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First published online 7 February 2007
doi: 10.1242/dev.02797
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Baylor Breast Center and Department of Molecular and Cellular Biology, Room N1210; MS:BCM600, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
* Author for correspondence (e-mail: mtlewis{at}breastcenter.tmc.edu)
Accepted 3 January 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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50% of ductal carcinoma in
situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically
expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors
and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67
proliferation marker. Our data suggest that altered hedgehog signaling may
contribute to breast cancer development by stimulating proliferation, and by
increasing the pool of division-competent cells capable of
anchorage-independent growth.
Key words: Hedgehog signaling, Stem cell, Progenitor cell, Invasive breast cancer, Ductal carcinoma in situ
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hooper and Scott, 2005;
Lewis and Veltmaat, 2004;
Nusse, 2003). Genetic analyses
in mice indicate a role in mammary ductal morphogenesis, and recent data
suggest a role in human mammary epithelial stem cell self-renewal
(Liu et al., 2006). In
addition to hedgehog functions in normal development, altered hedgehog
signaling is now implicated in approximately 20-25% of all cancers
(Briscoe and Therond, 2005),
and there is increasing evidence to suggest a role in breast cancer
(Chang-Claude et al., 2003;
Kubo et al., 2004;
Lewis et al., 2001;
Lewis et al., 1999;
Liu et al., 2006;
Mukherjee et al., 2006;
Naylor et al., 2005). However,
the specific roles hedgehog network genes play in normal mammary gland
development remain unclear, and no experiments have been performed to test
directly whether inappropriately activated hedgehog signaling has functional
consequences for gland development, or causes breast cancer.
Development of the mouse mammary gland begins in the embryo with formation
of a rudimentary ductal tree, but most development occurs after puberty
(Daniel and Silberstein, 1987;
Sakakura, 1987). At puberty,
ovarian steroids stimulate rapid and invasive ductal elongation and branching
morphogenesis. At the growing tips of elongating ducts are bulb-like
structures called terminal end buds (TEBs). Histologically, TEBs consist of
two highly proliferative cell compartments: an outer `cap cell' compartment,
and an inner `body cell' compartment consisting of four to six layers of
relatively undifferentiated luminal epithelial cells. As the TEB invades the
mammary fat pad, cap cells serve as a progenitor cell population and
differentiate into myoepithelial cells that line the basement membrane
(Williams and Daniel, 1983),
whereas body cells are thought to give rise to all luminal epithelial cell
subtypes of the mature duct, including new multipotent mammary stem cells. As
ducts elongate, they are surrounded by a periductal stroma consisting of
fibroblasts, macrophages, eosinophils and vascular cells, within the confines
of the mammary fat pad.
Hedgehog signaling involves two types of cells: a signaling cell expressing
a member of the hedgehog family of secreted ligands [sonic hedgehog (SHH),
indian hedgehog (IHH), or desert hedgehog (DHH)], and a responding cell
expressing one or more patched family hedgehog receptors [patched-1 (PTCH1)
and patched-2 (PTCH2)]. In the absence of ligand, PTCH1 can function to
inhibit downstream signaling via the smoothened (SMO) transmembrane effector
protein. Under these conditions, expression of hedgehog target genes is
inhibited by repressor forms of one or more members of the Gli family of
transcription factors (GLI2 or GLI3). In the presence of ligand, PTCH1
releases inhibition of SMO, which leads to induction of target genes by
transcriptional activator forms of Gli transcription factors (GLI1, GLI2 or
GLI3). In addition to its signal transduction activities, PTCH1 can also
function to sequester hedgehog ligand, thereby restricting the range over
which free ligand can signal (reviewed by
Hooper and Scott, 2005).
Finally, there is evidence to suggest that PTCH1 can function as a
`dependence receptor' to induce apoptosis in cell types dependent on
ligand-bound PTCH1 for survival (Chao,
2003; Guerrero and Ruiz i
Altaba, 2003; Thibert et al.,
2003).
Our laboratory previously demonstrated crucial functions for Ptch1
and Gli2 in mammary ductal development
(Lewis et al., 2001;
Lewis et al., 1999;
Lewis and Veltmaat, 2004).
Heterozygous mutation of Ptch1 (
Ptch1/+) led to
ductal dysplasia in virgin mice characterized by multiple epithelial cell
layers within the ducts (Lewis et al.,
1999). Similarly, transplantation rescue of whole mammary glands
from homozygous Gli2-null mouse embryos yielded ductal dysplasias
(Lewis et al., 2001). However,
for both Ptch1 and Gli2, transplantation of mutant
epithelium into a wild-type stroma failed to recapitulate the phenotype
observed in intact glands, suggesting that these two genes function primarily
in the stroma to regulate epithelial cell behavior. Thus, despite
developmentally regulated hedgehog network gene expression in the epithelial
compartment (Lewis et al.,
2001), a role for activated hedgehog signaling in the epithelium
has not been demonstrated during ductal development
(Gallego et al., 2002;
Michno et al., 2003), and the
consequences of inappropriate hedgehog signaling in the epithelium remain
unknown.
