Departments of Cancer Biology, Cell and Developmental Biology, Medicine, and The Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA
Author for correspondence (e-mail:
chodosh{at}mail.med.upenn.edu)
Accepted 9 December 2004
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SUMMARY |
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Key words: Myc, Mammary, Differentiation, Stat5, Tgfß3, Caveolin 1, Mouse
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Introduction |
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Similar to the ability of the developmental state of the mammary gland to
modulate its susceptibility to carcinogenesis, in vitro studies indicate that
cellular environment can modulate oncogene action. For example, the
proto-oncogene Myc has long been known to stimulate cellular proliferation.
However, when constitutively expressed in growth factor-deprived cells, Myc
triggers apoptosis rather than proliferation
(Askew et al., 1991;
Evan et al., 1992
). These and
other findings suggest that cellular context plays an important role in
determining the consequences of Myc activation in vitro and are consistent
with the hypothesis that the effects of aberrant oncogene activation depend
upon the specific developmental context in which activation occurs.
Beyond its in vitro effects, MYC is amplified in 5-15% of human
breast cancers and is associated with aggressive tumor behavior and poor
outcome (Berns et al., 1996;
Chrzan et al., 2001
;
Deming et al., 2000
;
Watson et al., 1993
).
Consistent with a role in human breast cancer, MYC results in the development
of invasive mammary adenocarcinomas when aberrantly activated in the mammary
glands of transgenic mice (Andres et al.,
1988
; D'Cruz et al.,
2001
; Schoenenberger et al.,
1988
; Stewart et al.,
1984
). In addition, constitutive expression of Myc from
the mouse mammary tumor virus long-terminal repeat (MMTV-LTR) has been shown
to induce abnormal lobuloalveolar development in virgin mice and to inhibit
postpartum lactation (Andres et al.,
1988
; Stewart et al.,
1984
). Indeed, MMTV-myc transgenic mice exhibit a defect
in lactation that results in pup death within 24 hours of parturition. Thus,
the ability of Myc to simultaneously disrupt normal mammary gland development
and promote tumorigenesis again underscores the fundamental relationship
between these two processes.
The mechanisms by which normal developmental events modulate cancer
susceptibility are unknown. Understanding these mechanisms will undoubtedly
require a more complete understanding of the interaction between development,
reproductive history and oncogenic pathways than currently exists. To test the
hypothesis that the effects of oncogene activation depend upon the
developmental state of the breast at the time of exposure, we have generated a
novel bitransgenic mouse model system that permits the rapidly inducible,
spatially homogeneous expression of oncogenes in the mammary epithelium of
bitransgenic mice treated with doxycycline
(Gunther et al., 2002). This
system allows regulatory molecules to be inducibly expressed in the mammary
epithelium for a defined period of time, at a desired level, and during any
desired developmental stage. Transgene expression is mammary specific, can be
titrated over a wide range of expression levels, and is essentially
undetectable in the uninduced state. Together, these properties make this
system ideally suited for expressing oncogenes in a spatially and temporally
restricted manner during defined stages of mammary development.
We have used this conditional transgenic model for MYC action to identify the specific developmental window of susceptibility that is responsible for the ability of MYC to inhibit postpartum lactation, as well as to determine the molecular mechanism for this phenotype. Surprisingly, we have found that MYC blocks postpartum lactation not by inhibiting differentiation, but rather by accelerating mammary epithelial differentiation and promoting precocious lactation during pregnancy. This in turn, results in milk stasis and premature involution of the gland mediated by the activation of Stat3 and Tgfß3. We further show that epithelial differentiation is only accelerated when MYC is expressed within a discrete 72-hour period during mid-pregnancy, and that this developmental stage-specific effect is tightly linked to the ability of MYC to downregulate caveolin 1 expression and activate Stat5. These findings serve as a novel example of the ability of MYC to promote rather than inhibit differentiation, constitute the first in vivo example of a developmental stage-specific effect of aberrant oncogene activation, and provide the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.
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Materials and methods |
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Morphological analysis
Mammary glands were fixed in 4% paraformaldehyde in 1 x
phosphate-buffered saline overnight and then transferred to 70% ethanol. Fixed
mammary glands were embedded in paraffin and sectioned for histological
staining as described (D'Cruz et al.,
2001). Sections (5 µm) were cut, applied to glass slides and
stained with Hematoxylin and Eosin.
Immunohistochemistry and TUNEL analysis
Mice were injected with 1 mg BrdU per 20 g body weight 2 hours prior to
tissue harvest. Mammary gland number 4 was removed and fixed overnight in 4%
paraformaldehyde, transferred to 70% ethanol and embedded in paraffin wax.
Sections (5 µm) were prepared and BrdU immunohistochemistry performed as
described (D'Cruz et al.,
2001). Terminal deoxynucleotidyltansferase-mediated dUTP nick
end-labeling (TUNEL) analysis was performed using the Apoptag Peroxidase Kit
(Intergen, Purchase, New York) according to the manufacturer's instructions as
previously described (D'Cruz et al.,
2001
). BrdU and TUNEL quantitation was performed using ImagePro
software to determine the percentage of positive nuclei within a
representative section. Six fields of view, and a minimum of 700 nuclei were
counted per section. The standard error of the mean (s.e.m.) was calculated
and a two-tailed T-test was performed to determine whether samples
were statistically different.
