Estrogen expands breast cancer stem-like cells through
paracrine FGF/Tbx3 signaling
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Citation
Fillmore, C. M., P. B. Gupta, J. A. Rudnick, S. Caballero, P. J.
Keller, E. S. Lander, and C. Kuperwasser. “Estrogen expands
breast cancer stem-like cells through paracrine FGF/Tbx3
signaling.” Proceedings of the National Academy of Sciences
107, no. 50 (December 14, 2010): 21737-21742.
As Published
http://dx.doi.org/10.1073/pnas.1007863107
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Wed May 25 19:02:29 EDT 2016
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http://hdl.handle.net/1721.1/84629
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Detailed Terms
Estrogen expands breast cancer stem-like cells
through paracrine FGF/Tbx3 signaling
Christine M. Fillmorea,b, Piyush B. Guptac,1, Jenny A. Rudnickb,d, Silvia Caballerob,d, Patricia J. Keller b,d, Eric S. Landerc,e,
and Charlotte Kuperwassera,b,d,2
a
Genetics Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111; bMolecular Oncology Research
Institute, Tufts Medical Center, Boston, MA 02111; cBroad Institute of MIT/Harvard and Department of Biology, Massachusetts Institute of Technology,
Cambridge, MA 02142; dDepartment of Anatomy and Cellular Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine,
Boston, MA 02111; and eDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115
Edited by Geoffrey M. Wahl, Salk Institute for Biological Studies, La Jolla, CA, and accepted by the Editorial Board October 26, 2010 (received for review June
17, 2010)
M
ore than 70% of breast cancers express high levels of the
estrogen receptor (ERα), and many of these tumors require estrogen for sustained growth and progression. In recent
years, multiple reports have shown that subpopulations of socalled cancer stem cells (CSCs; also called stem-like cells or
tumor-initiating cells) are also required for sustained tumor
growth and progression, and may be responsible for cancer recurrence and metastasis (1). Whether such CSCs in ERα+ breast
cancers are sensitive to estrogen is currently unknown.
Breast CSCs, which are operationally defined based on the
number of self-renewing cells required to initiate a tumor and
drive long-term tumor growth when transplanted into mice, can
be isolated from primary tumor tissue or cultured cells lines (2–7).
In human breast cancers, CSCs appear to be enriched within cell
subpopulations with a CD44+/CD24−/low/ESA+ surface marker
profile, are better able to form colonies, or “tumorspheres,” under
low-adherence conditions, and display increased resistance to
chemotherapeutic compounds (2–7).
The molecular mechanisms that regulate breast CSC frequency,
localization, and maintenance remain poorly understood. However, a fair amount is known about the spatio-temporal signaling
dynamics that govern the specification and maintenance of normal
mammary gland stem cells. Embryonic development of the mouse
mammary gland begins when Wnt and FGF signaling proteins,
which are secreted by the underlying mesenchyme, induce placode
formation and localize mammary epithelial fate specification (8).
FGF ligands, acting through cognate receptors, activate the Tbx3
transcription factor in both the mesenchymal and mammary
placodes. Tbx3, in a positive-feedback loop, activates additional
FGF secretion and also Wnt signaling (9–12).
During puberty, estrogen is responsible for maturation of the
mammary gland by mediating ductal elongation (9–13). Interestingly, there is significant evidence to suggest that estrogen
signaling does not act directly on adult mammary epithelial stem
www.pnas.org/cgi/doi/10.1073/pnas.1007863107
cells but, rather, activates their proliferation through paracrine
signaling (14, 15). These data imply a two-component mammary
stem cell niche in which estrogen signaling in the ERα+ nonstem cell compartment stimulates the proliferation of cells within
the ERα− stem cell compartment.
In breast cancer, it is unclear whether stem-like cells are also
regulated by specific hormone-growth factor paracrine signaling pathways. In this study, we discovered that estrogen regulates
breast CSC numbers through the FGF/Tbx3 signaling pathway, which happens also to regulate normal embryonic breast
stem cells.
Results
Estrogen Stimulation Induces Expansion of Breast Cancer Stem-Like
Cell Subpopulations. To study the signaling pathways that regulate
breast CSC expansion and maintenance, we needed an experimental system that allowed for consistent modulation of breast
CSC numbers through defined signaling perturbations. Tumor
initiation by the MCF7 cell line appears to rely on estrogen
signaling; these cells are very poor at forming tumors in ovariectomzed mice (16). However, we and others have found that
MCF7 cells can proliferate in vitro in the absence of estrogen
(E2) if serum (even charcoal-stripped serum) is supplemented in
high enough concentrations (Fig S1A). MCF7 cells grown under
these conditions maintain a low percentage of CSCs as gauged
by flow cytometry (Fig. S1B) and are likewise poor at forming
tumors in ovariectomized mice. Yet, intact ovaries or estrogen
supplementation allows even an estrogen-deprived MCF7 line to
form tumors, suggesting that estrogen induces the survival or
expansion of MCF7 CSCs.
To determine whether estrogen could indeed induce CSC expansion, we treated MCF7 cells as well as other estrogen receptorpositive (ER+) cell lines (T47D, HCC1428) with 1 nM 17-βestradiol or ethanol (vehicle control) for 6 d, and evaluated the
proportion of stem-like cells by flow cytometry and sphere formation assays. We found that after estrogen stimulation, the
proportion of CD44+/CD24−/ESA+ stem-like cells was nearly
eightfold higher in ERα+ cultures, whereas no significant change
in the proportion of CD44+/CD24−/ESA+ cells was observed
when the same culture conditions were imposed on cells that
lacked ER expression (Fig. 1A and Fig. S1C). When we challenged
Author contributions: C.M.F., P.B.G., J.A.R., and C.K. designed research; C.M.F., P.B.G.,
J.A.R., S.C., and P.J.K. performed research; C.M.F., P.B.G., and E.S.L. contributed new
reagents/analytic tools; C.M.F., P.B.G., J.A.R., S.C., and C.K. analyzed data; and C.M.F.,
P.B.G., P.J.K., and C.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. G.M.W. is a guest editor invited by the Editorial
Board.
1
Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
and Department of Biology, Massachusetts Institute of Technology, Cambridge MA
02139.
2
To whom correspondence should be addressed. E-mail: charlotte.kuperwasser@tufts.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1007863107/-/DCSupplemental.
PNAS | December 14, 2010 | vol. 107 | no. 50 | 21737–21742
MEDICAL SCIENCES
Many tumors contain heterogeneous populations of cells, only
some of which exhibit increased tumorigenicity and resistance to
anticancer therapies. Evidence suggests that these aggressive
cancer cells, often termed “cancer stem cells” or “cancer stem-like
cells” (CSCs), rely upon developmental signaling pathways that are
important for survival and expansion of normal stem cells. Here
we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast
CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast
cancer cell lines and freshly isolated breast cancer cells, whereas
cotreatment of cells with tamoxifen or a small molecule inhibitor
of FGFR signaling was sufficient to prevent the estrogen-induced
expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene
expression was able to abrogate tumorsphere formation, whereas
ectopic Tbx3 expression increased tumor seeding potential by 100fold. These findings demonstrate that breast CSCs are stimulated
by estrogen through a signaling pathway that similarly controls
normal mammary epithelial stem cell biology.
these MCF7 cultures to form tumorspheres, we found that the estrogen pretreated cultures formed sevenfold more tumorspheres
than the ethanol-pretreated cultures (Fig. 1B). Moreover, we
observed that the addition of the potent estrogen antagonist, 4hydroxy tamoxifen (4OHT), could prevent the 17-β-estradiol
induced expansion of CD44+/CD24−/ESA+ cells and sphere formation, indicating that these changes in marker expression and
sphere formation were mediated through ER signaling.
We next evaluated the ability of estrogen-pretreated MCF7
cultures to form tumors by injecting cells pretreated with estrogen
in vitro for 6 d into the mammary glands of ovariectomized NOD/
SCID mice in dilution series. Estrogen-pretreated cells were able
to form tumors in mice 100-fold more efficiently when compared
with the vehicle (EtOH+DMSO) treated cells (P = 0.001, Fig.