Our published working model for hedgehog signaling in mammary ductal
development (Lewis and Veltmaat,
2004; Lewis and Visbal,
2007) proposes that hedgehog signaling may function transiently in
the body cell layer of the TEB, but that signaling must be prevented in
differentiated ducts. Consistent with this hypothesis, Liu and colleagues
(Liu et al., 2006) showed that
treatment of human breast epithelium with recombinant hedgehog ligand
increased both primary and secondary mammosphere formation [in vitro assays of
anchorageindependent growth, and of self-renewal of both stem and progenitor
cell types (Dontu et al.,
2003; Dontu and Wicha,
2005)], whereas treatment with the hedgehog signaling antagonist
cyclopamine decreased mammosphere formation. In this paper, we examine the
effect of sustained SMO-mediated hedgehog signaling during ductal elongation
in virgin transgenic mice, and correlate our results with altered PTCH1 and
SMO expression in human breast cancer. Taken together, our results are
consistent with a role for hedgehog signaling in mammary epithelial progenitor
cell regulation, and suggest that ectopic hedgehog signaling may contribute to
human breast cancer by stimulating proliferation, and by increasing the pool
of division-competent cells capable of anchorage-independent growth.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Mice
were maintained in accordance with the NIH Guide for the Care and Use of
Experimental Animals with approval from our Institutional Animal Care and Use
Committee.
Screening MMTV-SmoM2 transgenic founder lines
Purified tail DNA from founders was used for PCR analysis for the presence
of the MMTV-SmoM2 transgene. Primers used were: forward,
5'-GAGCTGCAGAAGCGCCTGGGCC-3'; reverse,
5'-GGTATTGGTTCCTCTCTTTCCTG-3'. Cycling conditions were: 94°C
for 35 seconds, 62°C for 40 seconds, and 72°C for 50 seconds.
Detection of a 450 bp product indicated presence of the transgene. Of
seven transgenic founder lines, five yielded progeny expressing the
MMTV-SmoM2 transgene by RT-PCR.
Five female mice per genotype per line were screened at 5 and 10 weeks of age for expression of Smo mRNA and protein, as well as for changes in ductal patterning, histology, proliferation, apoptosis and expression of steroid hormone receptors. One line showing a representative ductal phenotype and proliferation rate [designated Tg(MMTV-SmoM2)724Mtl], and a line showing an identical ductal phenotype but elevated proliferation rate [designated Tg(MMTV-SmoM2)732Mtl] were chosen for follow-up analysis (hereafter referred to as lines 724 and 732, respectively). Unless otherwise indicated, all data shown are for line 724.
Whole gland morphological analysis
Mammary glands #1-5 were harvested from the right side of at least ten
female mice at 5 and 10 weeks of age, as well as at greater than 50 weeks of
age (palpated weekly after 52 weeks of age to assay for tumor development),
fixed in ice-cold 4% paraformaldehyde in PBS, and examined as whole-mount
preparations using a Neutral Red staining protocol. After fixation, fat was
removed in three changes of acetone (1 hour each) and glands were stained in
Neutral Red staining solution (0.01% Neutral Red in 100% ethanol acidified to
pH 5.0 with glacial acetic acid) overnight with constant stirring. Glands were
destained in two changes of 100% ethanol, cleared in two changes of xylenes (1
hour each), and stored in xylenes.
Average TEB number per gland was quantified using the #3 mammary gland at 5 weeks of age. Branch-point analysis and evaluation of retained TEB-like structures were conducted using the #3 mammary gland at 10 weeks of age by direct counting of all branch points. Phenotypic analysis of MMTV-SmoM2 mice at other phases of gland development (e.g. pregnancy, lactation, involution) will be presented elsewhere.
Immunohistochemistry and immunofluorescence analysis
For histological analysis, the #2 and #3 mammary glands from the left side
of the animal were fixed in 4% paraformaldehyde in PBS, embedded in paraffin,
sectioned, and either stained with Hematoxylin-Eosin or used for
immunolocalization studies. Antibodies used for immunolocalization studies are
listed in Table 1.
Immunostaining was performed with antigen retrieval in 0.1 M Tris-HCl (pH 9.0)
with 10% Tween-20, or in TRS (DakoCytomation), by heating to 120°C for 10
minutes in a pressure cooker. For immunohistochemistry, detection was by
standard peroxidase staining using the ABC system (Vector Laboratories). For
immunofluorescence, sections were counterstained with DAPI in Mounting Medium
(Vectashield).
