997
). They
can induce growth arrest and apoptosis of tumor cells, and inhibit
angiogenesis and tumor cell invasion into the stroma. However, the changes in
gene expression that occur in the myoepithelial cells in malignant breast
tumors can significantly modify their properties so that they enhance the
proliferation, migration and invasion of the tumor
(Allinen et al., 2004
).
Significant changes in the p63 gene expression were observed in
K5-N57-ßcat mouse glands. Upregulation of
Np63 expression
was accompanied by a decrease in TAp63 transcript levels in pregnancy and,
particularly, in the basal-type mammary hyperplasia observed in the 12- to
18-month-old mice. Ablation of the p63 gene in mice results in an absence of
mammary glands and other epidermal appendages
(Mills et al., 1999
;
Yang et al., 1999
). However,
the functions of the p63 variants,
N and TAp63, in mammary epithelium
are not known.
Np63 was suggested to be associated with proliferation
and appears to accumulate in some tumors
(Michael and Oren, 2002
;
Westfall and Pietenpol,
2004
).
The shift towards Np63 variant expression observed in
K5-
N57-ßcat mouse glands may have important consequences on
crosstalk between the epithelial and the stromal mammary gland components.
Whereas TAp63 is inhibitory,
Np63 stimulates the expression of factors
essential for angiogenesis (Senoo et al.,
2002
; Wu et al.,
2003
). Therefore, activation of ß-catenin signaling in
myoepithelial cells, either in response to physiological stimuli during normal
development, or in cancer, may lead to a shift towards
Np63 expression
and the stimulation of angiogenesis in the adjacent stroma. The decrease in
Timp3 expression observed in basal-type mammary hyperplasia might also favor
angiogenesis, as Timp3 is an angiogenesis inhibitor
(Qi et al., 2003
). Therefore,
the role of myoepithelial cells in the control of angiogenesis during normal
mammary development, as well as in mammary tumors, deserves to be investigated
further.
Undifferentiated progenitors with basal characteristics: possible origin of basal-type mammary carcinoma
Transgenic mice expressing Wnt1 or N-terminally truncated ß-catenin
under the control of the MMTV promoter, in luminal epithelial cells, develop
adenocarcinoma containing both luminal and myoepithelial cells, as estimated
by cell-type-specific keratin and -SM-actin expression
(Li et al., 2003
;
Liu et al., 2004
). These
tumors contain numerous K6-positive cells, and are characterized by elevated
expression of Tcf/ß-catenin target genes, cyclin D1 and Myc
(Imbert et al., 2001
). By
contrast, mammary hyperplastic lesions observed in nulliparous
K5-
N57-ßcat mice are composed of K5/K14-positive cells lacking
luminal and myoepithelial lineage markers and negative for K6. In addition,
these cells express p63, a known marker of the basal cell layer in the
stratified epithelia (Yang et al.,
1998
). Although Myc expression is elevated in these hyperplastic
areas, expression of another Tcf/ß-catenin target gene, cyclin D1, was
found to be upregulated in only one out of four tumors. It has been suggested
that the mammary tumors found in MMTV-Wnt1 and MMTV-
N-ß-catenin
mice originate from bipotent mammary progenitor cells in the luminal
compartment (Li et al., 2003
;
Liu et al., 2004
).
Hyperplastic lesions observed in nulliparous K5-
N57-ßcat mice
contained only one cell type - basal, undifferentiated - suggesting that they
originate from a different population of mammary progenitors, those possessing
basal characteristics. Presumably, these progenitors are able to amplify and
remain undifferentiated as long as the K5-promoter is active and
N-ß-catenin is expressed. The Wnt/ß-catenin signaling pathway
has been shown to play a central role in the control of the
proliferation/differentiation switch and in tumorigenesis in humans as well as
in mouse models. The amplification of undifferentiated progenitors has been
reported to occur upon stimulation of this pathway in other tissues
(Giles et al., 2003
).