1C). Histological examination of tissue sections revealed that
MCF7 cells pretreated in vitro with estrogen formed invasive
ductal carcinomas (Fig. 1D). We also examined the injection sites
of MCF7 cells pretreated with ethanol that had not formed
tumors and observed viable cells within the mammary glands that
formed only benign epithelial structures, suggesting that lack of
tumor growth was not due to immune clearance of cells or increased cell death. These results indicate that estrogen-induced
expansion of cancer stem-like cells in vitro leads to a functional
increase in breast CSCs and tumorigenic phenotypes in vivo.
Estrogen Expands Breast CSCs via Paracrine-Acting Protein Factors.
No. cells injected
1.25
EtOH +
DMSO
1 nM E2
1 nM E2 +
10 M PD173074
0.75
1x106
1/4
3/4
2/4
0.25
0
1x105
1x104
1/10
2/10
5/10 *
6/10 *
3/10
1/10
EtOH
16
14
12
10
8
6
4
*
1nM E2
1nM E2
+100nM
4OHT
P < 0.0001
*
P = 0.001
D
In vitro pre-treatment
17- -estradiol
1x106
EtOH
2
0 EtOH
100nM
4OHT
untreated
1nM E2
1nM E2
+100nM
4OHT
1nM E2 pre-treated
-
Cells injected
Spheres per 1000 cells
In vitro pre - treatment
1.75
untreated
B
C
P < 0.0001
*
2.25
1x105
A
MCF7
% CD44+/CD24-/ESA+
Because ER activity appeared necessary for the expansion of
breast CSCs in response to estrogen, we next examined the levels
of ERα expression in the CD44+/CD24−/ESA+ stem-like MCF7
subpopulation. Using both immunofluorescence on freshly sorted cytospun cells and four-color flow cytometry, we found that
≥70% of the cells in the bulk fraction (CD44+/CD24+/ESA+)
were strongly ERα+, whereas only 20–25% of the CD44+/
CD24−/ESA+ stem-like cells had detectable nuclear ERα staining (Fig. 2 A and B and Fig. S2A). We also observed that ERα+
cells in the CD44+/CD24−/ESA+ population had much lower
100µm
100µm
100µm
Fig. 1. Estrogen increases cancer stem cells in ERα+ cell lines. (A) Average
percentage of CD44+/CD24−/ESA+ cells in the ERα+ cell line MCF7 following 6d treatment with either 1 nM 17-β-estradiol (E2) or vehicle (EtOH); n = 5 biological replicates. Data are mean ± SEM. (B) MCF7 tumorsphere formation
presented as the average number of spheres per 1,000 cells plated ± SEM; n =
3 biological replicates. Representative phase contrast micrographs of MCF7
spheres are shown. (C) Tumor formation of MCF7 cells pretreated with EtOH
and DMSO (vehicles), E2, or E2 and PD173074 injected in limiting dilution
into ovariectomized mice. *Nonparametric χ2 statistic was used to test the expected vs. observed frequencies of tumor formation at limiting dilution with
a level of 0.001 (critical value, 10.83). (D) Representative H&E-stained sections
of mammary glands injected with EtOH-pretreated or 1 nM estrogen (E2)pretreated MCF7 cells are shown.
21738 | www.pnas.org/cgi/doi/10.1073/pnas.1007863107
levels of staining than ERα+ cells from the bulk fraction (Fig.
2B, histogram).
Given these results, we hypothesized that in analogy to the
normal mammary gland, paracrine factors released by the ERα+
cells in response to estrogen stimulation might induce the expansion of CD44+/CD24−/ESA+ stem-like cells. To evaluate this
hypothesis, we harvested conditioned media from MCF7 cells
that were pretreated with either vehicle (EtOH) or 1 nM 17β-estradiol. We observed that MCF7 cultures fed estrogenconditioned media for 6 d contained 20-fold more CD44+/
CD24−/ESA+ cells than matched cultures fed vehicle conditioned
media (Fig. S2B, P < 0.002). In addition, expansion of this subpopulation was significantly attenuated if the conditioned medium was boiled before treatment of recipient lines (P < 0.05),
indicating that the factors promoting stem-like cell expansion
were heat labile and thus likely to be secreted proteins.
We tested whether conditioned media from estrogen-treated
MCF7 cells could increase CSC numbers in three ERα− breast
cancer lines, SUM149, SUM159, and BT-20. We observed that
exposure to conditioned media from estrogen-treated MCF7 cells
induced a statistically significant expansion of the CD44+/CD24−/
ESA+ stem-like cells in all three cell lines, yielding cultures that
were more efficient at forming tumorspheres (Fig. 2 C and D). In
sum, these data suggest that estrogen acts to induce secretion of
paracrine acting proteins, which in turn increase percentages of
CD44+/CD24−/ESA+ populations and corresponding cancer
stem-like cell properties in many breast cancer cell lines.
Estrogen Induces FGF9/FGFR3 Signaling to Increase Cancer Stem-Like
Numbers. To identify the secreted proteins mediating breast
cancer stem-like cell expansion following estrogen treatment, we
examined the conditioned media from either 17-β-estradiol–
treated or vehicle-treated MCF7 cells and quantitatively assayed
for 164 secreted growth factors and cytokines using an antibodybased protein array. In addition to known estrogen-induced
factors, we observed that the secretion of every assayed FGF
family member (FGF2/bFGF, FGF4, FGF6, FGF7, and FGF9)
was increased at least twofold upon estrogen treatment compared with ethanol-treated controls (Fig. S2C). Notably, FGF9,
which is induced by estrogen in endometriosis and during embryonic mammary placode formation (17), was increased 14-fold
following estrogen treatment of MCF7 cells.
We next tested whether FGF signaling was necessary for the
estrogen-induced expansion of the breast CSC-enriched subpopulation. Accordingly, we treated MCF7 cells with a chemical
inhibitor of FGFR signaling, PD173074, together with either estrogen or conditioned medium from estrogen-pretreated cells. By
flow cytometry, we observed that inhibition of FGF signaling
prevented either estrogen or conditioned medium from estrogenpretreated cells to elicit an increase in CD44+/CD24−/ESA+ cells
(Fig. 3A). In contrast, the addition of recombinant FGF9 to serumfree cultures was sufficient to increase the CD44+/CD24−/ESA+
subpopulation (Fig. S2D) and to promote tumorsphere formation
to levels comparable to those in estrogen-treated sphere cultures
(Fig. 3B). FGF9 and estrogen appeared to have a synergistic effect
on increasing MCF7 CSCs (Fig. 3A). When we tested two other
ligands from the FGF family, FGF2 and FGF10, we saw that although these factors did not increase the basal levels of CD44+/
CD24−/ESA+ cells in the cell line, they were able to slightly increase the effect of estrogen (Fig. S2E). In contrast, feeding candidate growth factors, including EGF, HRG, IGFII, BMP6, and
SDF1β, failed to increase the proportion of CSCs in the presence
or absence of estrogen supplementation (Fig. S2E).
There are four FGF receptors, and MCF7 cells express high
levels of FGFR3 (Fig. S3A), which binds with high affinity to
FGF9 (17). To rule out a nonspecific effect of the PD173074
compound, we examined whether the knockdown of FGFR3 in
MCF7 cells might also abolish estrogen-induced expansion of
the breast cancer stem-like cell populations. Accordingly, we
inhibited FGFR3 expression using lentiviral infection with targeted shRNAs. We observed a 76% reduction in FGFR3 protein
Fillmore et al.
B
CD44+/CD24 -/ESA+
ER
ESA
CD44
Negative
Bulk
Stem
10µm
Bulk
Stem
Shpere formation
% CD44+/CD24-/ESA+
700
EtOH
Conditioned Medium
ER
D
*
MCF7
BT-20
SUM149
SUM159
None
CD24
E2
SUM159
Shpere formation
C
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Gated on ESA+
ER
ESA
600
500
400
300
200
100
0
None
EtOH
E2
Conditioned Medium
expression in MCF7 cultures transduced with shFGFR3 (Fig.
S3B). Similar to treatment with PD173074, inhibition of FGFR3
expression in MCF7 cells led to a fourfold reduction in the
proportion of CD44+/CD24−/ESA+ cells and a twofold reduction in sphere formation in response to estrogen treatment
(Fig. S3C and Fig. 3C) without reducing estrogen-induced proliferation in adherent cultures (Fig. S3D).
