1 Genetics of Development and Disease Branch, NIDDK, NIH, 10/9N105, 10 Center
Drive, Bethesda, MD 20892, USA
3 Laboratory of Genetics and Physiology, NIDDK, NIH, 10/9N105, 10 Center Drive,
Bethesda, MD 20892, USA
2 Department of Surgery, Neuroscience Program, Upstate Medical University,
Syracuse, NY 13210, USA
Author for correspondence (e-mail:
chuxiad{at}bdg10.niddk.nih.gov)
Accepted 27 August 2003
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SUMMARY |
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Key words: Smad4/Dpc4, TGFß, Transdifferentiation, Keratinocytes, Neoplasia
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Introduction |
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Mammalian TGFßs constitute a superfamily of over 40 secreted signaling
molecules, which function in diverse developmental processes by regulating
proliferation, differentiation and apoptosis (reviewed by
Derynck et al., 2001;
Wakefield et al., 2001
;
Wakefield and Roberts, 2002
).
Members of a TGFß subgroup (TGFß1-TGFß3) and their receptors
are expressed in the mammary epithelium and terminal endbuds during branching
morphogenesis and have been identified as important regulators of mammary
epithelial cell proliferation, differentiation and transformation
(Buggiano et al., 2001
;
Gorska et al., 1998
;
Joseph et al., 1999
;
Nguyen and Pollard, 2000
).
TGFß signals have been implicated in breast cancer formation. Breast
cancer cell lines, primary breast cancers and invasive carcinomas show
increased expression of TGFß1
(Chakravarthy et al., 1999).
Consistently, TGFß1 was also found to induce both estrogen-dependent and
-independent tumorigenicity of human breast cancer cells in nude mice
(Arteaga et al., 1993
), whereas
inhibition of TGFß signals inhibits breast cancer cell tumorigenicity
(Muraoka et al., 2002
;
Yang et al., 2002b
). However,
ectopic expression of TGFß1 in transgenic mice represses 7,
12-dimethylbenz[a]anthracene-induced mammary tumor formation
(Pierce et al., 1995
). The
decreased incidence of mammary tumor was correlated with the inhibition of
TGFß on the proliferative activity of mammary epithelial cells and
mammary stem cells (Boulanger and Smith,
2001
). Consistently, a dominant-negative form of TGFß type II
receptor (TGFß-DNIIR), which blocks TGFß responsiveness, has been
found to cause mammary tumor formation in response to carcinogen
(Bottinger et al., 1997a
).
Therefore, it was proposed that TGFß has biphasic actions on tumors
cells, i.e. it is an important negative growth effector at an early stage, but
later enhances the malignant conversion and invasion, primarily through the
induction of epithelial-mesenchymal transformation (EMT)
(Oft et al., 2002
;
Piek et al., 1999b
). After
EMT, tumor cells lose cell-cell contact and become more invasive because of
the increased migration ability (Akhurst
and Balmain, 1999
; Cui et al.,
1996
; Ellenrieder et al.,
2001
; Portella et al.,
1998
).
TGFß signals are transduced into nuclei by intracellular mediator
SMADs. Based on their functions in the TGFß signaling pathway, SMADs are
divided into three subtypes, including receptor activated SMADs
(MADHsHuman Gene Nomenclature Database), SMAD1, SMAD2, SMAD3, SMAD5 and
SMAD8; inhibitory SMADs, SMAD6 and SMAD7; and a common SMAD, SMAD4 (reviewed
in Heldin et al., 1997;
Massague, 1998
).
SMAD4 was cloned as a tumor suppressor gene, deleted in pancreatic
cancer (DPC4) (Hahn et al.,
1996a
). Loss-of-function mutations of SMAD4 are
frequently detected in pancreatic cancer, colon cancer, and gastric polyposis
and adenocarcinomas (Friedl et al.,
1999
; Hahn et al.,
1996b
; Howe et al.,
1998
; Tamura et al.,
1996
).
Targeted disruption of Smad (MadhMouse Genome Informatics) genes in
mice has revealed multiple essential roles of these proteins in mammalian
development (reviewed by Weinstein et al.,
2000). It was shown that loss of Smad4 results in
lethality at embryonic (E) days 6-7 because of impaired extra-embryonic
membrane formation and decreased epiblast proliferation
(Sirard et al., 1998
;
Yang et al., 1998
). To
overcome the early lethality and to study functions of Smad4 during later
stages, especially during mammary gland development and neoplasia, we
performed a mammary epithelium specific knockout of Smad4 using the
Cre-loxP system. Our data showed that the disruption of Smad4 in
mammary epithelium overall does not disrupt normal development of mammary
glands. However, it results in the formation of squamous cell carcinoma and
mammary abscesses primarily caused by transdifferentiation of mammary
epithelium to squamous epithelium caused by the loss of TGFß
responsiveness. These observations uncover a role of Smad4 in cell fate
maintenance during mammary gland development and mammary cycle
progression.