Diverse, often invasive, lesions were observed in multiparous transgenic
mice. They included squamous metaplasia presenting cutaneous epithelial
lineage markers, as described by others
(Miyoshi et al., 2002a;
Miyoshi et al., 2002b
) and
invasive carcinoma composed essentially of basal (K5/K14-positive) cells.
Although most human breast carcinomas express phenotypic markers suggestive of
a luminal origin, cDNA microarray analysis has revealed a distinct subclass of
tumors that strongly express the genes encoding basal keratins, ECM proteins,
integrins and other markers of basal mammary cells
(Perou et al., 2000
;
Sorlie et al., 2003
). These
tumors are associated with a poor clinical outcome. Expression of the entire
set of basal cell markers by this subclass of breast tumors suggests that they
may originate from mammary cell progenitors with the molecular characteristics
of basal cells. The K5-
N57-ßcat transgenic mouse line provides a
model with which to study the induction and progression of basal mammary
carcinomas.
In conclusion, our study demonstrates that activation of ß-catenin signaling in basal mammary epithelial cells (1) affects the growth, survival and differentiation of luminal cells, altering the entire process of the postnatal mammary gland development; and (2) leads to amplification of undifferentiated basal cells, resulting in the development of basal-type non-invasive and invasive mammary tumors. These data suggest that myoepithelial cells play an important role in the control of cell-ECM interactions and in crosstalk between the mammary epithelium and stroma, and indicate that the basal cell layer may harbor undifferentiated progenitors able to give rise to mammary tumors.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, C. M., Howard, E. W., Bissell, M. J. and Werb, Z. (1996). Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J. Cell Biol. 135,1669 -1677.[Abstract]
Allinen, M., Beroukhim, R., Cai, L., Brennan, C., Lahti-Domenici, J., Huang, H., Porter, D., Hu, M., Chin, L., Richardson, A. et al. (2004). Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 6,17 -32.[CrossRef][Medline]
Alonso, L. and Fuchs, E. (2003). Stem cells in
the skin: waste not, Wnt not. Genes Dev.
17,1189
-1200.
Bocker, W., Moll, R., Poremba, C., Holland, R., van Diest, P. J., Dervan, P., Burger, H., Wai, D., Ina Diallo, R., Brandt, B. et al. (2002). Common adult stem cells in the human breast give rise to glandular and myoepithelial cell lineages: a new cell biological concept. Lab. Invest. 82,737 -746.[Medline]
Bradbury, J. M., Edwards, P. A., Niemeyer, C. C. and Dale, T. C. (1995). Wnt-4 expression induces a pregnancy-like growth pattern in reconstituted mammary glands in virgin mice. Dev. Biol. 170,553 -563.[CrossRef][Medline]
Brisken, C., Heineman, A., Chavarria, T., Elenbaas, B., Tan, J.,
Dey, S. K., McMahon, J. A., McMahon, A. P. and Weinberg, R. A.
(2000). Essential function of Wnt-4 in mammary gland development
downstream of progesterone signaling. Genes Dev.
14,650
-654.
Buhler, T. A., Dale, T. C., Kieback, C., Humphreys, R. C. and Rosen, J. M. (1993). Localization and quantification of Wnt-2 gene expression in mouse mammary development. Dev. Biol. 155,87 -96.[CrossRef][Medline]
Celso, C. L., Prowse, D. M. and Watt, F. M.
(2004). Transient activation of beta-catenin signalling in adult
mouse epidermis is sufficient to induce new hair follicles but continuous
activation is required to maintain hair follicle tumours.
Development 131,1787
-1799.
Courtois, S., de Fromentel, C. C. and Hainaut, P. (2004). p53 protein variants: structural and functional similarities with p63 and p73 isoforms. Oncogene 23,631 -638.[CrossRef][Medline]
Deugnier, M. A., Faraldo, M. M., Janji, B., Rousselle, P.,
Thiery, J. P. and Glukhova, M. A. (2002a). EGF
controls the in vivo developmental potential of a mammary epithelial cell line
possessing progenitor properties. J. Cell Biol.