Northern hybridization
Total RNA isolation and northern hybridization was performed using 3 µg
of total RNA from snap-frozen number 3 and number 5 mammary gland tissues as
described (Marquis et al.,
1995). Blots were hybridized with radiolabeled cDNA probes
specific to exons 2 and 3 of human MYC, exon 1 of mouse Myc,
epsilon-casein (Csnd; nucleotides 83-637), Pip (nucleotides
34-429), cyclin A (Ccna2; nucleotides 182-739), Tgfb3
(nucleotides 2114-2568), pai-1 (Serpine1; nucleotides
1467-1980), Cdk4 (nucleotides 458-838), Fbl (nucleotides
39-540), Shmt1 (nucleotides 108-546), Cav1 (nucleotides
1414-1846), Cis1 (Cish; nucleotides 188-501) and
CK18 (nucleotides 589-1287).
Western analysis
Protein lysates were prepared from mammary glands by dounce homogenization
in EBC buffer as described (Gardner et
al., 2000). Equivalent amounts of each extract were
electrophoresed on 10% SDS-PAGE gels and transferred overnight to
nitrocellulose membranes. Following visualization by Ponceau staining to
verify equal protein loading and transfer, membranes were incubated with PBS
blocking solution consisting of 5% nonfat dry milk and 0.05% Tween-20
(PBST-MILK). Primary antibody incubation was performed at room temperature for
1 hour with the following antibodies in PBST-MILK solution:
anti-phospho-Stat5a/b (Y694/Y699) clone 8-5-2, 2.0 µg/ml (Upstate
biotechnology); anti-Stat5, 1:250 dilution (BD Biosciences);
anti-ß-tubulin, 0.1 µg/ml (InnoGenex); anti-phospho-Stat3 (Y705),
clone 9E12, 0.1 µg/ml (Upstate Biotechnology); anti-Stat3, 1:2500 dilution
(Transduction Laboratories); and anti-Caveolin-1, 1:1000 dilution, clone 2297
(BD Transduction Laboratories). For milk protein westerns, membranes were
blocked in a solution consisting of 3% BSA, 1 x TBS and 0.05% Tween-20
(TBST-BSA). Polyclonal rabbit antiserum to mouse milk-specific proteins
(Nordic Immunological Laboratories) was used at a 1:40,000 dilution in
PBST-BSA. Following primary antibody incubations, blots were washed three
times in blocking solution, and incubated with horseradish
peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies
at 1:10,000 dilutions (Jackson ImmunoResearch) in blocking solution for 30
minutes at room temperature. Following three washes in blocking solution and
two washes in 1 x PBS, blots were developed using the ECL Plus system
according to the manufacturer's instructions (Amersham Pharmacia) followed by
exposure to film (Kodak XAR-5).
Oligonucleotide microarray hybridization
Approximately 30 µg total RNA from the mammary glands of three separate
animals were used at each developmental time point. Biotinylated cRNA was
generated and hybridized to Affymetrix Mu6500 GeneChips. Raw data collection
and gene expression analysis was performed as described previously
(Master et al., 2002).
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Results |
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First, to determine whether the inducible MTB/TOM model that we have previously described phenocopies the lactation defect observed in MMTV-myc mice, we induced MYC expression in nulliparous MTB/TOM mice by administration of doxycycline beginning at 3 weeks of age. When these mice were subsequently mated at 6 weeks of age and allowed to undergo pregnancy, all pups were observed to die within 24 hours of parturition. Thus, constitutive expression of MYC in the mammary glands of MTB/TOM mice recapitulates the lactation defect observed in the constitutive MMTV-myc transgenic mouse model.
Deregulated expression of MYC has been demonstrated to be incompatible with
terminal differentiation in multiple cell types, including myoblasts,
erythroleukemia cells, adipocytes and B-lymphocytes
(Brandvold et al., 2001;
Coppola and Cole, 1986
;
Freytag, 1988
;
Heath et al., 2000
;
Miner and Wold, 1991
).
Consequently, we hypothesized that one mechanism by which deregulated MYC
expression might inhibit postpartum lactation would be by inhibiting terminal
differentiation of the mammary epithelium during this phase of development.
Surprisingly, although induction of MYC during lactation did eventually result
in pup death, the timing of pup mortality was markedly delayed compared with
litters from MMTV-myc mice or MTB/TOM mice induced throughout
pregnancy. Similarly, deregulated MYC expression in virgin mice from 3 weeks
to 7 weeks of age had no effect on pup survival in mice subsequently allowed
to undergo pregnancy. These findings suggest that MYC overexpression during
ductal morphogenesis or lactation is not responsible for the rapid postpartum
death of pups born to MMTV-myc mice.
In contrast to the lack of effect of MYC overexpression during virgin development or lactation, induction of MYC expression in MTB/TOM mice from day 0.5 (D0.5) of pregnancy through day 1 postpartum (D1PP) resulted in pup death within 24 hours of parturition in a manner identical to that observed in MMTV-myc mice. Pups did not have milk in their stomachs, implying a maternal defect in lactation. Importantly, pups from MMTV-myc or MTB/TOM mothers fostered with wild-type dams survive and develop normally, indicating that the defect lies in the ability of MMTV-myc or MTB/TOM mice to nurture their young and not with the pups themselves. Thus, deregulated MYC expression during pregnancy is both necessary and sufficient to block postpartum lactation and mimics the phenotype observed in MMTV-myc mice.