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Materials and methods |
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Northern blots, TUNEL assay and whole-mount staining of mammary
glands
RNA was isolated from the mammary glands of female mice during different
developmental stages using RNA Tet-60 based on the protocol recommended by the
manufacturer (Tel-Test `B, Friendswood, TX). About 20 µg of total RNA from
each sample was loaded on 1% agarose gel and transferred to a Gene-Screen
filter after electrophoreses. TUNEL assay was carried as recommended by the
manufacturer (Intergen Company, Purchase, NY). Whole-mount staining of mammary
glands was carried out as described
(Robinson and Hennighausen,
1997).
Histology, immunohistochemical staining and western blotting
For histology, tissues were fixed in 10% formalin, blocked in paraffin wax,
sectioned, stained with Hematoxylin and Eosin, and examined by light
microscopy. Detection of primary antibodies was performed using the ZYMED
HistomouseTM SP Kit according to the manufacturer's instructions. Western
analysis was performed using standard procedures. Cyclin D1, ErbB2 and Smad4
antibodies were purchased from Santa Cruz Biotechnology. Antibodies for K14,
K10 and BrdU were purchased from Covance; and ß-catenin and E-cadherin
were from BD Transduction Laboratories.
Generation of cell lines and TGFß treatment
Generation and maintaining of cell lines from mammary tissues, tumors and
abscesses were as described (Brodie et al.,
2001). Cells (2x106) were plated into 10 cm plate
for morphogenic transformation analysis and 1x105 cells were
seeded on 22x22 mm glass slides for immunofluorescence analysis. The
next day, cells were treated with 2 ng/ml of TGFß1 for various time
points as indicated in Fig. 7
before being harvested for analysis.
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Results |
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Cre mediated excision of exon 8 in different tissues isolated from
Smad4Co/CoWAP-Cre mouse was evaluated by PCR and Southern
blot analyses. PCR analysis on skin, heart, lung, liver, thymus, ovary,
pancreas, brain, skeletal muscle, kidney, spleen and mammary gland
demonstrated that the excision occurred exclusively in mammary tissues (not
shown). Southern blot analysis of mammary tissue of
Smad4Co/CoWAP-Cre mice (n>15) at several
stages of development, including virgin, pregnancy (P) day 16.5, lactation (L)
day 2 and 10, and involution (I) day 10, revealed extensive Cre-mediated
recombination, which peaked to 60% at L10
(Fig. 1I). The presence of an
unrecombined allele reflects to a large extent the presence of mammary stroma,
which is not targeted by the WAP-Cre transgene. We detected no
recombination in virgin glands, as WAP-Cre is not expressed at this stage.
Because mammary tissue consists not only of secretory epithelial cells but
also of fat and stroma cells that do not express the WAP-Cre
transgene, the amount of epithelial cells that undergo recombination may be
higher. Indeed, northern blot analysis demonstrated that Smad4
transcripts were reduced to about 10 and 20% of original levels in mammary
tissue from P16.5 (n=5) and L10 (n=5)
Smad4Co/CoWAP-Cre mice, respectively
(Fig. 1J). Our
immunohistochemical staining using an antibody to Smad4 on mutant mammary
glands (n=5) isolated from different developmental stages confirmed
that majority of mammary epithelial cells (over 90%) were negative for the
staining (Fig. 1G and not
shown). Collectively, these observations indicate that WAP-Cre has
achieved high efficiencies in deleting Smad4 in mammary epithelium.
Absence of Smad4 does not compromise mammary gland development and
function
Mammary tissues of Smad4Co/CoWAP-Cre and
Smad4Co/CoMMTV-Cre mice isolated from different
developmental stages, including virgin, P11.5, P14.5, P16.5, L2, L10, I2 and
I10, were carefully examined using whole-mount staining and histological
sections under microscope. Normal mammary development was observed in all
mutant mice examined (n>30) during the first two to three
pregnancies and dams were able to nurse their litters (data not shown). As
Smad4 is expressed in mammary epithelium during all stages of
development and Cre-mediated recombination occurred in majority of cells, we
conclude that the absence of Smad4 does not interfere with normal
development of the mammary gland.
Mammary tumor and abscess formation in the absence of Smad4
Lack of TGFß signals results in mammary tumor formation (reviewed by
Derynck et al., 2001;
Wakefield et al., 2001
). We
therefore continuously bred Smad4Co/CoWAP-Cre dams and monitored
for the appearance of mammary tumors. Whole-mount staining revealed dense
areas in mammary glands of mutant mice after multiple pregnancies
(Fig. 2A-C). Starting at 5
months of age, some Smad4Co/CoWAP-Cre mice developed visible tumor
masses in their mammary glands (Fig.