159,453
-463.
Deugnier, M. A., Teuliere, J., Faraldo, M. M., Thiery, J. P. and Glukhova, M. A. (2002b). The importance of being a myoepithelial cell. Breast Cancer Res. 4, 224-230.[CrossRef][Medline]
Dontu, G., Abdallah, W. M., Foley, J. M., Jackson, K. W.,
Clarke, M. F., Kawamura, M. J. and Wicha, M. S.
(2003). In vitro propagation and transcriptional profiling of
human mammary stem/progenitor cells. Genes Dev.
17,1253
-1270.
Gat, U., DasGupta, R., Degenstein, L. and Fuchs, E. (1998). De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95,605 -614.[Medline]
Gavin, B. J. and McMahon, A. P. (1992). Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. Mol. Cell. Biol. 12,2418 -2423.[Abstract]
Giles, R. H., van Es, J. H. and Clevers, H. (2003). Caught up in a Wnt storm: Wnt signaling in cancer. Biochim. Biophys. Acta 1653, 1-24.[Medline]
Gudjonsson, T., Villadsen, R., Nielsen, H. L., Ronnov-Jessen,
L., Bissell, M. J. and Petersen, O. W. (2002).
Isolation, immortalization, and characterization of a human breast epithelial
cell line with stem cell properties. Genes Dev.
16,693
-706.
Ha, H. Y., Moon, H. B., Nam, M. S., Lee, J. W., Ryoo, Z. Y.,
Lee, T. H., Lee, K. K., So, B. J., Sato, H., Seiki, M. et al.
(2001). Overexpression of membrane-type matrix
metalloproteinase-1 gene induces mammary gland abnormalities and
adenocarcinoma in transgenic mice. Cancer Res.
61,984
-990.
He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da
Costa, L. T., Morin, P. J., Vogelstein, B. and Kinzler, K. W.
(1998). Identification of c-MYC as a target of the APC pathway.
Science 281,1509
-1512.
Hsu, W., Shakya, R. and Costantini, F. (2001).
Impaired mammary gland and lymphoid development caused by inducible expression
of Axin in transgenic mice. J. Cell Biol.
155,1055
-1064.
Imbert, A., Eelkema, R., Jordan, S., Feiner, H. and Cowin,
P. (2001). Delta N89 beta-catenin induces precocious
development, differentiation, and neoplasia in mammary gland. J.
Cell Biol. 153,555
-568.
Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van der Valk, M. and Berns, A. (2001). Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29,418 -425.[CrossRef][Medline]
Koster, M. I., Kim, S., Mills, A. A., DeMayo, F. J. and Roop, D.
R. (2004). p63 is the molecular switch for initiation of an
epithelial stratification program. Genes Dev
18,126
-131.
Lakhani, S. R. and O'Hare, M. J. (2001). The mammary myoepithelial cell-Cinderella or ugly sister? Breast Cancer Res. 3,1 -4.[CrossRef][Medline]
Lane, T. F. and Leder, P. (1997). Wnt-10b directs hypermorphic development and transformation in mammary glands of male and female mice. Oncogene 15,2133 -2144.[CrossRef][Medline]
Li, Y., Welm, B., Podsypanina, K., Huang, S., Chamorro, M.,
Zhang, X., Rowlands, T., Egeblad, M., Cowin, P., Werb, Z. et al.
(2003). Evidence that transgenes encoding components of the Wnt
signaling pathway preferentially induce mammary cancers from progenitor cells.
Proc. Natl. Acad. Sci. USA
100,15853
-15858.
Liu, B. Y., McDermott, S. P., Khwaja, S. S. and Alexander, C.
M. (2004). The transforming activity of Wnt effectors
correlates with their ability to induce the accumulation of mammary progenitor
cells. Proc. Natl. Acad. Sci. USA
101,4158
-4163.