To functionally assess whether inhibition of FGF signaling in the
presence of estrogen affected tumor formation, MCF7 cells were
pretreated for 6 d in vitro with estrogen in the presence of
PD173074 and injected into mice. Tumor-initiating potential
conferred by 17-β-estradiol pretreatment alone was abolished in
the presence of FGFR-inhibition (Fig. 1C, P = 0.001). These data
indicate that estrogen expands breast cancer stem cell numbers at
least in part through the FGF/FGFR signaling pathway.
To determine whether the FGF signaling pathway also regulates stem-like cell populations in ERα− breast cancer cell lines,
we added either recombinant FGF9 or PD173074 to SUM149,
SUM159, and BT-20 cultures. Treatment with FGF9 induced an
average 2.5-fold expansion of the stem-like cells and enhanced
tumorsphere formation, whereas inhibition of FGFR signaling
with PD173074 decreased the proportion of CD44+/CD24−/
ESA+ stem-like cells by eightfold and reduced sphere formation
(Fig. 3D and Fig. S3E). In addition, SUM159 cells pretreated in
vitro with PD173074 or FGF9 were injected orthotopically into
immunocompromised mice to evaluate tumor initiation. Indeed,
PD173074 pretreatment significantly inhibited SUM159-derived
tumor growth in vivo (P < 0.02, Fig. 3E).
We also isolated patient-derived breast carcinoma cells, and
treated these cells with either FGF9 or PD173074 in sphere
culture. We observed a modest 1.2-fold increase in tumorsphere
formation in response to treatment with FGF9 but a statistically
significant twofold reduction in sphere formation in the presence
of PD173074 (P < 0.01, Fig. 3F). Similarly, when we dissociated
freshly isolated tumors from human-in-mouse tumor generated
tissues (SI Materials and Methods), we found that these cells grew
significantly fewer sphere colonies in the presence of PD173074
than in the presence of FGF9 (Fig. S3F). Collectively, these data
demonstrate that FGF/FGFR signaling is an important regulator
of breast cancer stem-like cells.
Estrogen and FGF Signaling Induce Tbx3 Expression. The Tbx3
transcription factor has been reported to activate FGF signaling
but also act downstream of FGF signaling, where it is required
for propagation of FGF and Wnt signals in the rudimentary
mammary epithelium (8–10). Therefore, we wanted to determine
whether levels of Tbx3 correlated with estrogen or FGF signalFillmore et al.
200
180
160
140
120
100
80
60
40
20
0
SUM149
None
EtOH
E2
Conditioned Medium
Fig. 2. Paracrine factors produced in response to estrogen expand ERα− breast
CSCs. (A) Immunofluorescence of sorted
cytospun MCF7 cells for ERα (green) and
ESA/EpCAM (red) expression, counterstained for nuclei with DAPI (blue). (B)
Cytometric plots of ERα expression in
CD44+/CD24+/ESA+ cells (red, bulk), and in
CD44+/CD24−/ESA+ stem-like cells (green),
which comprise 2% of the culture. (C) Average percentage of CD44+/CD24−/ESA+
cells in ERα− SUM149, SUM159, and BT20
cultures following treatment with conditioned media from either ethanol (EtOH)
or E2-pretreated MCF7. MCF7 cells are
shown for reference, *P < 0.0001, n = 4
biological replicates. Data are mean ±
SEM. (D) Tumorsphere-forming potential
of SUM149 or SUM159 cultures described
in C; n = 4 biological replicates. Data are
mean ± SEM.
ing. Accordingly, we examined Tbx3 expression in MCF7 cultures treated with combinations of estrogen, tamoxifen, FGF9,
or PD173074. Indeed, Tbx3 mRNA and protein expression were
increased in MCF7 cells treated with estrogen and further increased by FGF9 stimulation (Fig. 4 A and B). This induction
was effectively inhibited by 4OHT or PD173074. Tbx3 protein
was also visualized by immuno-fluorescence, revealing nuclear
localization for the Tbx3 transcription factor in 60% of the
MCF7 culture following estrogen treatment (Fig. 4C).
We also examined the FGF-Tbx3 signaling axis in ERα−
SUM149, SUM159, and BT-20 breast cancer cells treated with
recombinant FGF9 or PD173074. Consistent with the findings in
MCF7 cells, Tbx3 mRNA and protein expression was induced in
response to FGF9 treatment (Fig. 4 D and E). Although treatment of the cultures with PD173074 did not affect the basal Tbx3
mRNA levels, protein levels appeared to be modestly decreased.
Taken together, these data indicate that: (i) estrogen stimulates
expansion of tumorigenic breast cancer cells in part through
FGF signaling, (ii) inhibition of FGF/FGFR signaling decreases
tumorigenic breast stem-like cells, and (iii) estrogen causes induction of Tbx3 expression breast cancer cells and is a likely
mechanism through which FGF signaling is perpetuated.
Tbx3 Expression Is Sufficient for Breast CSC Expansion. Because Tbx3
is known to be necessary for the specification and expansion of
normal mammary stem cells, we next examined whether Tbx3
might also be necessary for the expansion of breast cancer stemlike cells. Using an RNAi knock-down approach in three different
breast cancer cell lines (MCF7, SUM149, and SUM159), we were
able to reduce endogenous Tbx3 mRNA and protein levels 60–
85% (Fig. S4 A and B). It is known that the DUSP6 phosphatase is
activated following FGF signaling, and that the spatiotemporal
expression pattern of DUSP6 in the developing mammary gland is
similar to that of Tbx3 (10). Therefore, we also assayed expression
of DUSP6 and found that DUSP6 mRNA expression was reduced
an average of 2.4-fold in the shTbx3 transduced cultures. These
results are analogous to observations in embryonic mammary
epithelial cells showing that FGF signaling is required for Tbx3 and
DUSP6 expression and that Tbx3 expression is important for
further FGF production and signal propagation (8–10).
To assess the role of Tbx3 in breast CSC maintenance, we
performed flow cytometry and tumorsphere assays on cells
exhibiting the greatest inhibition of Tbx3 expression. We found
no significant difference in the proliferation rates of SUM149,
SUM159, or MCF7 breast cancer cells upon Tbx3 knockdown
(Fig. S4 C and D). However, the ability of MCF7 cells transduced
with shTbx3 to increase the proportion of CD44+/CD24−/ESA+
PNAS | December 14, 2010 | vol. 107 | no. 50 | 21739
MEDICAL SCIENCES
CD44+/CD24+/ESA+
A
60
40
20
Average tumor volume (mm^3)
s
hG
700
600
500
7
6
5
FGF9
Vehicle
e
FGF9
PD173074
E2 + PD
*
*
4
3
*
2
**
FG
D
**
SUM159
SUM149
sh
F
300
200
100
*
0
3
4
Time (weeks)
5
E2+
4OHT 4OHT
PD
E2+
PD
C
E2+
FGF9
ESA
Tbx3
DAPI
EtOH
10µm
2.5
2
1.5
1
0.5
0
-0.5
-1
-1.5
-2
-2.5
1nM E2
E2
4OHT
E2+
4OHT
PD
E2+
PD
E2+
FGF9
**
1
BT20
400
2
B
E2
EtOH
FR
SUM159
untreated
FGF9 pre-treat
PD173074 pre-treat
E2
Actin
0
3
FP
EtOH+
DMSO
Percent Tbx3+ Nuclei
80
A
Tbx3
140
Patient-derived tumor
sphere fomration
Sphere formation
100
*
*
mRNA level (log2)
D
120
0
E
E2
conditioned
medium
450
400
350
300
250
200
150
100
50
0
120
mRNA level (log2)
*
1nM E2
C
B
**
PD173074
Sphere formation
no inhibitor
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
% CD44+/CD24-/ESA+
% CD44+/CD24-/ESA+
A
3
Tbx3
2.5
2
1.5
1
0.5
0
SUM149 SUM149
+ FGF9
+ PD
100
80
*
60
E
1.6
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1.4
SUM159
-
1.2
1
+FGF9
+PD
Tbx3
0.8
0.6
-Actin
0.4
0.2
SUM149
0
SUM159 SUM159
+ FGF9 + PD
-
-0.2
-0.4
BT20
+ FGF9
BT20
+ PD
+FGF9
+PD
Tbx3
-Actin
40
20
0
untreated DMSO
PD173074 FGF9
6
Fig. 3. FGFR signaling is necessary for estrogen CSC expansion. (A) Average
percentage of CD44+/CD24−/ESA+ cells in MCF7 cultures treated with 1 nM 17β-estradiol (E2) or E2-conditioned medium in the presence of the FGFR inhibitor PD173074. n = 6 Biological replicates for fresh media, *P < 0.0001; n = 4
biological replicates for conditioned media, **P < 0.005, Data are mean ± SEM.