During normal mammary gland development, Myc expression peaks at day 6 of pregnancy and remains elevated during the proliferative phase of lobuloalveolar development through day 12.5 of pregnancy (Fig. 1A). MYC expression levels subsequently return to baseline by day 18.5 of pregnancy. To identify a developmental window within pregnancy that is disrupted by aberrant Myc activity, we induced MYC expression for discrete periods during pregnancy and assayed pup viability at 24 hours postpartum. Using this approach, we identified a period from D12.5-D15.5 of pregnancy during which MYC expression is necessary and sufficient to prevent postpartum lactation, and which results in the death of all pups within 24 hours of parturition (Fig. 1B). Restriction of MYC expression to 24 or 48 hours within this developmental window was not sufficient to cause pup death postpartum, although it did have a modest inhibitory effect on postpartum pup growth. Expression of MYC for extended periods of time from early to mid-pregnancy (D0.5-D11.5) resulted only in mild effects on postpartum pup growth and had no impact on pup survival. Remarkably, expression of MYC during late (D15.5-D18.5) pregnancy had no impact on pup survival or postpartum growth. Thus, we conclude that the 72-hour period from day 12.5 to 15.5 of pregnancy represents a discrete developmental window of susceptibility to the effects of MYC; overexpression of MYC during this period is both necessary and sufficient to block postpartum lactation and cause pup death.
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To investigate patterns of apoptosis induced in the mammary gland by MYC activation, we performed TUNEL analysis at increasing intervals following doxycycline induction of MTB/TOM mice. Uninduced MTB and MTB/TOM mice at D12.5 of pregnancy exhibited equivalent rates of apoptosis (Fig. 7A,E) (MTB/TOM 3.2±1.1% compared with MTB 2.9±0.67%; P=0.82). Consistent with the known pro-apoptotic effects of MYC, induction of MYC from D12.5-D15.5 of pregnancy resulted in a marked increase in the proportion of TUNEL-positive mammary epithelial cells within 24 hours of doxycycline treatment (Fig. 7B,F; MTB/TOM 4.1±0.35% compared with MTB 1.0±0.39%; P=0.0002). This increased apoptotic rate persisted throughout the period of MYC induction (Fig. 7C,G; MTB/TOM 6.5±1.44% compared with MTB 0.62±0.17%; P=0.0023) (Fig. 7D,H; MTB/TOM 4.3±0.60% compared with MTB 1.00±0.45%; P=0.0012) and up until 1 day post-deinduction (Fig. 7I,M; MTB/TOM 12.6±1.90% compared with MTB 1.44±0.62%; P=0.0002). By day 17.5 of pregnancy, 2 days post-doxycycline withdrawal, apoptotic rates in MTB/TOM mice (Fig. 7J; 0.53±0.36%) had returned to the level of MTB control mice (Fig. 7N; 0.16±0.16%; P=0.38). Intriguingly, at D18.5 of pregnancy, 3 days post-doxycycline withdrawal, apoptosis rates were again markedly elevated in MTB/TOM mice (Fig. 7K; 4.8±1.64%) compared with MTB controls (Fig. 7O; 0.07±0.07%; P=0.017), and remained elevated at D1PP (Fig. 7L,P; MTB/TOM 8.97±1.50% compared with MTB 0.38±0.24%; P=0.0002).
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MYC promotes premature activation of Stat3 and upregulation of Tgfß3
Mammary involution at the time of pup weaning is triggered by milk stasis
and occurs as a consequence of activation of the Stat3 and Tgfb3 signaling
pathways (Chapman et al.,
1999; Humphreys et al.,
2002
). Given that MYC-induced precocious lactation during
pregnancy would be expected to result in milk stasis, we hypothesized that the
second wave of apoptosis observed in MTB/TOM mice might occur via Stat3 and
Tgfb3-mediated pathways. Consistent with this prediction, induction of MYC
expression from D12.5-D15.5 of pregnancy in MTB/TOM mice resulted in a
dramatic increase in the level of Stat3 Y705 phosphorylation in mammary tissue
extracts beginning at D15.5 of pregnancy, peaking at D1PP at a level similar
to that observed at day 2 of involution in wild-type mice
(Fig. 8A).
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MYC-induced Stat5 activation and precocious lactation are developmental stage-specific
To determine whether MYC-induced precocious lactation is dependent on the
developmental stage of the mammary gland at the time at which MYC is
expressed, we compared the effects of MYC induction from D12.5-D15.5 of
pregnancy with those from D6.5-D9.5 of pregnancy. In contrast to the
precocious alveolar distension and luminal secretion observed when Myc was
expressed from D12.5-D15.5 of pregnancy, mammary epithelial expression of Myc
from D6.5-D9.5 of pregnancy resulted in no discernable increase in
lobuloalveolar development within the mammary fat pad by D9.5 of pregnancy
(Fig. 9A). Similarly, there
were no detectable morphological differences between the mammary glands of
doxycycline-induced MTB/TOM and MTB mice 3 days post-doxycycline induction at
D12.5 of pregnancy (Fig.
9A).
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To determine whether MYC-induced Stat5 hyperactivation also occurs in a developmental stage-specific manner, we assessed Stat5a/b Y695/Y699 phosphorylation in response to MYC expression from D6.5-D9.5 of pregnancy. Induction of MYC in MTB/TOM mammary glands from D6.5-D7.5, D6.5-D8.5 or D6.5-D9.5 of pregnancy failed to result in a detectable difference in Stat5 tyrosine-phosphorylation as compared with MTB controls (Fig. 9C). This is in contrast to the elevated Stat5 tyrosine-phosphorylation observed when MYC was expressed from D12.5-D13.5, D12.5-D14.5 or D12.5-D15.5 of pregnancy (Fig. 9C). Though subtle, this effect has consistently been observed in multiple independent experiments. Thus, the developmental stage-specific ability of MYC to induce precocious lactation is tightly correlated with its ability to induce Stat5 activation.