Michael, D. and Oren, M. (2002). The p53 and Mdm2 families in cancer. Curr. Opin. Genet. Dev. 12, 53-59.[CrossRef][Medline]
Michaelson, J. S. and Leder, P. (2001). beta-catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland. Oncogene 20,5093 -5099.[CrossRef][Medline]
Mills, A. A., Zheng, B., Wang, X. J., Vogel, H., Roop, D. R. and Bradley, A. (1999). p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398,708 -713.[CrossRef][Medline]
Miyoshi, K., Rosner, A., Nozawa, M., Byrd, C., Morgan, F., Landesman-Bollag, E., Xu, X., Seldin, D. C., Schmidt, E. V., Taketo, M. M. et al. (2002a). Activation of different Wnt/beta-catenin signaling components in mammary epithelium induces transdifferentiation and the formation of pilar tumors. Oncogene 21,5548 -5556.[CrossRef][Medline]
Miyoshi, K., Shillingford, J. M., le Provost, F., Gounari, F.,
Bronson, R., von Boehmer, H., Taketo, M. M., Cardiff, R. D., Hennighausen, L.
and Khazaie, K. (2002b). Activation of beta-catenin signaling
in differentiated mammary secretory cells induces transdifferentiation into
epidermis and squamous metaplasias. Proc. Natl. Acad. Sci.
USA 99,219
-224.
Nelson, W. J. and Nusse, R. (2004). Convergence
of Wnt, beta-catenin, and cadherin pathways. Science
303,1483
-1487.
Pechoux, C., Gudjonsson, T., Ronnov-Jessen, L., Bissell, M. J. and Petersen, O. W. (1999). Human mammary luminal epithelial cells contain progenitors to myoepithelial cells. Dev. Biol. 206,88 -99.[CrossRef][Medline]
Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., Pollack, J. R., Ross, D. T., Johnsen, H., Akslen, L. A. et al. (2000). Molecular portraits of human breast tumours. Nature 406,747 -752.[CrossRef][Medline]
Polakis, P. (2000). Wnt signaling and cancer.
Genes Dev. 14,1837
-1851.
Qi, J. H., Ebrahem, Q., Moore, N., Murphy, G., Claesson-Welsh, L., Bond, M., Baker, A. and Anand-Apte, B. (2003). A novel function for tissue inhibitor of metalloproteinases-3 (Timp3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat. Med. 9,407 -415.[CrossRef][Medline]
Ramirez, A., Bravo, A., Jorcano, J. L. and Vidal, M. (1994). Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development. Differentiation 58, 53-64.[CrossRef][Medline]
Rowlands, T. M., Pechenkina, I. V., Hatsell, S. J., Pestell, R.
G. and Cowin, P. (2003). Dissecting the roles of
beta-catenin and cyclin D1 during mammary development and neoplasia.
Proc. Natl. Acad. Sci. USA
100,11400
-11405.
Sancho, E., Batlle, E. and Clevers, H. (2003). Live and let die in the intestinal epithelium. Curr. Opin. Cell Biol. 15,763 -770.[CrossRef][Medline]
Senoo, M., Matsumura, Y. and Habu, S. (2002). TAp63gamma (p51A) and dNp63alpha (p73L), two major isoforms of the p63 gene, exert opposite effects on the vascular endothelial growth factor (VEGF) gene expression. Oncogene 21,2455 -2465.[CrossRef][Medline]
Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D'Amico,
M., Pestell, R. and Ben-Ze'ev, A. (1999). The cyclin
D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl.
Acad. Sci. USA 96,5522
-5527.
Smalley, M. and Ashworth, A. (2003). Stem cells and breast cancer: a field in transit. Nat. Rev. Cancer 3,832 -844.[CrossRef][Medline]
Smalley, M. J., Titley, J., Paterson, H., Perusinghe, N.,
Clarke, C. and O'Hare, M. J. (1999). Differentiation of
separated mouse mammary luminal epithelial and myoepithelial cells cultured on
EHS matrix analyzed by indirect immunofluorescence of cytoskeletal antigens.