(B) MCF7 cells pretreated E2, FGF9 (100 ng/mL), or E2 and PD173074 were
seeded for tumorspheres and resulting spheres, Data are mean ± SEM, n = 4
biological replicates. *P = 0.01 either E2 or FGF9 vs. EtOH. (C) Sphere formation
of estrogen-pretreated MCF7 cultures transduced with indicated small hairpins. (D) Flow-cytometric analysis of CD44+/CD24−/ESA+ cells in ERα− SUM149,
SUM159, BT20 cultures following treatment with either recombinant human
FGF9 or the FGFR inhibitor, PD173074. MCF7 cells treated with E2 are shown as
reference. Data are mean ± SEM; n = 4 biological replicates. *P < 0.004 FGF9 vs.
vehicle; **P < 0.0005 PD vs. vehicle. (E) Tumor formation of 104 SUM159 cells
pretreated with DMSO, FGF9, or PD173074 injected orthotopically into mice;
n = 12 for each treatment. *P < 0.02 DMSO vs. PD. Data are mean ± SEM. (F)
Tumorsphere formation of breast cancer cells isolated from a primary human
breast cancer (TUM177) treated with FGF9 or the FGFR inhibitor PD173074,
*P = 0.01 DMSO vs. PD. Data are mean ± SEM.
cells or increase sphere formation following estrogen was significantly attenuated (Fig. 5 A and B). Likewise, a 20–50%
reduction in cancer stem-like cells and tumorsphere formation
was observed in shTbx3 transduced SUM149 and SUM159 lines
(Fig. 5 A and B). Furthermore, Tbx3 was inhibited using two
different hairpins in patient-derived cancer cells and also resulted in a significant reduction in sphere formation (Fig. 5C).
Notably, we were unable to maintain efficient knockdown of
Tbx3 in any cell line for more than two passages following selection; therefore, we could not assess in vivo tumor seeding
ability of shTbx3 cells. Consequently, we took an alternative
approach and ectopically overexpressed Tbx3 in normal human
mammary epithelial cells (HMECs) and MCF7 cancer cells to
determine whether Tbx3 expression would suffice to promote
stem-like cell behavior. Indeed, expression of Tbx3 resulted in a
∼twofold increase in the number of spheres formed by HMEC
cells and increased the proportion CD44+/CD24−/ESA+ cancer
stem-like cells in MCF7 cells by ninefold (Fig. 5D and Fig. S4E).
Furthermore, overexpression of Tbx3 in MCF7 cells led to a robust
twofold increase in tumorsphere formation (Fig. 5D). Consistent
with the expansion of cancer stem-like cells, overexpression of
Tbx3 in MCF7 cells resulted in a 100-fold increase in tumor21740 | www.pnas.org/cgi/doi/10.1073/pnas.1007863107
Fig. 4. FGF/Tbx3 signaling is intact human breast cancer cells. (A) Western blot
of Tbx3 in MCF7 cultures treated with vehicle (EtOH+DMSO), 1 nM E2, 100 ng/mL
FGF9, 100 nM 4OHT, or FGFR inhibitor 10 μM PD173074. (B) Quantitative RT- PCR
of Tbx3 expression in the same MCF7 cells assayed in A. Data are represented as
average delta (deltaCt) ± SEM; n = 4 experiments. (C) Immunofluorescence of
MCF7 cells treated with 1 nM E2 or EtOH vehicle; ESA/EpCAM (green), Tbx3 (red),
and DAPI (blue) show nuclear localization of Tbx3. Quantification is shown
below. (D) Quantitative RT-PCR analysis of DUSP6 and Tbx3 expression in
SUM149, SUM159, and BT-20 cultures treated with FGF9 or PD173074 relative to
expression in cultures treated with DMSO. Data are represented as average
delta (deltaCt) ± SEM; n = 4 biological replicates. (E) Western blot analysis of
Tbx3 expression in SUM149 and SUM159 cells described in D.
initiation compared with control cells (Fig. 5E, P = 0.001).
Collectively, these findings indicate that Tbx3 is sufficient to promote normal and cancer stem-like cell phenotypes.
Expression of FGFR3 and Tbx3 in Human Breast Cancers. Our results
suggest that paracrine FGF signaling mediated through Tbx3 is
important in regulating the proportion of CSCs within cultured
breast cancer populations. To determine whether this mechanism
might also operate within the context of primary human breast
cancers, we queried a gene expression database that encompasses
more than 18,000 human cancer gene expression microarrays (18,
19) for FGFR3 and TBX3 expression. We found that TBX3 was
highly expressed in many subtypes of breast cancer when compared with normal tissue, and that Tbx3 expression correlated with
ER-positive tumors. Furthermore, Tbx3 expression was highly
correlated with metastatic recurrence at both 3 and 5 y, whereas
Stage III tumors had a high correlation with genomic amplification
of the Tbx3 locus (Figs. S5 and S6 A and B). These data are consistent with and support other recent findings that TBX3 is upregulated in human breast cancers (16). In addition, we found that
breast tumors that responded to chemotherapy expressed significantly lower levels of Tbx3 than nonresponders, and that cell lines
that are sensitive to chemotherapies likewise have much lower
Tbx3 expression relative to chemotherapy-resistant cell lines (Fig.
S6 C and D). Furthermore, ERα expression levels were strongly
correlated with FGFR3 expression in a majority of primary tumor
samples (P = 0.001, Fig. S5). These data are compatible with the
notion that the E2/FGF/Tbx3 signaling axis is activated in many
primary breast cancers.
Discussion
Here, we identify the estrogen/FGF/Tbx3 signaling axis as an
important modulator of CSC properties both in vitro and in vivo.
Fillmore et al.
MCF7
Empty vector
80
60
*
**
20
# of cells injected
100µm
1x10
1x104
5
2/8
1/8
8/8 *
7/8 *
1x103
1x102
2/8
0/8
3/8
0/8
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)
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100µm
sh
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M
14
9
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)
0
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100µm
40
350
300
250
200
150
100
50
0
*
EV
Tbx3
100µm
MCF7 sphere formation
20
100
300
250
200
150
100
50
0
While much of our data were collected using the experimental
system of the ERα+ MCF7 cell line, we were able to observe upregulation of Tbx3 in many different primary human tumor
datasets, suggesting the relevance of this pathway in primary
tumor samples. In addition, we observed conservation of the
FGF/FGFR3/Tbx3 signaling pathway in basal-type ERα− cell
lines, as well in freshly dissociated patient tissue, indicating that
this pathway may be important for growth of many subtypes of
breast cancers other than the common ERα+ subtype.
The experiments described here also demonstrate that the
regulation of breast CSCs are influenced by the same regulatory
pathways that control stem cells in the developing mammary
gland. Although the underlying basis for this connection is unclear, several other groups have observed a conservation or reexpression of developmental signaling programs in cancers and
cancer stem cells (20–22). Based on the results described here,
we propose a model in which Tbx3 expressing cancer cells promote the expansion of CSCs through paracrine FGF signaling
(Fig. S7). In ERα+ breast cancer cell lines, estrogen (E2) binds
to the estrogen receptor to induce FGF9 secretion and Tbx3
expression in the non-CSC compartment. Expression of Tbx3
leads to further expression of Wnts and FGFs to perpetuate
signaling, which ultimately leads to expansion of the CSC pool.
In breast cancers that do not express ERα, Tbx3 expression
stabilizes paracrine FGF and Wnt signaling to regulate cancer
stem cell (CSC) subpopulations. This model is consistent with
prior work showing that Wnt signaling is necessary for maintenance
of subpopulations with stem-like properties in normal mammary
tissues and breast cancer (23). Clearly, further studies will be required to determine whether Tbx3 expression is induced by Wnt
signaling in cancer and whether inhibition of this pathway will have
a clinical impact.