2D,F). By 12 months, more than 60% of Smad4Co/CoWAP-Cre
mice developed tumor masses. The majority of mutant mice contained multiple
tumor masses of varying sizes per gland
(Fig. 2C,D). By 16 months of
age, all Smad4Co/CoWAP-Cre mice had developed mammary tumor masses
(Fig. 2F). Examination of the
majority of these mice at autopsy revealed no sign of tumor metastasis. Our
examination on Smad4Co/CoMMTV-Cre mice under same mating condition
obtained a very similar result (Fig.
2F). Because MMTV-Cre is also expressed in virgin mice and causes
gene deletion to lesser extent than it does during pregnancy (not shown), we
next studied virgin Smad4Co/CoMMTV-Cre (n=20) mice and
found that they all exhibited similar phenotypes with reduced multiplicity
compared with continuously mated group (data not shown). Considering that the
MMTV-Cre is expressed less efficiently in virgin mice, this observation
suggests that pregnancy related hormones, such as estrogen, do not have an
obvious influence on squamous metaplasia and tumorigenesis.
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TGFß has been known as a potent factor that promotes EMT of cultured
mammary epithelial cells (Piek et al.,
1999a). The existence of Smad4-null epithelial cells provides an
excellent opportunity to study the role of Smad4 in this process. Thus, we
treated the cells with TGFß1 at multiple time points, ranging from 0, 2,
4, 6, 12, 24 and 48 hours. We found that both Smad4-null cells lost the
TGFß responsiveness and showed no signs of EMT
(Fig. 7C). By contrast, EMT was
readily observed in four Smad4+/+ cell lines tested
(Fig. 7D and not shown),
indicating that Smad4 is essential for TGFß induced cell fate
transformation.
We have also followed progression of EMT and dynamic changes of ß-catenin upon TGFß treatment in wild-type cells. Our result indicated that the morphological transformation became apparent 12 hours after TGFß treatment and the EMT was more obvious at later time points (Fig. 7D). A slightly lower level of ß-catenin in the TGFß treated than untreated cells was observed at 6 hours and a further reduction of ß-catenin occurred at 12 hours and later points (Fig. 7D). Thus, the downregulation of ß-catenin correlates with the onset of cell fate transformation, suggesting that ß-catenin may play an active role in TGFß-induced EMT.
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Discussion |
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Although Smad4-null embryos die at E6.5 and display defects in cell
proliferation (Sirard et al.,
1998; Yang et al.,
1998
), the loss of Smad4 in mammary epithelium does not disrupt
mammary development during the first a few pregnancies. It is possible that
the TGFß/Smad4 signals do not contribute to the proliferation and
differentiation of the mammary epithelium, or that TGFß/Smad4 signals can
be compensated for by the presence of other factors. As Smad4 serves as a
central mediator for TGFß signals (reviewed by
Heldin et al., 1997
;
Massague, 1998
), we favor the
second possibility. Moreover, recent studies with cultured cells revealed that
TGFß signals can be relayed through a number of additional mediators,
such as MAPK, EGF and HGF (Engel et al.,
1999
; Hocevar et al.,
1999
; Kretzschmar et al.,
1999
; Mulder,
2000
). Thus, in the absence of Smad4, TGFß signals possibly
activate some of their downstream targets in Smad4-null mammary
epithelium.
The absence of Smad4 in the mammary epithelium reproducibly led to
hyperplastic foci with increased cell proliferation. This suggests that the
Smad4-mediated TGFß signaling pathways serve as a negative regulator for
mammary epithelial cell proliferation. The long latency in tumor formation may
reflect the complexity of growth regulation inherent to the mammary gland. It
is likely that TGFß/Smad4 signals interact with other growth pathways,
and that the loss of Smad4 enables the mutant cells to gradually escape growth
regulation, which subsequently results in increased proliferation. However,
the first burst of proliferation was restricted to the periphery of the
developing tumor masses, while the cells in the center underwent
transdifferentiation and formed squamous metaplasia, leading to the
establishment of squamous cell carcinomas. Notably, the expression of a
TGFß DNIIR transgene alone, which specifically blocks the
signals of the TGFß subfamily, only causes hyperplasia and squamous cell
carcinoma initiation after carcinogen treatment
(Bottinger et al., 1997b). The
phenotype observed upon deletion of the Smad4 gene in mammary epithelium was
more profound than the overexpression of the TGFß DNIIR
transgene, which is consistent with the view that Smad4 is a common mediator,
which not only mediates TGFß subfamily, but also the BMP and activin
pathways as well.