MYC down-regulates caveolin 1 expression in a developmental stage-specific manner
Downregulation of negative regulators of the Prlr-Jak2-Stat5 signaling
pathway have been proposed to play a vital role in the transition of the
mammary gland from pregnancy to lactation. Genetic deletion of Socs1
or Cav1, for example, have been shown to result in premature Stat5
activation and precocious lactation during pregnancy
(Lindeman et al., 2001;
Park et al., 2002
).
Interestingly, experiments in NIH3T3 and Rat1 cells have shown that MYC can
directly repress Cav1 transcription
(Park et al., 2001
;
Timme et al., 2000
).
Therefore, we hypothesized that the developmental stage-specific ability of
MYC to activate Stat5 might be due to the ability of MYC to downregulate
Cav1 expression in the mouse mammary gland in a developmental
stage-specific manner.
Indeed, northern blot analysis demonstrated rapid downregulation of Cav1 mRNA levels when MYC was induced from D12.5-D15.5, but not D6.5-D9.5, of pregnancy (Fig. 10A). By contrast, MYC did not downregulate expression of the suppressor of cytokine signaling gene, Cish, when induced from D6.5-D9.5 or D12.5-D15.5 of pregnancy (Fig. 10A). Western blot analysis demonstrated that Cav1 protein expression was markedly downregulated following 48 hours of MYC induction from D12.5-D14.5, but not D6.5-D8.5, of pregnancy (Fig. 10B). Thus, the developmental stage-specific ability of MYC to downregulate mammary expression of Cav1 in vivo is closely linked with its ability to promote Stat5 activation and precocious lactation.
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Discussion |
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We have demonstrated that the ability of MYC to inhibit postpartum lactation is entirely due to its activation within a specific 72-hour window during mid-pregnancy, and that this developmental stage-specific window of susceptibility correlates tightly with the ability of MYC to downregulate Cav1 and activate Stat5 in a developmental stage-specific manner. MYC expression during a defined 72 hour window in mid-pregnancy is both necessary and sufficient to inhibit postpartum lactation. Surprisingly, MYC expression during this period does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by accelerating mammary epithelial differentiation and inducing mice to lactate during pregnancy. MYC-induced precocious lactation results in milk stasis, aberrant Stat3 and Tgfb3 activation, and premature involution of the gland, thereby accounting for the inability of mice to lactate postpartum. These findings provide new insights into the mechanism by which the developmental state of the mammary gland modulates its response to oncogene exposure.
In the vast majority of cell types studied, deregulated MYC expression is
incompatible with the terminally differentiated state
(Brandvold et al., 2001;
Freytag, 1988
;
Heath et al., 2000
;
Miner and Wold, 1991
). This is
typically ascribed to the role of MYC as a potent promoter of cellular
proliferation through its ability to transcriptionally activate and repress
genes vital to cell cycle regulation
(Bouchard et al., 1998
).
Contrary to this paradigm, however, are studies by Gandarillas and Watt,
demonstrating that Myc expression in epidermal stem cells actually promotes
terminal differentiation by driving cells into the transit amplifying
compartment (Gandarillas and Watt,
1997
). These and other observations suggest the intriguing
possibility that in cell types in which proliferation is an important aspect
of the differentiation process, Myc may actually promote, or even be required
for terminal differentiation (Arnold and
Watt, 2001
; Battaglino et al.,
2002
; Gandarillas and Watt,
1997
).
In light of the above findings, the ability of MYC to promote rather than
inhibit mammary epithelial differentiation may stem from the fact that normal
differentiation of the mammary gland proceeds through a series of
proliferative steps. During early pregnancy, side-branches arise from existing
ducts as a subset of ductal epithelial cells proliferate in response to
progesterone (Brisken, 2002).
As pregnancy progresses, continuous proliferation of side branches gives rise
to alveolar clusters in a Prl-dependent mechanism known as lobuloalveolar
development (Brisken, 2002
).
It is only after lobuloalveolar development that Prl-dependent lactogenesis
can occur (Neville et al.,
2002
). We have shown that when expressed in the mammary epithelium
from D12.5-D15.5 of pregnancy, MYC induces proliferation and accelerates
lobuloalveolar hyperplasia. By accelerating lobuloalveolar development during
pregnancy, we speculate that MYC brings the mammary epithelium to a state in
which it is competent to undergo precocious terminal differentiation.
Stat5 plays a vital role in mammary development, as demonstrated by the
fact that homozygous deletion of Stat5a and Stat5b inhibits
both lobuloalveolar development and lactation
(Miyoshi et al., 2001;
Teglund et al., 1998
).
Downstream of Prlr, Stat5a and Stat5b are activated by Jak2 tyrosine
kinase-mediated phosphorylation of residues Y694 and Y699, respectively. This
event is tightly linked to mammary differentiation and lactation, peaking late
in pregnancy during normal mammary gland development
(Liu et al., 1996
). Expression
of MYC from D12.5-15.5 of pregnancy results in a striking shift in the peak of
Stat5 tyrosine-phosphorylation from D18.5 to D14.5 of pregnancy. The tight
link between Stat5 hyperactivation and lactation strongly suggests that this
is the mechanism by which MYC promotes precocious lactation.