J. Histochem. Cytochem.
47,1513
-1524.
Smith, G. H. and Boulanger, C. A. (2003). Mammary epithelial stem cells: transplantation and self-renewal analysis. Cell Prolif. 36,3 -15.[CrossRef][Medline]
Smith, G. H. and Medina, D. (1988). A morphologically distinct candidate for an epithelial stem cell in mouse mammary gland. J. Cell Sci. 90,173 -183.[Abstract]
Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J.
S., Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S. et
al. (2003). Repeated observation of breast tumor subtypes in
independent gene expression data sets. Proc. Natl. Acad. Sci.
USA 100,8418
-8423.
Sternlicht, M. D., Kedeshian, P., Shao, Z. M., Safarians, S. and Barsky, S. H. (1997). The human myoepithelial cell is a natural tumor suppressor. Clin. Cancer Res. 3,1949 -1958.[Abstract]
Sternlicht, M. D., Lochter, A., Sympson, C. J., Huey, B., Rougier, J. P., Gray, J. W., Pinkel, D., Bissell, M. J. and Werb, Z. (1999). The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98,137 -146.[Medline]
Sympson, C. J., Talhouk, R. S., Alexander, C. M., Chin, J. R., Clift, S. M., Bissell, M. J. and Werb, Z. (1994). Targeted expression of stromelysin-1 in mammary gland provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression. J. Cell Biol. 125,681 -693.[Abstract]
Tepera, S. B., McCrea, P. D. and Rosen, J. M.
(2003). A beta-catenin survival signal is required for normal
lobular development in the mammary gland. J. Cell Sci.
116,1137
-1149.
Tetsu, O. and McCormick, F. (1999). Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398,422 -426.[CrossRef][Medline]
Tsukamoto, A. S., Grosschedl, R., Guzman, R. C., Parslow, T. and Varmus, H. E. (1988). Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell 55,619 -625.[Medline]
Van Mater, D., Kolligs, F. T., Dlugosz, A. A. and Fearon, E.
R. (2003). Transient activation of beta-catenin signaling in
cutaneous keratinocytes is sufficient to trigger the active growth phase of
the hair cycle in mice. Genes Dev.
17,1219
-1224.
Weber-Hall, S. J., Phippard, D. J., Niemeyer, C. C. and Dale, T. C. (1994). Developmental and hormonal regulation of Wnt gene expression in the mouse mammary gland. Differentiation 57,205 -214.[CrossRef][Medline]
Welm, B. E., Tepera, S. B., Venezia, T., Graubert, T. A., Rosen, J. M. and Goodell, M. A. (2002). Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev. Biol. 245,42 -56.[CrossRef][Medline]
Westfall, M. D. and Pietenpol, J. A. (2004).
p63: molecular complexity in development and cancer.
Carcinogenesis 25,857
-864.
Wiseman, B. S. and Werb, Z. (2002). Stromal
effects on mammary gland development and breast cancer.
Science 296,1046
-1049.
Witty, J. P., Wright, J. H. and Matrisian, L. M. (1995). Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development. Mol. Biol. Cell 6,1287 -1303.[Abstract]
Wu, G., Nomoto, S., Hoque, M. O., Dracheva, T., Osada, M., Lee,
C. C., Dong, S. M., Guo, Z., Benoit, N., Cohen, Y. et al.
(2003). DeltaNp63alpha and TAp63alpha regulate transcription of
genes with distinct biological functions in cancer and development.
Cancer Res. 63,2351
-2357.
Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M. D., Dotsch, V., Andrews, N. C., Caput, D. and McKeon, F. (1998). p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2,305 -316.[Medline]
Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin, C., Sharpe, A., Caput, D., Crum, C. et al. (1999). p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398,714 -718.[CrossRef][Medline]