The experiments described here demonstrate that estrogen can
also influence the representation of breast CSCs within cancer cell
populations, in part through its effects on the extracellular signaling milieu. Similar observations have been made in the normal
mouse mammary gland, in which epithelial stem cell function is
controlled in part through RANKL and Wnt4, which are secreted
in response to both estrogen and progesterone (24, 25).
Fillmore et al.
**
EV
Tbx3
Fig. 5. Tbx3 is necessary and sufficient for breast CSC expansion. (A) (Left) Average percentage of CD44+/CD24−/ESA+ cells
in MCF7 cultures transduced with lentiviruses encoding short
hairpins targeting a scrambled sequence (Cntrl), GFP, or Tbx3
and treated with 1 nM 17-β-estradiol (E2) or vehicle (EtOH).
*P < 0.0015. (Right) Average percentage of CD44+/CD24−/ESA+
cells in SUM149 and SUM159 cultures transduced with lentiviruses encoding short hairpins targeting Tbx3 and treated with
recombinant FGF9. Data are mean ± SEM; n = 4 biological
replicates. *P < 0.003; **P < 0.007. (B) Normalized tumorsphere-forming potential of SUM159, SUM149, or MCF7 cultures transduced with hairpins targeting a scramble sequence
(Cntrl) or Tbx3. Data as mean ± SEM; n = 4 experiments. *P <
0.001; **P < 0.005; ***P < 0.02. (C) Normalized tumorsphere
formation of breast cancer cells isolated from a primary human
breast cancer (TUM177) transduced with lentiviruses containing two different short hairpin sequences targeting Tbx3. *P <
0.002; **P < 0.0008. (D) Normalized sphere-forming ability of
immortalized human mammary epithelia cells (HMEC) or MCF7
cells ectopically overexpressing human Tbx3; n = 4 experiments, 2 biological replicates. *P = 0.002; **P = 0.003. (E) Tumor formation of MCF7 cells overexpressing Tbx3 or empty
vector (EV) injected in limiting dilution into NOD/SCID mice.
*Nonparametric χ2 statistic was used as described in Fig. 1.
The experiments described here as well as in other studies
have demonstrated that normal and cancer breast stem cell pools
lack abundant ERα expression (24–26, 12). This suggests that the
successes of tamoxifen and aromatase inhibitors, such as letrozole, for the treatment estrogen-sensitive breast tumors may be
attributed to the inhibition of paracrine factors released by
ERα+ breast cancer cells but not to the eradication of ERnegative CSCs. Indeed, residual breast cancer cells in tumor
tissues treated with letrozole exhibited a pronounced enrichment
of cells exhibiting CSC phenotypes (27). This observation,
combined with our findings, suggests that resistance to antiestrogen therapies and recurrence of ERα+ breast cancers could
arise from genetic or epigenetic alterations that allow for the
acquisition of FGF/Tbx3 activity in the absence of continued
estrogen stimulation. In support of this notion, tamoxifen resistance by breast cancer cells is accompanied by increases in
DUSP6 expression (28), as well as mesenchymal transdifferentiation (29). Furthermore, studies have shown that overexpression
of FGF ligands subverts the requirement for estrogen to drive
tumor formation (30–32).
Although our experiments focused on breast cancer CSC expansion stimulated by FGF9, we found that other FGF ligands
are also capable of influencing CSC numbers. We did not address here whether other FGFRs in addition to FGFR3 can
contribute to breast CSC expansion. Nevertheless, an important
prediction of our model is that the acquisition of resistance to
anti-hormone therapies might be accompanied by an increase in
FGF/FGFR/Tbx3 signaling and a concomitant increase in the
proportion of CSCs. Therefore, targeting the FGF/FGFR/Tbx3
pathway may be a useful therapeutic strategy for hormonetherapy refractory luminal (ERα+) breast cancers.
Materials and Methods
Detailed methods are described in SI Materials and Methods.
Cells and Tissue Culture. Cell line procurement and culture is described in
SI Materials and Methods.
All human breast tissue was obtained in compliance with the laws and
institutional guidelines, as approved by the institutional institutional review
board committee from Tufts University School of Medicine. An ER+, Her2−
tumor was obtained from discarded material, and noncancerous breast tissue
PNAS | December 14, 2010 | vol. 107 | no. 50 | 21741
MEDICAL SCIENCES
rl
sh
sh
Tb
x3
nt
C
rl
nt
sh
sh
D
Tbx3
***
M
15
9
SUM149
HMEC
HMEC sphere formation
40
Patient-derived
sphere fomration
**
SU
*
120
80
SU
*
C
Tb
x3
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sh
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C
100
*
**
SUM159
shCntrl
shTbx3
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FP
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0
untreated
+ FGF9
7
6
5
4
3
2
1
0
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%CD44+/CD24-/ESA+
2
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**
*
EtOH
E2
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nt
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ce
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A
was obtained from patients undergoing elective reduction mammoplasty
at Tufts Medical Center. Cells were manipulated as described in SI Materials
and Methods.
L-glutamine and 10% charcoal-dextran–stripped FBS and fed to naive cells
for a total of 6 d, with media changed every 2 d, after boiling for 5 min
where specified.
Flow Cytometry. Antibodies used are EpCAM (ESA)-FITC (clone VU-ID9, AbD
Serotec), CD24-PE (clone ML5, BD Pharmingen), and CD44-APC (clone G44-26,
BD Pharmingen). When staining for ERα-FITC (clone SP1, Abcam) cells were
stained sequentially with EpCAM (clone VU- ID9, Abcam), rat–anti-mouse
PerCP (BD Pharmingen) and CD24-PE/CD44-APC (BD Pharmingen) before
cells were fixed in 4% paraformaldehyde and 0.1% Saponin and incubated
with ERα-FITC.
Western Blot and Immunofluorescence. Antibodies used for IF were ERα-FITC
(clone SP1, Abcam), EpCAM (clone B29.1, Abcam), and Tbx3 (rabbit, Aviva).
Antibodies used for Western blotting were Tbx3 (mouse, Abcam), FGFR3
(rabbit, Sigma), and β-actin (clone mAbcam 8226, Abcam).
Tumorsphere Assays. Cells were trypsinized and mechanically separated and,
when necessary, passed through 40-μm filters to obtain single cell suspensions
that were plated at less than 10,000 cells per mL in super–low-attachment
plates in normal growth media (with supplements where indicated). Quantification of mammosphere and tumorsphere numbers was accomplished using
a Multisizer 3 Coulter Counter (Beckman-Coulter) that provided number and
size distributions of particles between 40 μm and 336 μm.
Isolation of RNA, Microarray, and Quantitative RT-PCR. Cells were harvested by
trypsinization of fluorescence-activated cell sorting and pelleted by centrifugation, and RNA isolation was performed using the RNAeasy kit (Qiagen) in
accordance with the manufacturer’s protocol. The RNA samples were then
reverse transcribed using the iScript cDNA kit (Bio-Rad), and quantitative
PCR was performed with Sybr green (Bio-Rad) on a Bio- Rad iCycler. Primers
are listed in SI Materials and Methods.
Conditioned Medium Experiments. Subconfluent MCF7 cultures grown in
standard phenol red containing DMEM with 10% FBS were washed and
switched to phenol-red–free DMEM + 10% charcoal-dextran stripped FBS
supplemented with 1 nM 17-β-estradiol or EtOH for 6 d. Cultures were then
washed five times with PBS and incubated with fresh serum-free phenolred–free DMEM. Conditioned medium (CM) was harvested 72 h later, passed
through a 0.2-μm filter, and frozen at −80°C. For each experiment, at least
three distinct batches of CM were combined and supplemented with 2 mM
ACKNOWLEDGMENTS. We thank Ina Klebba and Dr. Lisa Arendt for surgical
assistance and maintenance of the animal colony. We thank Allen Parmelee
and Steve Kwok for expert technical assistance with cell sorting. We thank
Josh LaBaer at Harvard Medical School (Boston, MA) for generously providing us with human Tbx3 cDNA. This work was supported grants from the
American Cancer Society–New England Division–Broadway on Beachside
Postdoctoral Fellowship PF-08-101-01-CSM (to P.J.K.), Breast Cancer Research
Foundation (to C.K. and C.M.F.), Raymond and Beverly Sackler Foundation
(to C.K.), and National Institutes of Health/National Cancer Institute Grant
R01CA125554 (to C.K.). C.K. is a Raymond and Beverly Sackler FoundationScholar.