Though Prolactin-receptor binding and activation provide a Stat5 activating
signal, negative regulation of Prolactin-receptor-Stat5 signaling pathways
plays an equally important role in the regulation of Stat5 activity. In
particular, the suppressor of cytokine signaling (SOCS) family of molecules,
as well as the scaffolding protein, Cav1, have emerged as important negative
regulators of cytokine-receptor-mediated Stat5 activation
(Kile and Alexander,
2001).
Cav1 negatively regulates Prlr-Jak2-Stat5 signaling in the mammary gland
and plays an essential role in the transition from pregnancy to lactation.
This is convincingly demonstrated by the fact that Cav1-/-
mice exhibit increased Jak2 kinase activity, hyperactivation of Stat5 and
precocious lactation during pregnancy
(Park et al., 2002). Indeed,
mammary expression of Cav1 mRNA is downregulated during late
pregnancy and has been proposed to be the impetus for the transition of the
mammary gland to a lactogenic state (Park
et al., 2002
). Intriguingly, reports by Lisanti et al. have
identified Cav1 as a direct target of Myc-mediated transcriptional
repression in vitro (Park et al.,
2001
). We have confirmed that MYC expression for as little as 48
hours in vivo, from D12.5-D14.5 of pregnancy, results in the dramatic
downregulation of Cav1 expression in the mammary gland. Though it cannot be
ruled out that repression of Cav1 expression is a consequence of Myc-induced
precocious lactation and not the cause, our data support a model in which
MYC-mediated repression of Cav1 expression results in upregulated Jak2 kinase
activity, premature Stat5 hyperactivation and precocious lactation.
We have demonstrated that Stat3 activation, downregulation of Stat5
activity, and upregulation of Tgfb3 expression occur shortly after
induction of the precocious lactogenic state engendered in MTB/TOM mice by
expression of MYC from D12.5-D15.5 of pregnancy. These molecular changes
parallel events observed during the early stages of mammary involution in
response to milk stasis. Normal involution occurs in two distinct phases.
Stage 1 is reversible and initiated within hours of forced weaning or teat
sealing. This stage is characterized by a rapid increase in Tgfb3
expression (Nguyen and Pollard,
2000), activation of Stat3, and decreased activation of Stat5
(Chapman et al., 1999
).
Importantly, both Tgfb3 and Stat3 are required for normal
involution to occur, as is downregulation of Stat5 activity
(Chapman et al., 1999
;
Iavnilovitch et al., 2002
;
Nguyen and Pollard, 2000
).
These molecular changes precede apoptosis of alveolar cells, which is
initiated within 24 and 48 hours of milk stasis
(Quarrie et al., 1996
). The
second stage of involution, which begins between involution day 3 and day 4,
is irreversible and is accompanied by tissue restructuring in the form of
alveolar collapse and replacement with adipose tissue
(Quarrie et al., 1996
). We
observe similar molecular, cellular and morphological alterations in MTB/TOM
mouse mammary glands that have been induced to undergo precocious lactation.
Taken together, our findings support a model in which MYC expression from
D12.5-D15.5 of pregnancy induces precocious lactation, which results in milk
stasis, premature involution and the inability of mice to nurse their pups
postpartum.
The observation that MYC activates the established anti-apoptotic survival
factor Stat5 has important implications for MYC-induced mammary tumorigenesis.
Aberrant MYC expression induces apoptosis in a number of cell types, including
mammary epithelial cells (D'Cruz et al.,
2001). Interestingly, the latency for MYC-induced tumor formation
is reduced dramatically in the setting of concurrent activation of
anti-apoptotic pathways, such as the Bcl2 family member Bcl-xL
(Pelengaris et al., 2002
). Our
findings provide evidence that deregulated MYC expression upregulates a known
anti-apoptotic survival pathway in vivo. It is tempting to speculate that MYC
may require activation of Stat5 as a mechanism to avoid apoptosis in the
processes of tumor initiation and progression. Recent genetic studies have
suggested a similar role for Stat5 in TGF
and SV40 large T
antigen-induced mammary tumorigenesis
(Humphreys and Hennighausen,
1999
; Ren et al.,
2002
). A role for the Prlr and Stat5 in MYC-induced mammary
tumorigenesis has yet to be determined. Genetic studies with
Prlr-null and Stat5a/b-null mice should help elucidate the
function of this important mammary developmental signaling pathway in
MYC-induced mammary tumor formation.