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Primary mammary epithelial cell isolation
Primary mammary epithelial cells were isolated from freshly dissected
mammary glands by enzymatic dissociation overnight in DMEM-Ham's F12 medium (5
ml per mouse) containing collagenase (1 mg/ml), hyaluronidase (100 U/ml),
penicillin-streptomycin (100 U/ml), and gentamycin (50 µg/ml), essentially
as described (Smith, 1996).
Resulting cell pellets were treated with 1 ml 0.25% trypsin-EDTA at 37°C
for 5 minutes. Trypsin was inactivated with 10 ml HBSS containing 5% serum.
Cells were centrifuged and washed three times in HBSS containing 5% serum.
After the final wash, each preparation was filtered through a 40 µm
strainer to yield a single-cell suspension. Single-cell suspensions were used
directly in mammosphere-formation assays and limiting-dilution transplantation
assays.
Mammosphere-formation and transplantation assays
Primary mammary epithelial cells derived from five paired sets of wild-type
or MMTV-SmoM2 mice (three to four mice per genotype per set) were
plated in triplicate wells of six-well, ultra-low attachment plates (2 ml per
well) at a concentration of 30,000 cells/ml as described previously
(Chen et al., 2007;
Dontu et al., 2003;
Youn et al., 2005). Cells were
fed every 3-4 days for 10-14 days. Primary mammospheres were counted for each
genotype, and the percentage of mammosphere-forming cells was calculated as a
measure of mammosphere-forming efficiency.
To demonstrate that mammospheres contained stem cells capable of
regenerating ductal trees, single mammospheres derived from wild-type and
MMTV-SmoM2 mice were transplanted into contralateral cleared fat pads
of 3-week-old recipient FVB female mice
(Deome et al., 1959), and
allowed to grow for 8 weeks. Glands were then excised, fixed and stained as
whole-mount preparations.
Limiting-dilution cell transplantation assays
Primary mammary epithelial cells derived from five paired sets of wild-type
or MMTV-SmoM2 mice (three to four mice per genotype per set) were
counted on a hemocytometer, resuspended at the desired concentration in a 1:1
solution of PBS:Matrigel (BD Biosciences, 354234), and kept on ice until
transplantation. Cells of each genotype were injected at limiting dilutions
(1000, 500, 200, 100, 50 and 25 cells per gland, in a total volume of 10
µl) into contralateral cleared fat pads of #4 mammary glands of 21-day-old
female wild-type mice using a 25G needle attached to a 50 µl Hamilton glass
syringe (Deome et al., 1959).
Seven weeks after transplantation, #4 glands and a #3 host control gland were
excised and stained as whole-mounts. Glands showing at least 5% fat pad
filling were scored as a positive `take'.
Human breast clinical samples
Low density tissue arrays comprising archival formalin-fixed,
paraffinembedded samples of either ductal carcinoma in situ (DCIS), invasive
breast cancer (IBC), or normal tissue derived from patients with breast cancer
were used. All tissue was obtained and used with approval from our
Institutional Review Board. DCIS samples had been scored previously for
histological grade. Both DCIS and IBC samples had been scored previously for
expression of estrogen receptor alpha (ER
; ESR1 - Human Gene
Nomenclature Database), ERBB2 (HER2) and p53 (TP53-Human Gene Nomenclature
Database) by the method of Allred (Allred
et al., 1998; Harvey et al.,
1999), in which a total score (0-8) is assigned as the sum of the
proportion score (0-5) and an intensity score (0-3) for the expression of a
given gene.
Quantitative RT-PCR
Total RNA was extracted using Trizol Reagent (Invitrogen). Total RNA (100
ng per sample, in triplicate) was reverse-transcribed (M-MLV Reverse
Transcriptase, Invitrogen) following the manufacturer's protocol. The
resulting cDNA was analyzed using an Applied Biosystems 7500-Fast thermocycler
for TaqMan quantitative PCR (Q-PCR) using standard conditions. TaqMan Assay On
Demand primers and probes were purchased from Applied Biosystems. Product
accumulation was evaluated using the comparative Ct method (Ct
method), with beta-actin (ActB) as an endogenous control for
normalization (Livak and Schmittgen,
2001).
Statistical analysis
Comparisons of gene expression levels across wild-type and transgenic lines
724 and 732 were assessed by one-way ANOVA and pairwise t-tests.
Spearman correlation coefficients were calculated to assess associations
between human SMO and the expression of mouse hedgehog network genes. Changes
in hedgehog network gene expression in Ptch1/+ animals
relative to wild type were evaluated using a t-test. For protein
expression analyses, the average percentage of cells expressing a given
protein in a given genetic background was compared with corresponding
wild-type controls using the Wilcoxon rank-sum test.