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21742 | www.pnas.org/cgi/doi/10.1073/pnas.1007863107
Fillmore et al.
Supporting Information
Fillmore et al. 10.1073/pnas.1007863107
SI Materials and Methods
Cells and Tissue Culture. SUM cell lines were obtained from Dr.
Stephen Ethier (Karmanos Institute, Detroit) and are commercially available (Asterand). MCF7, HCC1428, T47D, and BT-20
cell lines were purchased from ATCC. MCF7, HCC1428, T47D,
and BT-20 cells were cultured in DMEM with 10% calf serum or
in phenol-red–free DMEM, 2 mM L-glutamine, and 10% charcoal-dextran–stripped FBS for experiments with estrogen stimulation. SUM149PT and SUM159PT cells were cultured in
Ham’s F-12 medium with 5% calf serum, insulin (5 μg/mL), and
hydrocortisone (1 μg/mL). All cell lines were grown at 37 °C in
a 5% CO2 incubator. Estrogen (17-β-estradiol) was dissolved in
ethanol to a stock concentration of 1 μM; PD173074 (Sigma) was
dissolved in DMSO to a stock concentration of 10 mM. All
treatments, including those with conditioned media, lasted 6 d.
All human breast tissue procurement for these experiments was
obtained in compliance with the laws and institutional guidelines,
as approved by the institutional institutional review board committee from Tufts University School of Medicine. An ER+, Her2−
tumor was obtained from discarded material, and noncancerous
breast tissue was obtained from patients undergoing elective
reduction mammoplasty at Tufts Medical Center. Breast tissues
were minced and enzymatically digested overnight with a mixture
of collagenase and hyaluronidase as previously described (1, 2).
Digested cells were plated briefly in serum (1–2 h) to deplete
mammary fibroblasts from the organoid fraction of mammary
fibroblasts. The organoids were dissociated to a single cell suspension by trypsinization and filtered with a 40-μm filter (BD
Biosciences) to remove residual clustered cells. Immediately
after dissociation, cells were assayed for mammospheres formation or were infected with lentivirus and then assayed. For the
human-in-mouse tumors, mammary epithelial cells from three
different patient samples were spin infected with lentivirusencoding MyrP110α, kRasG12V, p53R175H, and CCND1 and
implanted into humanized mouse mammary glands (1).
Flow Cytometry. Subconfluent cultures were trypsinized into single
cell suspension, counted, washed with PBS, and stained with
antibodies specific for the following human cell-surface markers:
EpCAM (ESA)-FITC (clone VU-ID9, AbD Serotec), CD24-PE
(clone ML5, BD Pharmingen); and CD44-APC (clone G44-26,
BD Pharmingen). For each staining reaction, 100,000 cells were
incubated with 4 μL of each antibody for 15 min at room temperature. Unbound antibody was washed off and cells were analyzed on a BD FACSCaliber no more than 1 h poststaining.
Isotype controls included mouse IgG1-FITC, mouse IgG2aκ-PE,
and mouse IgG2bκ-APC (BD Pharmingen). When staining for
ERα-FITC (clone SP1, Abcam) cells were stained sequentially
with EpCAM (clone VU-ID9, Abcam), rat–anti-mouse PerCP
(BD Pharmingen), and CD24-PE/CD44-APC (BD Pharmingen)
before cells were fixed in 4% paraformaldehyde and 0.1% Saponin and incubated with ERα-FITC.
Animals and Surgery. All animal procedures were conducted in
accordance with relevant national and international guidelines
and according to the animal protocol approved by the Tufts
University Institutional Animal Care and Use Committee. NOD/
SCID mice were purchased from Jackson Labs. Female mice 5–7
wk of age were ovariectomized and allowed to recover for 4 wk
before tumor cell injection. For tumor-seeding studies, the indicated numbers of MCF7 cells pretreated for 6 d with vehicle
(EtOH), 1 nM 17-β-estradiol (E2), or 1 nM 17-β-estradiol and
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
the FGFR inhibitor PD173074 (E2+ PD) were suspended in 1:1
(vol/vol) culture medium: Matrigel (BD Biosciences) mixture
and injected into the fourth inguinal mammary gland. For
SUM159 pretreatment experiments, intact 8-wk-old female
NOD/SCID mice were injected into the fourth inguinal mammary gland (n = 12 for each group) with 10,000 cells pretreated
for 6 d with PD173074 or with FGF9.
Tumorsphere Assays. Viable dissociated single cells (∼30,000/mL)
were plated in 6-cm ultra–low-attachment plates (Corning) in the
indicated media. Tumorspheres and mammospheres were allowed
to form for 5 or 8 d, respectively, after which spheres were collected
for analysis. Quantification of mammosphere and tumorsphere
numbers was accomplished using a Multisizer 3 Coulter Counter
(Beckman-Coulter) that provided number and size distributions
with an overall sizing range of 40 μm to 336 μm. Tumorspheres and
mammospheres were collected and pelleted at 800 rpm for 5 min
and resuspended in 1 mL freshly filtered growth media, diluted in
20 mL 6:4 isoton II:glycerol diluent (Beckman-Coulter), and run in
triplicate on the Multisizer 3.
Conditioned Medium Experiments. Subconfluent MCF7 cultures
were treated with 1 nM 17-β-estradiol or EtOH for 6 d in phenolred–free DMEM and 10% charcoal-dextran–stripped FBS (Invitrogen). Cultures were washed five times with PBS and incubated
with fresh serum-free, phenol-red–free DMEM. Conditioned
medium (CM) was harvested 72 h later, passed through a 0.2-μm
filter, and frozen at −80 °C. For each experiment, at least three
distinct batches of CM were combined and supplemented with
2 mM L-glutamine and 10% charcoal-dextran stripped FBS and
fed to naive cells for a total of 6 d, with media changed every 2 d,
after boiling for 5 min where specified.
Cytokine Array and Quantification. Serum-free CM was collected as
described above. Human cytokine arrays (2000 series, RayBiotech) were exposed to conditioned medium from MCF7 cultures
pretreated with either ethanol (vehicle) or estrogen and processed in accordance with the manufacturer’s protocols. Exposed
films of chemiluminescence signal obtained from dot blots were
scanned, and the pixel intensity for each cytokine was quantified
and normalized to IgG loading controls using ImageJ software
(National Institutes of Health).
Western Blot and Immunofluorescence. For immunfluorescence
(IF), cells were fixed in 4% paraformaldehyde and 0.1% saponin
and permeabilized with 0.1% BSA and 0.25% Triton-X, both in
PBS. Coverslips were mounted with Vectashield mounting medium plus DAPI (Vector Labs). Antibodies used for IF were
ERα-FITC (clone SP1, Abcam), DUSP6 (clone 3G2, Novus),
EpCAM (clone B29.1, Abcam), and Tbx3 (rabbit, Aviva). For
Western blotting, 25 μg protein extract per sample denatured
with heat and reducing agents, separated on a 4–12% acrylamide
gel, and transferred to nitrocellulose. Antibodies used for
Western blotting were Tbx3 (mouse, Abcam), FGFR3 (rabbit,
Sigma), and β-actin (clone mAbcam 8226, Abcam).