The developmental stage-specific manner in which MYC downregulates Cav1,
drives premature Stat5 hyperactivation, and induces precocious lactation has
important implications for our understanding of the developmental
stage-specific effects of oncogene action. Previously, in vitro studies have
suggested that the effects of deregulated MYC expression may be dependent on
the cellular context in which it is expressed. For example, Evan et al. have
demonstrated that access to serum determines whether Rat1 fibroblasts
proliferate or undergo apoptosis in response to MYC expression
(Evan et al., 1992). This
suggests that cellular access to nutrients and growth factors may determine
the consequence of MYC activation. The effects of aberrant MYC expression have
also been shown to vary with cell type. Expression of MYC in suprabasal
keratinocytes or lymphocytes drives proliferation, but results in little
detectable apoptosis (Felsher and Bishop,
1999
; Pelengaris et al.,
1999
). By contrast, targeted MYC expression in pancreatic
ß-cells results in overwhelming apoptosis with little detectable
proliferation (Pelengaris et al.,
2002
). Our results indicate that the developmental state of a cell
at the time of MYC exposure plays a crucial role in determining the biological
effects of aberrant MYC activation. This is the first known developmental
stage-specific effect of aberrant oncogene activation and suggests that
MYC-induced mammary tumorigenesis may be similarly affected by the mammary
developmental stage at which this oncogene is expressed.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Andres, A. C., van der Valk, M. A., Schonenberger, C. A., Fluckiger, F., LeMeur, M., Gerlinger, P. and Groner, B. (1988). Ha-ras and c-myc oncogene expression interferes with morphological and functional differentiation of mammary epithelial cells in single and double transgenic mice. Genes Dev. 2,1486 -1495.[Abstract]
Arnold, I. and Watt, F. M. (2001). c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny. Curr. Biol. 11,558 -568.[CrossRef][Medline]
Askew, D. S., Ashmun, R. A., Simmons, B. C. and Cleveland, J. L. (1991). Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 6,1915 -1922.[Medline]
Bain, C., Willett, W., Rosner, B., Speizer, F. E., Belanger, C. and Hennekens, C. H. (1981). Early age at first birth and decreased risk of breast cancer. Am. J. Epidemiol. 114,705 -709.[Abstract]
Battaglino, R., Kim, D., Fu, J., Vaage, B., Fu, X. Y. and Stashenko, P. (2002). c-myc is required for osteoclast differentiation. J. Bone Miner. Res. 17,763 -773.[Medline]
Berns, E. M., Klijn, J. G., Smid, M., van Staveren, I. L., Look, M. P., van Putten, W. L. and Foekens, J. A. (1996). TP53 and MYC gene alterations independently predict poor prognosis in breast cancer patients. Genes Chromosomes Cancer 16,170 -179.[CrossRef][Medline]
Boice, J. D., Jr, Preston, D., Davis, F. G. and Monson, R. R. (1991). Frequent chest X-ray fluoroscopy and breast cancer incidence among tuberculosis patients in Massachusetts. Radiat. Res. 125,214 -222.[Medline]
Bouchard, C., Staller, P. and Eilers, M. (1998). Control of cell proliferation by Myc. Trends Cell Biol. 8,202 -206.[CrossRef][Medline]
Brandvold, K. A., Ewert, D. L., Kent, S. C., Neiman, P. and Ruddell, A. (2001). Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas. Oncogene 20,3226 -3234.[CrossRef][Medline]
Brisken, C. (2002). Hormonal control of alveolar development and its implications for breast carcinogenesis. J. Mammary Gland Biol. Neoplasia 7, 39-48.[CrossRef][Medline]
Chapman, R. S., Lourenco, P. C., Tonner, E., Flint, D. J.,
Selbert, S., Takeda, K., Akira, S., Clarke, A. R. and Watson, C. J.
(1999). Suppression of epithelial apoptosis and delayed mammary
gland involution in mice with a conditional knockout of Stat3.
Genes Dev. 13,2604
-2616.
Chrzan, P., Skokowski, J., Karmolinski, A. and Pawelczyk, T. (2001). Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue. Clin. Biochem. 34,557 -562.[CrossRef][Medline]
Coppola, J. A. and Cole, M. D. (1986). Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment. Nature 320,760 -763.[Medline]
D'Cruz, C. M., Gunther, E. J., Boxer, R. B., Hartman, J. L., Sintasath, L., Moody, S. E., Cox, J. D., Ha, S. I., Belka, G. K., Golant, A., Cardiff, R. D. and Chodosh, L. A. (2001). c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations. Nat. Med. 7, 235-239.[CrossRef][Medline]
Deming, S. L., Nass, S. J., Dickson, R. B. and Trock, B. J. (2000). C-myc amplification in breast cancer: a meta-analysis of its occurrence and prognostic relevance. Br. J. Cancer 83,1688 -1695.[CrossRef][Medline]
Dores, G. M., Metayer, C., Curtis, R. E., Lynch, C. F., Clarke,
E. A., Glimelius, B., Storm, H., Pukkala, E., van Leeuwen, F. E., Holowaty, E.
J. et al. (2002). Second malignant neoplasms among long-term
survivors of Hodgkin's disease: a population-based evaluation over 25 years.
J. Clin. Oncol. 20,3484
-3494.
Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z. and Hancock, D. C. (1992). Induction of apoptosis in fibroblasts by c-myc protein. Cell 69,119 -128.[Medline]
Facchini, L. M., Chen, S., Marhin, W. W., Lear, J. N. and Penn, L. Z. (1997). The Myc negative autoregulation mechanism requires Myc-Max association and involves the c-myc P2 minimal promoter. Mol. Cell Biol. 17,100 -114.[Abstract]
Felsher, D. W. and Bishop, J. M. (1999). Reversible tumorigenesis by MYC in hematopoietic lineages. Mol. Cell 4,199 -207.[Medline]
Freytag, S. O. (1988). Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol. Cell. Biol. 8,1614 -1624.[Medline]
Gandarillas, A. and Watt, F. M. (1997). c-Myc
promotes differentiation of human epidermal stem cells. Genes
Dev. 11,2869
-2882.
Gardner, H. P., Belka, G. K., Wertheim, G. B., Hartman, J. L.,
Ha, S. I., Gimotty, P. A., Marquis, S. T. and Chodosh, L. A.
(2000). Developmental role of the SNF1-related kinase Hunk in
pregnancy-induced changes in the mammary gland.
Development 127,4493
-4509.
Gunther, E. J., Belka, G. K., Wertheim, G. B., Wang, J.,
Hartman, J. L., Boxer, R. B. and Chodosh, L. A. (2002). A
novel doxycycline-inducible system for the transgenic analysis of mammary
gland biology. FASEB J.