Keratin pearls and squamous cell carcinoma are also observed in a number of
transgenic mice, including Pten+/
(Stambolic et al., 2000),
Apc474/+ (Sasai et
al., 2000
) and TGFß DNIIR
(Bottinger et al., 1997a
) mice.
However, mammary abscess formation caused by continuous epidermalization is
not a feature in these transgenic mice. It is unique that Smad4-associated
neoplasia undergoes transdifferentiation to such an extent that it eventually
turns into skin-like structures in the mammary glands. Mammary epithelium and
epidermis share a common origin as they both derive from the ectoderm. They
undergo distinct developmental outcomes as the mammary bud forms ductal
branches during embryogenesis (Cardiff et
al., 2000
). The loss of Smad4 in the mammary epithelium results in
a continuous epidermalization, suggesting that TGFß/Smad4 signals may be
normally involved in a process that positively regulates either the transition
of the mammary epithelium from embryonic epidermal cells or its maintenance.
Because the loss of Smad4 reverses this process, leading to the
epidermalization of mammary epithelium, we suggest that Smad4 is required to
maintain mammary epithelium and prevent them from undergoing
transdifferentiation. Thus, our findings may suggest that the correct dose of
TGFß/Smad4 signals is essential in maintaining normal development of
mammary epithelial cells. Both the activation and the inactivation of these
signals can cause abnormalities, i.e. induce transdifferentiation of mammary
cells to opposite differentiation pathways. This may provide a molecular basis
for the long observed dual functions of TGFß signals (reviewed by
Wakefield and Roberts, 2002
).
Therefore, we propose that TGFß signals act through Smad4 to inhibit
tumor initiation through their ability to inhibit epithelial cell
proliferation. When these inhibition signals are absent due to the lack of
Smad4, mammary epithelial cells increase proliferation leading to the
hyperplasia and tumor initiation. Meanwhile, Smad4 also plays a potent role in
determining the fate of the cells. Its absence unavoidably triggers
transdifferentiation of mammary epithelial cells. As a net result of losing
these functions, Smad4-null mammary epithelial cells undergo both
tumorigenesis and continuous transdifferentiation. This results in the
conversion of Smad4-null tumor cells into highly differentiated, yet less
malignant cells, leading to the mammary abscess formation. It was proposed
that TGFß signals promote tumor metastasis at later stages through
inducing EMT (Oft et al.,
2002
; Oft et al.,
1996
; Piek et al.,
1999a
). The mutation we introduced could not directly address
this, as it produces a loss-of-function, instead of activation, of TGFß
signals. However, our observation that the absence of Smad4 blocked
TGFß-induced EMT in cultured cells suggests that Smad4 may mediate this
action. Although it remains to be confirmed by in vivo studies, the lack of
metastasis in all Smad4Co/CoWAP-Cre and
Smad4Co/CoMMTV-Cre mice examined (n>50) is
consistent with this view.
We showed that the absence of TGFß/Smad4 signals results in increased
levels of ß-catenin in vivo, while an activation of these signals leads
to a decrease of ß-catenin in vitro. We demonstrated that the increase of
ß-catenin in Smad4-null mammary epithelium occurs prior to and during the
transdifferentiation, while in the TGFß treated cell, the decreased
ß-catenin occurs at onset of the morphogenic transition. These
observations suggest that ß-catenin could serve as one of the key
molecules that mediate TGFß/Smad4 signals in determining cell outcome.
Recently, it was shown that constitutive activation of ß-catenin in
mammary epithelium through the deletion of the N-terminal including the
GSK3ß phosphorylation sites results in the transdifferentiation of
mammary alveolar epithelium into epidermal structures
(Miyoshi et al., 2002).
However, our analysis failed to detect a mutation in ß-catenin,
suggesting that a different mechanism, rather than mutations that stabilize
the protein, is involved in the increased levels of ß-catenin. Of note,
alterations of ß-catenin were also observed in many human breast cancers
without detectable mutations (Candidus et
al., 1996
; Karayiannakis et
al., 2001
). Thus, it was proposed that the regulation of
ß-catenin might occur at transcriptional, translational and/or
post-translational levels (Karayiannakis
et al., 2001
). Our observation that the treatment of TGFß1
decreased ß-catenin levels in wild-type, but not the Smad4-null,
cells, is consistent with this view. It also suggests a potential interaction
between TGFß/Smad4 and Wnt signals in regulation of ß-catenin,
although the underlying mechanism is currently unknown and needs further
investigation.
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ACKNOWLEDGMENTS |
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Footnotes |
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