Isolation of RNA and Quantitative RT-PCR. Cells were harvested by
trypsinization, pelleted by centrifugation, and RNA isolation was
performed using the RNAeasy kit (Qiagen) in accordance with
the manufacturer’s protocol. The RNA samples were then reverse transcribed using the iScript cDNA kit (Bio-Rad), and
quantitative PCR was performed with Sybr green (Bio-Rad) on a
Bio-Rad iCycler. Primers used are: GAPDH F-GAGTCAAC1 of 8
isolated by miniprep (Qiagen). Lentiviral expression construct
for Tbx3 gene transduction was created using standard Gateway
cloning techniques into the self-inactivating pLenti6.2/V5DEST Gateway vector (Invitrogen). A WT human Tbx3 cDNA
clone (NM_016569.2–443) was generously provided by Josh
LaBaer (Harvard Institute of Proteomics, Harvard Medical
School. Boston, MA). The VSV-G-pseudotyped lentiviral vectors were generated by transient cotransfection of the above
vectors with the VSV-G-expressing construct pCMV-VSV-G
and the packaging construct pCMV DR8.2Dvpr (3), both generously provided by Inder Verma (Salk Institute), into 293T cells
with the FuGENE 6 transfection reagent (Roche). Viral supernatant was collected and introduced to subconfluent SUM149,
SUM159, MCF7, and HMEC cultures, or to patient-derived
breast cancer cells. Lentiviral integration was selected with 1 μg/
mL puromycin (for shRNAs), or with 10 μg/mL blasticidin (Tbx3)
for 7 d.
GGATTTGGTCGT R-GACAAGCTTCCCGTTCTCAG, Tbx3
F-TGGGGACCTCTGATGAGTCCT R-CCATGCTCCTCTTTGCTCTC, DUSP6 F-GCTATACGAGTCGTCGCACA RCGTCCTTGAGCTTCTTGAGC, Wnt5a F-GGGAGGTTGGCTTGAACATA R-GAATGGCACGCAATTACCTT, ERα FATTTGAAGTGGGCAGAGAACAT R-CAATACCAACATCAGCCAGAAA, FGFR3 F-ACTGGGGAACAGTGGATGTC
R-GGATGCCTGCATACACACTG, FGF9 F-TTTCTGGTGCCGTTTAGTCC R-GACTACCTGCTGGGCATCAA, Vimentin
F-AGATGGCCCTTGACATTGAG R-GGTCATCGTGATGCTGAGAA, N-Cadherin F-ACAGTGGCCACCTACAAAGG RCCGAGATGGGGTTGATAATG, E-Cadherin F-TGCCCAGAAAATGAAAAAGG R-GGATGACACAGCGTGAGAGA,
Zeb-1 F-GATCAACCACCAATGGTTCC R-TTGCGCAAGACAAGTTCAAG.
Lentiviral Constructs and Infection. Bacterial glycerol stocks of
MISSION shRNA were obtained (Sigma), and plasmid DNA was
1. Proia DA, Kuperwasser C (2006) Reconstruction of human mammary tissues in a mouse
model. Nat Protoc 1:206–214.
2. Wu M, et al. (2009b) Dissecting genetic requirements of human breast tumorigenesis in
a tissue transgenic model of human breast cancer in mice. Proc Natl Acad Sci USA 106:
7022–7027.
900
800
700
600
500
400
300
200
100
0
B
4
EtOH
% CD44+/CD24-/ESA+
Thousands of Cells
A
3. Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM (1998) Development of a selfinactivating lentivirus vector. J Virol 72:8150–8157.
E2
1
2
3
4
5
6
EtOH
3.5
E2
3
2.5
2
1.5
1
0.5
0
1
Days in Culture – Phenol Red Free DMEM
+ 10% Charcoal stripped FBS
2
3
4
5
6
Days in Culture – Phenol Red Free DMEM
+ 10% Charcoal stripped FBS
T47D
% CD44+/CD24-/ESA+
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
HCC1428
% CD44+/CD24-/ESA+
untreated
1nM E2
EtOH
*
20
18
16
14
12
10
8
6
4
2
0
1nM E2
+100nM
4OHT
% CD44+/CD24-/ESA+
*
C
p>0.5
2.5
2
p>0.5
1.5
1
0.5
0
EtOH
untreated
EtOH
1nM E2
SUM1315
SUM159
1nM E2
1nM E2
+10 nM
4OHT
Fig. S1. (A) MCF7 cells were seeded in six-well plates at 100,000 cells per well. The next day, the cultures were switched to phenol-red–free DMEM with 10%
charcoal-dextran FBS and either 1 nM estrogen or vehicle (EtOH). Each day, two wells per condition were trypsinized and counted. Average cell number per
well per day is shown. (B) Cells described in A were assayed daily for percentage of CD44+/CD24−/ESA+ cells by flow cytometry. (C) Average percentage of
CD44+/CD24−/ESA+ cells as measured by flow cytometry in the ERα+ cell lines T47D (*P < 0.01) and HCC1428 (*P < 0.0001), or in the ERα− cell lines SUM159 and
SUM1315, following 6-d treatment with either 1 nM 17-β-estradiol (E2) or vehicle (EtOH). Data are mean ± SEM; n = 5 biological replicates.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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E
4
3.5
3
2.5
2
1.5
1
0.5
0
*
EtOH
1nM E2
**
IGF-I
IGF-II
IGFBP-6
IGFBP-2
conditioned conditioned
medium medium boiled
EGF
F
IGFBP-4
fresh medium
boiled
FGF-9
fresh
medium
FGF-7
bFGF
bNGF
PBMP-7
PBMP-6
PBMP-5
PBMP-4
2
TGFb2
40
30
20
10
0
Fb
TGFb3
16
14
12
10
8
6
4
2
0
-2
-4
-6
-8
-10
p<0.0001
TGFa
% ER + cells
D
% CD44+/CD24-/ESA+
n relative
r
e tto EtOH
Fold Secretion
C
70
60
50
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
FGF-6
B
Bulk
% CD44+/CD24-/ESA+
Stem
90
80
FGF-4
A
*
**
EtOH
E2
FGF9
E2+FGF9 E2+PD
3.00
EtOH
E2
2.00
1.50
1.00
0.50
0.00
not assayed
not assayed
% CD44+/CD24-/ESA+
2.50
no growth 100ng/mL 100ng/mL 100ng/mL 100ng/mL 100ng/mL 100ng/mL 100ng/mL
factor
FGF2
FGF10
EGF
Heregulin
IGFII
BMP6
SDF1b
Fig. S2. (A) Quantification of ERα immunofluorescence staining on sorted and cytospun MCF7 cells. Graph represents percentage of ERα+ cells for from seven
fields per sort with an average of 56 nuclei per field ± SEM. (B) Average percentage of CD44+/CD24−/ESA+ cells as assayed by flow cytometry in MCF7 cultures
following 6-d treatment with fresh estrogen, vehicle (EtOH), or conditioned media from EtOH- or E2-pretreated MCF7 cells. *P < 0.001 E2 CM vs. EtOH CM.