For comparison of TEB number and branch-point number between glands of wild-type versus MMTV-SmoM2 mice, mean numbers per gland were compared using a t-test. Tumor incidence between wild-type and MMTV-SmoM2 mice was compared using Fisher's exact test.
For mammosphere-formation assays, mammosphere-formation efficiency values were log-transformed and compared between paired groups of wild-type and MMTV-SmoM2 animals using a paired t-test. Single mammosphere transplantation assays were performed to evaluate the frequency of mammospheres containing regenerative stem cells in wild-type versus MMTV-SmoM2 mice. Regeneration frequencies were compared using Fisher's exact test.
Limiting-dilution analysis was performed to estimate the frequency of
regenerative stem cells in primary mammary epithelial cell preparations, along
with 95% Wald confidence intervals [Smyth G. (2006) Statmod: Statistical
Modeling. R package version 1.2.4.
http://www.statsci.org/r].
The single-hit Poisson model (SHPM) was fitted to limiting-dilution data using
a complementary log-log generalized linear model
(Bonnefoix et al., 1996).
In human clinical samples, correlations between expression of PTCH1 or SMO and clinically-relevant markers in DCIS, as well as the correlation between PTCH1 and SMO expression in DCIS and IBC, were tested for statistical significance using Spearman's rank correlation. All of the variables in the correlation analysis were analyzed as continuous variables. Immunohistochemical total scores for expression of PTCH1 and SMO were compared between normal, DCIS, and IBC tissues using the Wilcoxon rank-sum test. Statistical analyses were performed with SAS (version 9.1), SPLUS (version 7.0), or R (version 2.2.1). P values of 0.05 or less were deemed statistically significant.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) selectively in mammary epithelium under the control of the
MMTV promoter [Tg(MMTV-SmoM2)].
In whole-mount analysis, glands of wild-type mice at 5 weeks of age showed
normal morphology of TEBs and subtending ducts
(Fig. 1A). By contrast, TEBs of
MMTV-SmoM2 mice frequently displayed excessive budding at the neck of
the TEB (Fig. 1B) and an
increase in TEB number (Fig.
1C) (P=0.05). In histological analyses, wild-type glands
showed normal TEB structure (Fig.
1D). By contrast, 30% of TEBs in MMTV-SmoM2 glands
showed disorganized cap and body cell layers
(Fig. 1E).
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At 10 weeks of age, ducts of wild-type glands
(Fig. 1I) showed normal
histoarchitecture. Ducts of MMTV-SmoM2 glands showed histology
consistent with increased side-budding, but appeared normal with respect to
the lumenal and myoepithelial cell layers
(Fig. 1J), and did not show
ducts having multiple layers of lumenal epithelium that are characteristic of
Ptch1/+ mice (Lewis et
al., 1999) (see also Fig.
3E,F). Lumenal and myoepithelial cell layer number were confirmed
by immunostaining for smooth muscle actin (SMA)
(Fig. 1K,L) and p63 (not
shown). Retained TEB-like structures showed histoarchitecture similar to TEBs,
but the body cell layer was generally only 2-4 cell layers thick
(Fig. 1J, inset).
In an interim analysis of an ongoing tumor-formation study, no tumors have been detected in a cohort of wild-type virgin mice over 52 weeks of age (n=84), or in a cohort of aged MMTV-SmoM2 mice from line 724 (n=43). Two tumors have been detected in a cohort of MMTV-SmoM2 mice from line 732 (n=80). However, this frequency of tumor formation is not statistically different from wild type (P=0.4). Thus, MMTV-SmoM2 expression in virgin mice does not lead to high frequency tumor formation.
Our initial screen suggested that only a small percentage of epithelial
cells in MMTV-SmoM2 mice expressed detectable levels of SMO protein.
Consistent with this observation, Q-PCR did not detect significant changes in
hedgehog network gene expression, with the possible exception of decreased
Ptch1 in line 732 (see Fig. S1 in the supplementary material). Gene
expression analysis in Ptch1/+ mice was consistent with
results in MMTV-SmoM2 mice. In Ptch1/+ mice, with the
exception of Hip1, whose expression by Q-PCR was elevated 2-fold
above wild type (P=0.049), Ptch1 heterozygosity was
also not sufficient to induce expression of other hedgehog network genes (see
Fig. S1 in the supplementary material).