16,283
-292.
Haggerty, T. J., Zeller, K. I., Osthus, R. C., Wonsey, D. R. and
Dang, C. V. (2003). A strategy for identifying transcription
factor binding sites reveals two classes of genomic c-Myc target sites.
Proc. Natl. Acad. Sci. USA
100,5313
-5318.
Heath, V. J., Gillespie, D. A. and Crouch, D. H. (2000). Inhibition of the terminal stages of adipocyte differentiation by cMyc. Exp. Cell Res. 254, 91-98.[CrossRef][Medline]
Helmrich, S. P., Shapiro, S., Rosenberg, L., Kaufman, D. W., Slone, D., Bain, C., Miettinen, O. S., Stolley, P. D., Rosenshein, N. B., Knapp, R. C. et al. (1983). Risk factors for breast cancer. Am. J. Epidemiol. 117,35 -45.[Abstract]
Hennighausen, L. G. and Sippel, A. E. (1982). Characterization and cloning of the mRNAs specific for the lactating mouse mammary gland. Eur. J. Biochem. 125,131 -141.[Abstract]
Hennighausen, L., Robinson, G. W., Wagner, K. U. and Liu, X. (1997). Developing a mammary gland is a stat affair. J. Mammary Gland Biol. Neoplasia 2, 365-372.[Medline]
Hildreth, N. G., Shore, R. E. and Dvoretsky, P. M. (1989). The risk of breast cancer after irradiation of the thymus in infancy. N. Engl. J. Med. 321,1281 -1284.[Abstract]
Humphreys, R. C. and Hennighausen, L. (1999).
Signal transducer and activator of transcription 5a influences mammary
epithelial cell survival and tumorigenesis. Cell Growth
Differ. 10,685
-694.
Humphreys, R. C., Bierie, B., Zhao, L., Raz, R., Levy, D. and
Hennighausen, L. (2002). Deletion of Stat3 blocks mammary
gland involution and extends functional competence of the secretory epithelium
in the absence of lactogenic stimuli. Endocrinology
143,3641
-3650.
Hynes, N. E., Cella, N. and Wartmann, M. (1997). Prolactin mediated intracellular signaling in mammary epithelial cells. J. Mammary Gland Biology and Neoplasia 2,19 -27.[CrossRef]
Iavnilovitch, E., Groner, B. and Barash, I.
(2002). Overexpression and forced activation of stat5 in mammary
gland of transgenic mice promotes cellular proliferation, enhances
differentiation, and delays postlactational apoptosis. Mol. Cancer
Res. 1,32
-47.
Kelly, P. A., Bachelot, A., Kedzia, C., Hennighausen, L., Ormandy, C. J., Kopchick, J. J. and Binart, N. (2002). The role of prolactin and growth hormone in mammary gland development. Mol. Cell. Endocrinol. 197,127 -131.[CrossRef][Medline]
Kile, B. T. and Alexander, W. S. (2001). The suppressors of cytokine signalling (SOCS). Cell Mol. Life Sci. 58,1627 -1635.[Medline]
Land, C. E., Tokunaga, M., Koyama, K., Soda, M., Preston, D. L., Nishimori, I. and Tokuoka, S. (2003). Incidence of female breast cancer among atomic bomb survivors, Hiroshima and Nagasaki, 1950-1990. Radiat. Res. 160,707 -717.[Medline]
Layde, P. M., Webster, L. A., Baughman, A. L., Wingo, P. A., Rubin, G. L. and Ory, H. W. (1989). The independent associations of parity, age at first full term pregnancy, and duration of breastfeeding with the risk of breast cancer. Cancer and Steroid Hormone Study Group. J. Clin. Epidemiol. 42,963 -973.[Medline]
Lindeman, G. J., Wittlin, S., Lada, H., Naylor, M. J.,
Santamaria, M., Zhang, J. G., Starr, R., Hilton, D. J., Alexander, W. S.,
Ormandy, C. J. and Visvader, J. (2001). SOCS1 deficiency
results in accelerated mammary gland development and rescues lactation in
prolactin receptor-deficient mice. Genes & Dev.
15,1631
-1636.
Liu, X., Robinson, G. W. and Hennighausen, L. (1996). Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol. Endocrinol. 10,1496 -1506.[Abstract]
MacMahon, B., Cole, P., Lin, T. M., Lowe, C. R., Mirra, A. P., Ravnihar, B., Salber, E. J., Valaoras, V. G. and Yuasa, S. (1970). Age at first birth and breast cancer risk. Bull. World Health Organ. 43,209 -221.[Medline]
MacMahon, B., Purde, M., Cramer, D. and Hint, E. (1982). Association of breast cancer risk with age at first and subsequent births: a study in the population of the Estonian Republic. J. Natl. Cancer Inst. 69,1035 -1038.[Medline]
Marquis, S. T., Rajan, J. V., Wynshaw-Boris, A., Xu, J., Yin, G. Y., Abel, K. J., Weber, B. L. and Chodosh, L. A. (1995). The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues. Nat. Genet. 11, 17-26.[Medline]
Master, S. R., Hartman, J. L., D'Cruz, C. M., Moody, S. E.,
Keiper, E. A., Ha, S. I., Cox, J. D., Belka, G. K. and Chodosh, L. A.
(2002). Functional microarray analysis of mammary organogenesis
reveals a developmental role in adaptive thermogenesis. Mol.