Where indicated, unconditioned or conditioned media was boiled before feeding the cells. For unconditioned media containing fresh 1 nM 17-β-estradiol, the
estrogen was added before boiling to show its relative heat stability. *P < 0.04 E2 CM vs. E2 CM boiled. Data are mean ± SEM; n = 4 biological replicates. (C)
Cytokine array quantification of proteins secreted by MCF7 cells in response to estrogen (E2). All data are normalized to the respective IgG controls, and the
fold increase in secretion is shown as the E2 pixel value divided by the EtOH pixel value for matched exposure lengths. (D) Average percentage of CD44+/CD24−/
ESA+ cells in MCF7 cultures treated with 1 nM 17-β-estradiol (E2), recombinant human FGF9 (100 ng/mL), or FGF9 and E2 in the absence of serum. *P < 0.05
E2+FGF9 vs. E2; **P < 0.001 E2 vs. E2+PD. Data are mean ± SEM; n = 6 biological replicates. (E) Average percentage of CD44+/CD24−/ESA+ cells in MCF7 cultures
following 6-d treatment with EtOH vehicle, 1 nM 17-β-estradiol (E2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 10 (FGF10), human epidermal
growth factor (EGF), recombinant human heregulin (HRG), insulin-like growth factor 2 (IGFII), bone morphogenic protein 6 (BMP6), or stromal-derived factor 1β (SDF1β). Data are mean ± SEM.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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A
E
Sphere formation
FGFR3
DAPI
10µm
B
SUM159
SUM149
300
*
*
250
200
150
100
50
0
untreated
FGF9
pre-treat
FGF9
PD173074
pre-treat
PD173074
FGFR3
-Actin
F
DMSO
PD173074
FGF9
*
160
140
sphere formation
EtOH
E2
2.5
2
1.5
1
*
120
100
40
Tumor 2
Tumor 3
sh
G
FP
sh
FG
FR
3
nt
C
sh
ce
pt
y
du
Tumor 1
em
ns
rl
0
ve
ct
or
20
tra
*
60
0
un
*
80
0.5
d
%CD44+/CD24-/ESA+
C
D
Relative cell n umber
R
E2/EtOH cultures
3
2.5
2
1.5
1
0.5
un
tra
ns
em duc
ed
pt
y
ve
c
s h to r
C
nt
rl
sh
G
F
sh P
FG
FR
3
0
MCF7
Fig. S3. (A) Immunofluorescence of FGFR3 (red) and DAPI (blue) of untreated MCF7 cultures. (B) Western blot of FGFR3 expression in MCF7 cells transduced
lentivirus containing a small-hairpin directed to FGFR3. (C) Average percentage of CD44+/CD24−/ESA+ cells in MCF7 cultures treated for 6 d with 1 nM 17β-estradiol (E2) in cultures transduced with indicated small hairpins. *P = 0.001 shFGFR3+E2 vs. shCntrl+E2. (D) Proliferation rates of FGFR3 knockdown MCF7
cell lines in response to 1 nM 17-β-estradiol (E2). Data are shown as total cell number in E2-treated cultures normalized to cell number in matched EtOH-treated
cultures; n = 4. (E) Tumorsphere formation of SUM159 or SUM149 cells pretreated with recombinant human FGF9 or FGFR inhibitor PD173074, or treated while
making spheres with FGF9 or PD173074. Data collected at 6 d after seeding, *P < 0.002 FGF9 vs. vehicle. (F) Three different tumors derived from primary
mammary epithelial cells transformed with MyrP110α, kRasG12V, p53R175H, and CCND1 and implanted into humanized mouse mammary glands were dissociated into single cells and plated at low dilution on super–low-attachment plates in filtered MEGM ± DMSO, 100 ng/mL FGF9, or 10 μM PD173074. Spheres
were quantified on a Becton Dickonson Multisizer 8 d after seeding, and sphere formation was normalized to the DMSO condition; n = 12.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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mRNA level (log2)
A
MCF7
mRNA level (log2)
mRNA level (log2)
MCF7
-1
EV
-2
sh
sh
GF
P
Tb
x3
Tbx3
-3
Actin
n
-4
SUM159
0
SUM159
-0.2
-0.4
sh
-0.6
-0.8
-1
Cn
trl
sh
GF
sh
P
Tb
x3
Tbx3
-1.2
TBX3
DUSP6
-1.4
-1.6
Actin
SUM149
SUM149
0
sh
-0.2
-0.4
-0.6
Tbx3
-0.8
-1
Actin
Cn
trl
sh
GF
sh
P
Tb
x3
3
Relative
el
cell number
C
B
0
2.5
2
EtOH
E
1.5
0.14%
1
1nM E2
2.06%
0.5
un
tra
ns
em duc
ed
pt
y
ve
ct
sh or
C
nt
rl
sh
G
FP
sh
Tb
x3
0
MCF7
EV
TBX3
4.5
D
0.17%
4
2.59%
3
CD44
Mean OD600
3.5
2.5
2
1.5
CD24
1
0.5
SUM159
trl
sh
Tb
x3
sh
Cn
sh
Cn
trl
sh
Tb
x3
0
SUM149
Fig. S4. RNAi-mediated knock down of Tbx3. (A) RT-PCR of Tbx3 and DUSP6 expression (relative to shCntrl). (B) Western blot of Tbx3 expression in MCF7,
SUM159, and SUM149 cultures transduced with lentivirus containing short hairpins targeting a scrambled sequence (Cntrl), GFP, or Tbx3. (C) Proliferation rates
of Tbx3 knockdown MCF7 cell lines in response to 1 nM 17-β-estradiol (E2). Data are shown as total cell number in E2-treated cultures normalized to the cell
number in matched EtOH-treated cultures; n = 4. (D) Proliferation rates of SUM149 and SUM159 Tbx3 knockdown cultures as measured by Crystal Violet
staining. Cells were stained 6 d after being equally plated, and OD595 was measured on a spectrophotometer. Data are mean ± SEM. (E) Representative flow
cytometric dot plots of CD44 and CD24 expression in MCF7 cultures over-\expressing Tbx3. Empty vector control-transduced cultures are shown for comparison.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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A
Radvanyi et al, 2005
(NV, N=9)
(NB, N=7)
(DCIS, N=7)
Minn et al, 2005
(IDBC, N=7)
(ILBC, N=7)
(IMBC, N=3)
(ER-, N=42)
(ER+,N=57)
Chin et al.,2006
(ER-, N=43)
(ER+,N=75)
Lu et al.,2008
(ER-, N=50)
(ER+,N=45)
B
Fig. S5. TBX3 expression in human breast cancers. (A) Oncomine (Compendia Bioscience) was used for analysis and visualization of TBX3 expression in
published microarray data sets. TBX3 overexpression was observed in many invasive breast cancer subtypes when compared with normal breast tissue, and
correlates very highly with ER expression. DCIS, ductal breast carcinoma in situ; IDBC, invasive ductal breast carcinoma; ILBC, invasive lobular breast carcinoma;
IMBC, invasive mixed breast carcinoma; NB, normal breast; NV, no value. (B) Gene expression correlation analysis in a data set shows that a large group of
primary breast tumors coexpress high levels of ERα and FGFR3.
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A
Loi et al
Loi et al
P-value: 4.20E-4
1. No Metastatic Recurrence at 3 Years (78)
2. Metastatic Recurrence at 3 Years (8)
P-value: 0.002
1. No Metastatic Recurrence at 5 Years (78)
2. Metastatic Recurrence at 5 Years (8)
J Clin Oncol 2007/04/02
J Clin Oncol 2007/04/02
B
P value: 0.022
Chin et al. P-value:
1. Stage I (119) 2. Stage II (34)
3. Stage III (16)
Genome Biol 2007/10/07
D
Gyorffy et al. P-value: 0.019
C
Hess et al. P-value: 4.20E
1. P/FAC Non-responder (90)
2. P/FAC Responder (32)
J Clin Oncol 2006/09/10
Wooster et al. P-value: 0.023
-5
Hoeflich et al. P-value: 1.37E-5
1. Doxorubicin Resistant (6)
1. Paclitaxel Resistant (7)
1. Erlotinib Resistant (18)
2. Doxorubicin Sensitive (20)
2. Paclitaxel Sensitive (153)
2. Erlotinib Sensitive (3)
Int J Cancer 2006/04/01
Not Published 2008/05/09
Clin Cancer Res 2009/07/15
Fig. S6. TBX3 expression in tumors following response to therapy. Oncomine (Compendia Bioscience) was used for analysis and visualization of TBX3 expression in published microarray data sets grouped as follows: (A) metastasic recurrence at 3 and 5 y; (B) genomic amplification of Tbx3 locus and tumor stage;
(C) pathological complete response to therapy; and (D) sensitivity of breast cancer cells to common breast cancer therapeutics in vitro.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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E2
E2
ERα
FGF/FGFR
TBX3
TBX3
FGF
wnt
Luminal/ERα + Cells
FGF9
FGFR3
FGF9 FGFR3
E2
ERα
ERα
E2
ERα
ERα
FGF
Wnt
TBX3
ERα
TBX3
Basal/EMT Cells
Cells
Basal/EMT
FGF
FGF
Wnt
TBX3
TCF4
Wnt
Wnt
TBX3
TCF4
DUSP6
DUSP6
TBX3
Fig. S7. Proposed model of paracrine signaling within breast cancer cell lines. In ERα+ tumors, estrogen (E2) binds to the estrogen receptor in the non-CSC
compartment to induce paracrine FGF9 secretion. FGF9 then binds to FGFR3 and induces Tbx3 expression. Expression of Tbx3 leads to further expression of
Wnts and FGF/FGF signaling which promote CSC phenotypes. In ERα− tumors, it appears that FGFR/Tbx3 is active in the EMT-like cells, leading to stabilized
paracrine signaling that regulates cancer stem cell (CSC) subpopulations.
Fillmore et al. www.pnas.org/cgi/content/short/1007863107
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