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). Whereas SMO was undetectable in ducts of wild-type glands at 10 weeks of age (Fig. 2A), PTCH1 was near-uniformly detected in punctate foci in the cytoplasm of epithelial cells by deconvolution microscopy (Fig. 2B,C). In MMTV-SmoM2 mice, ducts with altered morphology generally showed detectable SMO protein expression (Fig. 2D). Staining was mosaic, with a median of only 5.7% of all epithelial cells showing detectable SMO expression (Fig. 2F). In most SMO-expressing cells, PTCH1 expression appeared largely membrane-associated and was slightly to moderately elevated relative to adjacent SMO-negative cells, consistent with intermediate to high levels of signaling activation in SMO-positive cells (Fig. 2E,F).
PTCH1 loss and SMO activation increase proliferation in the mammary gland in vivo
We next compared BrdU incorporation rates and expression patterns for
cleaved caspase-3, estrogen receptor (ER) and progesterone receptor (PR),
using glands of 10-week-old MMTV-SmoM2, Ptch1/+ and
wild-type control mice (Fig. 3
and see Table S1 in the supplementary material). We detected no change in ER
or PR expression between wild-type and either MMTV-SmoM2 or
Ptch1/+ mice, but detected a significant increase in BrdU
incorporation in both MMTV-SmoM2 (4.4%) and Ptch1/+
mice (11.3%) relative to wild-type age-matched littermate controls (0.6%)
(Fig. 3, A versus C,E).
Elevated BrdU incorporation rates in MMTV-SmoM2 glands were
corroborated by staining for the proliferation marker Ki67 (Mki67 - Mouse
Genome Informatics), which was detected in just 6.2% of wild-type cells
(Fig. 3A, inset), but in 31.3%
of cells in MMTV-SmoM2 glands
(Fig. 3C, inset, and see Table
S1 in the supplementary material).
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Ptch1/+ phenotype, we expected to observe a compensatory
increase in cell death to offset increased proliferation. Wild-type glands
showed low levels of cleaved caspase-3 staining (<1%)
(Fig. 3B). Contrary to
expectations, MMTV-SmoM2 mice did not show increased
caspase-3-mediated apoptosis (Fig.
3D). Glands from Ptch1/+ mice also showed no
change in cleaved caspase-3 expression as a percentage of total epithelial
cells (Fig. 3F). Thus, the
reason proliferating cells accumulate in glands of Ptch1/+
mice, but do not accumulate in glands of MMTV-SmoM2 mice, remains
unclear but may be due to non-caspase-3-mediated apoptosis or autophagy.
MMTV-SmoM2 transgene expression and proliferation do not colocalize
The observation that SMO expression was limited, yet morphological defects
and expression of proliferation markers were widespread, led us to question to
what degree transgene expression correlated with proliferation and hormone
receptor status. We conducted dual immunofluorescence staining for SMO, Ki67,
and ER, and quantified co-expression in pairwise combinations. Overall, SMO
was expressed in 5.7% of all epithelial cells, whereas Ki67 was expressed in
31.3% of epithelial cells. However, SMO and Ki67 did not colocalize
(Fig. 4A). We also found that
SMO expression did not colocalize with ER
(Fig. 4B), with 33.0% of cells
showing ER expression exclusively. Unlike wild-type glands, ER and Ki67
colocalized at a low, but measurable, frequency (1.0%) in glands of transgenic
mice (Fig. 4C, and see Table S1
in the supplementary material). Proliferation in ER+ cells was
confirmed by co-staining for ER and BrdU
(Fig. 4C, inset).
MMTV-SmoM2 mice show altered epithelial cell differentiation
Keratin 6 (CK6; KRT6), a marker of primitive progenitor cells
(Grimm et al., 2006;
Stingl et al., 2005), is
expressed primarily in ER+ cells in the body cell layer of the TEB,
and only rarely in differentiated ducts of mature glands
(Grimm et al., 2006). As
expected, in mature ducts of 10-week-old wild-type mice, expression of SMO was
undetectable and CK6 was observed infrequently (7.5%), and at very low levels
(see Table S1 in the supplementary material). However, in 10-week-old
MMTV-SmoM2 mice, CK6 expression was readily detectable in 20% of
epithelial cells (Fig. 4D,E).
Co-staining of CK6 and ER demonstrated that the majority of CK6+
cells (82.0%) were also ER+
(Fig. 4D). There was no
colocalization of SMO with CK6 (Fig.
4E with inset). Thus, the MMTV-SmoM2 transgene was
expressed to detectable levels only in non-proliferative ER-
CK6- cells.