Endocrinol. 16,1185
-1203.
Master, S. R., Stoddard, A. J., Bailey, L. C., Pan, T-C., Dugan, K. D. and Chodosh, L. A. (2005). Genomic analysis of early murine mammary gland development. Genome Biol. (in press).
Miller, A. B., Howe, G. R., Sherman, G. J., Lindsay, J. P., Yaffe, M. J., Dinner, P. J., Risch, H. A. and Preston, D. L. (1989). Mortality from breast cancer after irradiation during fluoroscopic examinations in patients being treated for tuberculosis. New Engl. J. Med. 321,1285 -1289.[Abstract]
Miner, J. H. and Wold, B. J. (1991). c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation. Mol. Cell. Biol. 11,2842 -2851.[Medline]
Miyoshi, K., Shillingford, J. M., Smith, G. H., Grimm, S. L.,
Wagner, K. U., Oka, T., Rosen, J. M., Robinson, G. W. and Hennighausen, L.
(2001). Signal transducer and activator of transcription (Stat) 5
controls the proliferation and differentiation of mammary alveolar epithelium.
J. Cell Biol. 155,531
-542.
Murphy, W., Sarid, J., Taub, R., Vasicek, T., Battey, J., Lenoir, G. and Leder, P. (1986). A translocated human c-myc oncogene is altered in a conserved coding sequence. Proc. Natl. Acad. Sci. USA 83,2939 -2943.[Abstract]
Myal, Y., Robinson, D. B., Iwasiow, B., Tsuyuki, D., Wong, P. and Shiu, R. P. (1991). The prolactin-inducible protein (PIP/GCDFP-15) gene: cloning, structure and regulation. Mol. Cell. Endocrinol. 80,165 -175.[CrossRef][Medline]
Neville, M. C., McFadden, T. B. and Forsyth, I. (2002). Hormonal regulation of mammary differentiation and milk secretion. J. Mammary Gland Biol. Neoplasia 7, 49-66.[CrossRef][Medline]
Nguyen, A. V. and Pollard, J. W. (2000).
Transforming growth factor beta3 induces cell death during the first stage of
mammary gland involution. Development
127,3107
-3118.
Park, D. S., Razani, B., Lasorella, A., Schreiber-Agus, N., Pestell, R. G., Iavarone, A. and Lisanti, M. P. (2001). Evidence that Myc isoforms transcriptionally repress caveolin-1 gene expression via an INR-dependent mechanism. Biochemistry 40,3354 -3362.[CrossRef][Medline]
Park, D. S., Lee, H., Frank, P. G., Razani, B., Nguyen, A. V.,
Parlow, A. F., Russell, R. G., Hulit, J., Pestell, R. G. and Lisanti, M.
P. (2002). Caveolin-1-deficient mice show accelerated mammary
gland development during pregnancy, premature lactation, and hyperactivation
of the Jak-2/STAT5a signaling cascade. Mol. Biol. Cell
13,3416
-3430.
Pelengaris, S., Littlewood, T. D., Khan, M., Elia, G. and Evan, G. (1999). Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol. Cell 3,565 -577.[Medline]
Pelengaris, S., Khan, M. and Evan, G. I. (2002). Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 109,321 -334.[Medline]
Quarrie, L. H., Addey, C. V. and Wilde, C. J. (1996). Programmed cell death during mammary tissue involution induced by weaning, litter removal, and milk stasis. J. Cell Physiol. 168,559 -569.[CrossRef][Medline]
Ren, S., Cai, H. R., Li, M. and Furth, P. A. (2002). Loss of Stat5a delays mammary cancer progression in a mouse model. Oncogene 21,4335 -4339.[CrossRef][Medline]
Russo, J., Rivera, R. and Russo, I. H. (1992). Influence of age and parity on the development of the human breast. Breast Cancer Res. Treat. 23,211 -218.[Medline]
Schoenenberger, C. A., Andres, A. C., Groner, B., van der Valk, M., LeMeur, M. and Gerlinger, P. (1988). Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription. EMBO J. 7, 169-175.[Abstract]
Shore, R. E., Hempelmann, L. H., Kowaluk, E., Mansur, P. S., Pasternack, B. S., Albert, R. E. and Haughie, G. E. (1977). Breast neoplasms in women treated with x-rays for acute postpartum mastitis. J. Natl. Cancer Inst. 59,813 -822.[Medline]
Stampfer, M. R., Yaswen, P., Alhadeff, M. and Hosoda, J. (1993). TGF beta induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects. J. Cell Physiol. 155,210 -221.[Medline]
Stewart, T. A., Pattengale, P. K. and Leder, P. (1984). Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes. Cell 38,627 -637.[Medline]
Teglund, S., McKay, C., Schuetz, E., van Deursen, J. M., Stravopodis, D., Wang, D., Brown, M., Bodner, S., Grosveld, G. and Ihle, J. N. (1998). Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93,841 -850.[Medline]
Timme, T. L., Goltsov, A., Tahir, S., Li, L., Wang, J., Ren, C., Johnston, R. N. and Thompson, T. C. (2000). Caveolin-1 is regulated by c-myc and suppresses c-myc-induced apoptosis. Oncogene 19,3256 -3265.[CrossRef][Medline]
Watson, P. H., Safneck, J. R., Le, K., Dubik, D. and Shiu, R. P. (1993). Relationship of c-myc amplification to progression of breast cancer from in situ to invasive tumor and lymph node metastasis. J. Natl. Cancer Inst. 85,902 -907.[Abstract]
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