MMTV-SmoM2 increases the proportion of mammosphere-forming cells in mammary glands of transgenic mice in vivo
To determine whether MMTV-SmoM2 transgenic mice showed a change in
the frequency of stem and progenitor cell types relative to wild-type
controls, we conducted primary mammosphere-formation assays
(Dontu et al., 2003;
Dontu and Wicha, 2005). In
four of five independent, paired, primary cell preparations, cells derived
from MMTV-SmoM2 mice showed a 2-fold increase in the percentage
of cells capable of forming primary mammospheres (mean raw value=0.76%)
relative to cells isolated from wild-type littermate control mice (mean raw
value=0.38%) (P=0.02, paired t-test)
(Fig. 5A,B). There were no
differences in mammosphere size or shape between the two genotypes
(Fig. 5C).
To verify that primary mammospheres contained regenerative mammary epithelial stem cells, we transplanted single mammospheres derived from wild-type and MMTV-SmoM2 mice into contralateral cleared fat pads of 3-week-old host mice. Mammospheres derived from both genotypes showed regenerative potential, with 2 out of 13 (15%) wild-type mammospheres (Fig. 5D, left panel), and 5 out of 15 (33%) MMTV-SmoM2 mammospheres (Fig. 5D, right panel), capable of regenerating ductal trees. These regeneration frequencies were not statistically different from one another (P=0.40, Fisher's exact test). Duct morphology in MMTV-SmoM2 outgrowths was consistently altered as compared with wild type in a manner consistent with the phenotype observed in intact mice.
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For both genotypes, as few as 25 cells were capable of regenerating ductal
trees. However, the rate of successful transplantation ('take rate') was lower
in cells derived from MMTV-SmoM2 mice when fewer than 200 cells per
gland were injected. Using a single-hit Poisson distribution model we estimate
the frequency of regenerative stem cells in wild-type epithelium is 1 stem
cell per 106 cells (Fig. 5E).
The frequency of regenerative stem cells in MMTV-SmoM2 epithelium was
decreased 2.5-fold, to 1 stem cell per 255 cells. Again, duct morphology
in MMTV-SmoM2 outgrowths was consistently altered as compared with
wild type (Fig. 5F).
Hedgehog signaling is altered at high frequency in human breast cancer
To evaluate a potential role for PTCH1 and SMO in human breast cancer, we
conducted an immunohistochemical study for expression of PTCH1 and SMO in a
panel of normal, ductal carcinoma in situ (DCIS), and invasive breast cancer
(IBC) samples (Fig. 6). PTCH1
was detectable throughout the epithelium
(Fig. 6A), and in isolated
stromal cells of the normal breast. By contrast, PTCH1 expression was
decreased or absent in 50% of DCIS
(Fig. 6B) and IBC
(Fig. 6C). Conversely, SMO was
undetectable in normal breast (Fig.
6D), but ectopically expressed in 70% of DCIS and 30% of
IBC (Fig. 6E,F). For both
proteins, total scores between DCIS and IBC were significantly different from
each other (SMO, P=0.0003; PTCH1, P=0.0001; Wilcoxon
rank-sum test) (Fig. 6G). By
Spearman rank correlation analysis, expression of neither PTCH1 nor SMO
correlated with histological grade (DCIS only), nor with the expression of any
of the clinically-relevant markers tested. PTCH1 expression was not
significantly correlated with SMO expression in either DCIS or IBC.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Ptch1
heterozygosity, despite increased proliferation in both models. SMO expression
rarely colocalized with the Ki67 proliferation marker, and activation was not
sufficient to cause tumors at high frequency. SMO activation led to altered
differentiation, as well as to enhanced primary mammosphere-forming
efficiency. However, limiting-dilution transplantation analysis showed a
decrease in the frequency of regenerative stem cells in MMTV-SmoM2
epithelium relative to wild type. In human clinical samples, hedgehog network
gene expression is altered frequently, and early, in breast cancer
development. As in MMTV-SmoM2 mice, SMO rarely colocalized with Ki67
in breast tumors. Taken together, these data are consistent with our model
that hedgehog signaling must be prevented in mature ducts of virgin mice, and
suggest that it is also normally inactive in mature ducts in humans. Thus,
ectopic hedgehog signaling could contribute to early breast cancer development
by stimulating proliferation, and by increasing the pool of division-competent
cells capable of anchorage-independent growth.
In light of our transplantation results published previously
(Lewis et al., 1999), failure
of MMTV-SmoM2 to recapitulate the Ptch1/+
hyperplastic phenotype was not entirely unexpected. In these experiments, the
Ptch1/+ phenotype could be partially recapitulated, but only
when the entire mammary gland was transplanted - transplantation of epithelial
fragments into cleared fat pads of wild-type mice did not lead to ductal
dysplasia. These results led to the interpretation that the
Ptch1/+ phenotype was due primarily to loss-of-function in
mammary stroma. Since the MMTV-SmoM2 transgene is expressed
selectively in the mammary epithelium, stromal activation of hedgehog
signaling was not tested here. Thus, the two models are not directly
comparable. Regardless of these differences, our results clearly demonstrate
that Ptch1 heterozygosity is not functionally equivalent to
SMO activation solely in mammary epithelium.
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5.7% of all epithelial
cells expressed detectable levels of SMO protein. There are at least three
possible interpretations of these results.
First, the simplest interpretation is that SMO is, in fact, only expressed
and active in a small percentage of cells in MMTV-SmoM2 mice. This
interpretation then leads to the intriguing hypothesis that SMO activity in
these few cells promotes persistence of ER+CK6+ cells,
and stimulates proliferation indirectly via an undefined paracrine signaling
factor (or factors). We are currently testing this hypothesis in a series of
in vitro cell mixing and in vivo transplantation experiments, similar to those
used recently to demonstrate paracrine signaling functions for the estrogen
and progesterone receptors during mammary gland development
(Brisken et al., 1998;
Mallepell et al., 2006). In
addition, we are using alternative models in which SMO activity can be
manipulated in vivo (Du et al.,
2006; Jeong et al.,
2004).
A second possibility is that SMO is expressed and active in greater than
5.7% of cells, but that SMO protein expression is below the limit of
detection. Indeed, available antibodies must be used at relatively high
concentrations and fail to detect SMO in some tissues known to have hedgehog
signaling activity (e.g. E14 embryo, hair follicle, colon) (not shown). This
possibility cannot be excluded given currently available reagents. However, if
true, this interpretation requires that low-level activity is not sufficient
to induce expression of PTCH1 in most cells
(Hooper and Scott, 2005)
(Fig. 2 and see Fig. S1 in the
supplementary material).
A third interpretation is that the primary effect of ectopic SMO expression
occurs in, or near, the TEB and leads to a permanent alteration in cell fate.
This would require that the permanent alteration leads to inactivation of the
MMTV promoter, such that it is no longer expressed. This possibility is being
addressed using alternative models in which SMO activity can be controlled in
a temporally-regulated manner (Du et al.,
2006; Jeong et al.,
2004).
With respect to normal mammary development, our data and those of Liu et
al. (Liu et al., 2006) are
consistent with our working model in which hedgehog signaling may be active in
the growing TEB, but signaling is normally absent from mature ducts of
wild-type mice (Lewis and Veltmaat,
2004; Lewis and Visbal,
2007). Absence of activated hedgehog signaling in mature ducts is
supported by reduced levels of Ptch1 mRNA in differentiated ducts
relative to the TEB, the lack of detectable SMO protein expression, and the
failure to detect Gli gene function in mammary epithelium of postnatal animals
(Hatsell and Cowin, 2006;
Lewis et al., 2001;
Lewis and Veltmaat, 2004).
However, the definitive experiments required to determine whether active
hedgehog signaling functions in the epithelium during postnatal ductal
development have never been performed
(Lewis and Visbal, 2007). We
are currently analyzing conditional null mutants of both Ptch1 and
Smo, which should allow us to test our model conclusively
(Ellis et al., 2003;
Long et al., 2001).
Mammosphere-formation assays are a powerful new addition to our
experimental arsenal for the evaluation of stem and progenitor cell types
(Dontu et al., 2003;
Youn et al., 2005). However,
an important caveat to this assay is the fact that not all mammospheres are
derived from regenerative stem cells. Using normal human mammary epithelial
cells, some mammospheres are derived from multipotent cells, giving rise to
both myoepithelial and luminal cell types upon differentiation in culture.
These multipotent mammosphere-initiating cells are tacitly assumed to
represent regenerative stem cells. Other mammospheres are derived from
self-renewing, lineage-restricted progenitors that are capable of
differentiating into myoepithelial-only or luminal-only cell colonies. These
mammosphere-initiating cells are thought to represent more-differentiated
downstream progenitor cells that should not have regenerative potential.
Unfortunately, the regenerative potential of either of these populations
cannot be formally tested with existing xenograft technology; thus, the
frequency of regenerative stem cells in the normal human mammary gland is not
known.
Fortunately, the technical limitations inherent in the use of human mammary epithelial cells do not apply to mouse mammary epithelial cells, where it is possible to assay directly whether mammospheres contain (and are therefore likely to be derived from) regenerative stem cells using single mammosphere transplantation, as well as to estimate directly the proportion of regenerative stem cells present in the mammary gland. According to our results using mouse cells, the proportion of mammospheres containing regenerative stem cells is approximately 15-33%. Thus, the remaining 67-85% of mammospheres are likely to be derived from downstream progenitor cell types lacking regenerative potential. To our knowledge, these are the first data to demonstrate directly the regenerative capacity of single mammospheres.
Given that at least three sub-populations of mammosphere-initiating cells
are known to exist in humans, and that similar subsets of cells are likely to
exist in mice, we do not believe that our mammosphere-formation results
showing a 2-fold increase in mammosphere-formation efficiency, and our
limiting-dilution transplantation data showing a 2.5-fold decrease in the
frequency of regenerative stem cells in MMTV-SmoM2 mice, are
contradictory. Our interpretation of the mammosphere-formation versus
limitingdilution transplantation data is that expression of
MMTV-SmoM2 may favor differentiation of stem cells into downstream
proliferating progenitor cell pools. From our in vivo gene expression data, we
present evidence that we increase a pool of proliferative
ER-CK6- cells, as well as a pool of
ER+CK6+ cells that divides at a lower frequency. These
proliferating cell pools may be capable of mammosphere-formation, but be
incapable of regenerating a mammary gland upon transplantation.
A potential limitation of secondary mammosphere-formation assays for the
evaluation of changes in self-renewal capacity in MMTV transgenic mice was
revealed. In our hands, transgene expression was not detectable in primary
mammospheres by immunofluorescence (not shown). Because of this, secondary
mammosphere-formation assays to test for changes in stem cell selfrenewal as a
consequence of transgene expression during primary mammosphere culture were
not considered reliable. Thus, it is difficult to compare our mouse
mammosphere-formation data directly with those of Liu et al.
(Liu et al., 2006), who
demonstrated increased primary and secondary mammosphere formation in response
to treatment with recombinant SHH ligand using human cells. We are currently
repeating these experiments using mouse primary mammary epithelial cells to
reconcile these two datasets.
With respect to human breast disease, there are some data to suggest a role
for altered hedgehog signaling in mammary cancer. An early study found
Ptch1 mutations in two of seven human breast cancers
(Xie et al., 1997).
Additionally, a Ptch1 polymorphism was linked to increased breast
cancer risk associated with oral contraceptive use
(Chang-Claude et al., 2003).
More recently, array comparative genomic hybridization (CGH) analyses indicate
that genomic loss at the Ptch1 locus was the fourth most commonly
detected change among the tumor suppressor genes identified in the study,
occurring in 19% of human breast cancers and in 33% of breast cancer cell
lines (Naylor et al., 2005).
However, no mutations in other network components have been identified in
breast cancer (Vorechovsky et al.,
1999). A recent immunohistochemical staining study suggested that
hedgehog signaling is activated in a majority of human invasive breast cancers
based on ectopic expression of PTCH1 and nuclear GLI1
(Kubo et al., 2004). However,
two other studies show loss of PTCH1 in many cases, perhaps owing, in part, to
promoter methylation (Kubo et al.,
2004; Mukherjee et al.,
2006; Wolf et al.,
2007). Finally, the hedgehog signaling inhibitor cyclopamine
inhibited growth of some breast cell lines in vitro
(Kubo et al., 2004;
Mukherjee et al., 2006).
However, the specificity of cyclopamine at the doses required for growth
inhibition remains an open question
(Mukherjee et al., 2006).
Our data showing loss of PTCH1 protein expression in 50% of DCIS and
IBC are most consistent with the array CGH results, as well as with recent
immunostaining and methylation data (Kubo
et al., 2004; Mukherjee et
al., 2006; Wolf et al.,
2007), but differ significantly from the immunohistochemical study
by Kubo et al. (Kubo et al.,
2004). The reason for the discrepancies among these studies is
unclear, but may be related to the different antigen retrieval strategies
used. To date, the preponderance of the data indicates that PTCH1 is lost or
reduced in 50% of all breast cancers. Altered SMO expression in human
breast cancer has not been demonstrated previously. Given that SMO was not
detectable in normal human or mouse tissue, but was readily detectable in
MMTV-SmoM2 mice, we are confident of the specificity of the two
antibodies used.
Our observation that SMO protein expression does not colocalize frequently
with proliferation markers in either the MMTV-SmoM2 mouse model or in
human breast tumors was unexpected. These observations must be reconciled with
data suggesting a direct role for hedgehog signaling in normal human stem cell
self-renewal (Liu et al.,
2006), and with reports that hedgehog signaling activation appears
to increase proliferation directly in other cell types (e.g.
Detmer et al., 2005;
Hutchin et al., 2005;
MacLean and Kronenberg, 2005;
Palma et al., 2005). In any
case, the similarity in staining patterns of SMO relative to proliferation
markers in both the mouse and human models suggests that our
MMTV-SmoM2 transgenic model reflects important aspects of human
breast tumor biology, and that it will therefore be a useful model for
studying the underlying mechanism of hedgehog network regulation of
stem/progenitor cell behavior in mammary gland development and breast
cancer.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1231/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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